Gel Electrophoresis Size Marker - AppliChem · The different gel formats for agarose and polyacrylamide gel electrophoresis and the varying sensitivity of staining or detec-tion mean

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Gel Electrophoresis Size Marker

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Gel Electrophoresis

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Detergents

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Immunoassay Buffer

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Kontaminationen durch Nukleinsäurendurch Nukleinsäurendurch

Probleme & praktische Lösungen NukleinsäurenProbleme & praktische Lösungen Nukleinsäuren

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WichtigesWissenswertesWunderbaresWunderbares

aus Chemie & Biologie& Biologie&

AppliCationsDer Anteil von molekularbiologischen Nachweismethoden ist in den letzten Jahren erheblich gestiegen, besonders in den Bereichen Qualitätskontrolle, Forensik, klinischer Forschung und Diagnostik – insbesondere der Infektionsdiagnostik. Gerade für diese Applikationen werden hochsensitive und gleichzeitig zuverlässige PCR-Tests benötigt. Dafür bietet AppliChem nun optimierte PCR-Kits an und widmet sich explizit der Hintergrundproblematik, die durch DNA belastete Reagenzien und Arbeitsplätze entstehen kann.

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Nr.3AppliCationsDNA-freie Reagenzien und Mastermixe für die PCR

AppliCationsAppliCations

Freie Nukleinsäuren verursachen als Kontaminationen große Probleme im Forschungs- und molekularbiologisch-analytischen oder klinisch-diagnostischen Labor. Durch die extrem hohe Sensitivität von DNA-Nachweistests, können kleinste Verunreinigungen in PCR-Ansätzen zusätzliche Arbeit bedeuten und im schlimm-sten Fall Ergebnisse verfälschen. Mit Derma-ExitusPlus™ (HHDK) aus der Serie von ExitusPlus™-Produkten wird erstmals ein völlig neuer Anwendungsbereich erschlossen bzw. zusätzliche Kontaminationsquellen ausgeschlossen.

Eine der Hauptquellen für Kontaminationen mit Nukleinsäuren ist der Experimentator selbst. Die Nukleinsäuren stammen z.B. aus Hautschuppen, Haaren und Speichel oder von Mikroorganismen, die seine Haut besiedeln oder z.B. beim Niesen freigesetzt werden. Gelangen diese in die PCR-Ansätze oder PCR-Reagenzien, können sie entsprechend der eingesetzten Primer (besonders 16S rDNA für Bakterien) leicht nachgewiesen werden. Ausserdem besteht die Gefahr, dass beim Öffnen und

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Nr.5AppliCationsDekontamination der Haut und Hände von Nukleinsäuren

AppliCations AppliCationsSize-exclusion chromatography (SEC) is a popular method to separate biomolecules based on their size. Primarily, it is applied to the separation of biopolymers such as proteins and nucleic acids, i.e. water-soluble polymers. This system is also called gel filtration, typically with beads of dextran or agarose serving as gel matrix. Smaller molecules pass significantly slower through the column than larger molecules. Not to be mixed up with gel electrophoresis, there are big differences in terms of theseparation principle. SEC does not require electric current and the sieving effect will not separate small molecules first.

It is indeed correct that smaller molecules pass more slowly through the matrix than larger molecules. This is due to the longer path the smaller molecules must travel. The longer path arises from the pores of the beads. The smaller molecules can

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No.6AppliCationsSize-Exclusion Chromatography for purification of biomolecules

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Agarose-Gel-Elektrophorese AppliCations

Using ready-to-use ELISA kits from manufacturers is easy and convenient. Sometimes however, home-made ELISA is required because there is no kit available with the right antibodies or the characteristics of the available kits such as their limits of detection are not appropriate. Ready-to-use ELISA kits from good suppliers mastored for two years at 4°C without any problem. With home-made ELISA it is a completely different story. For any new measurement one has to coat a new plate, because after storage of some days the plates don’t perform as well as before.

Why is there such a great difference in storage between home-made ELISA and ELISA kits?The reason is that in professional ELISA kit production the plates are not only blocked after coating, but also stabilised. This easy to perform process has been an industry standard for thirty years. For stabilisation of a plate one has to coating stabiliser solution. It is just as simple as a “second blocking step”. But there were no such high lutions freely available in low volumes for use in research lab until now. AppliChem now offers a product for use in every researchin volumes starting as small as 50 ml, which is called the AppliCoat Plate Stabiliser (Cat. No. A7708). This stabiliser solution is easy-to-use and has a great advantage compared to almost any stabiliser used in industry. It gives better storage stability for coated antibodies and antigens than most other products do. And there is a second

Two benefits with one solutionWhen antibodies are coated onto ELISA plates, most of the antibodies are not active. When the antibodies (or any proteins) come into close contact to the plastics surface of the ELISA plate, conformational changes can occur due to surface-protein interactions. The result is that most antibodies coated on a plate are unfolded or inactive. Only around 2–8 % of all coated antibodies remain active and can bind to analytes and this is greatly variable depending on the surface characteristics of the ELISA plate, which can really differ from batch to batch or even from well to well.These differences from well to well can affect the variability of an assay, because the antibodies can be affected. If there waway of refolding antibodies and of preserving antibodies from conformational changes during storage, this could help to decrease such variabilities in assay performance. This is a key benefit of AppliCoat Plate Stabiliser. It assists antibodies and coated proteins to refold and then to preserve active conformation over a long time. Thus it has two benefits: 1. Refolding of antibody conformation of some of the coated antibodies and 2. Preserving correct conformation during storage. These benefits are used for production of high-quality ELISA kits as well as in research applications now. Even with AppliCoat Plate Stabiliser the percentage of active antibodies will still be in the range of 2–8 %. But the great difference is that the variability from well to well and from plate to plate can be minimised in most assays by using AppliCoat Plate Stabiliser. Such effects depend on the used antibodies, but when ELISA are validated (e.g. according to “Guidance for Industry: Bioanalytical Method Validation”, FDA, 2001) or according to other validation strategies, the difference can be measured in many assays.The positive effects of AppliCoat Plate Stabiliser are shown in Fig. 1. A sandwich ELISA with a monoclonal antibody has been

Keywords

Immunoassays

Antibody Stabilisation

ELISA Plates

Cross-reactivity

Interfering effects

Improving quality of ELISA

AppliCationsDie moderne Gentechnik zeigt, dass in vielen Fällen schon freie DNA-Moleküle für Infektionen, Rekombinationen oder biologische Transformationen ausreichen [1,2]. Zusätzlich werden die Nachweisverfahren für DNA-Moleküle immersensitiver. Daher wird die Detektion von Kontaminationen oder die Verhinderung von Amplifikations-Artefakten in der PCR für die Gentechnik, die Kriminalistik, die Biomedizin und die Hygiene immer wichtiger. Die vollständige Dekontamination von Geräten und Materialien von DNA-Molekülen wird so zu einem entscheidendenFaktor für die allgemeine biologische Sicherheit.

Alles oder Nichts: Erstaunliche ErkenntnisDas Mittel der Wahl zur Beseitigung von Kontaminationen durch NukleinDas Mittel der Wahl zur Beseitigung von Kontaminationen durch NukleinDas Mittel der Wahl zur Beseitigung von Kontamina säuren ist immer noch Chlorbleichlauge („bleach“) – ein Mittel das alles zerstört, nicht nur die Nukleinsäure. Dies hat uns veranlasst in Kooperation mit multiBIND Biotech, Köln, nach einer unschädlichen Alternative zu suchen und die molekulare Wirkungsweise der auf dem Markt befindlichen sonstigen DNA-Dekontaminationsmittel zu untersuchen. Hierfür wurde unter sehr hoher Belastung (großer DNA-Über-

suchen. Hierfür wurde unter sehr hoher Belastung (großer DNA-Über-suchen. Hierfür wurde unter sehr hoher Belastung (großer DNA-Überschuss) mit definierten DNA-Kontaminationen die Eigenschaften der konventionellen Mittel verglichen. Zwei Probleme werden offensichtlich: Erstens werden durch die konventionellen Mittel in keinem Fall die DNA-Moleküle effizient zerstört und zweitens enthalten diese Mittel Komponenten mit stark korrosiven oder giftigen Eigenschaften. Als Fazit daraus hat sich für uns die Notwendigkeit der Neuentwicklung einer effektiven Lösung zur DNA-Dekontamination ergeben, die wir hier als DNA-ExitusPlus™ und Autoclave-ExitusPlus™ vorstellen. Im Vergleich zu den herkömmlichen Produkten wird DNA und RNA schnell und effizient zerstört, ohne dass das Reagenz korrosive oder giftige Eigenschaften aufweist.Bei der DNA-Dekontamination unterscheidet man nach der molekularen Wirkungsweise der eingesetzten Mittel drei Grund-prinzipien zur Zerstörung oder Inaktivierung der genetischen Information: Modifikation, Denaturierung und Degradation.

prinzipien zur Zerstörung oder Inaktivierung der genetischen Information: Modifikation, Denaturierung und Degradation.

prinzipien zur Zerstörung oder Inaktivierung der genetischen Information: ModifikaJe nach Zusammensetzung der Mittel können diese drei Prinzipien einzeln oder in Kombination angewandt werden.Da nach den aktuellen Erkenntnissen zum biologischen Risikopotenzial von freien DNA-Molekülen für eine wirklich sichere DNA-Dekontamination die Zerlegung dieser DNA-Moleküle in möglichst kleine Fragmente die wirkungsvollste Methode ist, wurden die gängigen konventionellen Mittel mit unserer Neuentwicklung DNA-ExitusPlus™ im DNA-Degradationstest ver-tionellen Mittel mit unserer Neuentwicklung DNA-ExitusPlus™ im DNA-Degradationstest ver-tionellen Mittel mit unserer Neuentwicklung DNA-ExitusPlus™ im DNA-Degradationstest verglichen. Der DNA-Degradationstest erlaubt einen sensitiven, quantitativen Vergleich der Geschwindigkeit des DNA-Abbaus (Abb. 1 und 2).

Unerwarteter Weise haben wir festgestellt, dass einige der bekannten kommerziellen Mittel nur mit dem Prinzip der Modifi-kation oder Denaturierung der DNA-Moleküle arbeiten. Eine Zerlegung der DNA-Stränge erfolgt dabei nicht, sondern die genetische Information, für die diese DNA-Stränge kodieren, wird eigentlich nur maskiert. Eine genetische Information, für die diese DNA-Stränge kodieren, wird eigentlich nur maskiert. Eine genetische Informa

chemische Demaskierung der DNA-Moleküle durch Entfernung der blockierenden Gruppen würde die genetische Information wieder lesbar und am-plifizierbar machen. Nach dem heutigen Wissensstand zur Gentechnik und der Problematik der Neukombination von Erb-trägern sind solche Mittel eigentlich nicht mehr zeitgemäß. Aber auch die Mittel, die zu einer nachweisbaren Degradation

Keywords

Nukleinsäure-Dekontamination

DNA-DegradationstestPCR-Test

Autoklavieren von DNA

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Nr.1AppliCationsNukleinsäure-Dekontamination mit der ExitusPlus™-Technologie

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TransferMembranes

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SizeMarker•©2010AppliChem

Ever since the foundation of the firm, AppliChem has

investedextensivelyincommunicationandmarketing.The

freshandunusualappearanceattractedgreatattentionin

themarketfromtheverybeginning.Ourgrowthconfirms

therelevanceofthesemeasures.

Weofferourcustomersanextensivelibrary,withnumerous

brochuresandapplicationswhoseusemakeseverydaylife

inthelaboratoryeasier.

A comprehensive catalogue of products, with detailed

information provided by no other catalogue in the field,

isavailableinGermanandEnglish.

AppliChembringsadvantagesthroughknowledge.

“Thinkingwithoutknowledgemakeschancetheruler.”WernerKollath,Germanbacteriologist

©2010AppliChem•SizeMarker 1

contents 1 DNAmarker 2

1.1 Tips on the use of DNA length markers 2

1.2 AppliChem DNA marker overview 4

1.3 DNA marker 5

2 Dyesfornucleicacids 16

2.1 Overview 16

2.2 Methylene blue 16

2.3 Ethidium bromide 18

3 Proteinmarker 19

3.1 AppliChem protein marker overview 19

3.2 Precision protein markers 19

3.3 Prestained protein markers 21

4 Dyesforproteingels 24

4.1 Overview 24

4.2 Coomassie stain 24

4.3 Proteo-Dye 26

2 SizeMarker•©2010AppliChem

Tips on the use of DNA length markers

Correct storageDeproteinisedandlyophilisedDNAsamplesareextremelystable(>5years).ProblemsdonotusuallyoccurunlessDNAmarkersinsolutionarestored(>6weeks)atroomtemperature,theybecomecontaminatedwithbacteria,orarefrequent-lythawedandrefrozen(>20times).AfterdissolutionoftheDNA,wethereforerecommendaliquotingoftheDNAmarker in ready-to-usealiquots forstorageat -20°C(for2-4years).At+4°C,markers insolutionarestable forseveralweeks orevenmonths.Here,however,thereistheriskofbacterialornucleasecontamination.Thiscanbepreventedbystorageat-20°C.

End-labelingThankstotheirdeproteinisedandlyophilisedform,theseDNAmarkersaresuitableforuniversaluseforend-labelingwithmodifiednucleotides.LabelinginfourpositionsviatheterminalEcoRIgeneratedrecessedendsispossible,especiallywiththeDNAladder100bp(A3470)andtheDNALadderMix100-5000(A3660).ForthelabelingoftheDNA,theproductissimplydissolvedinTEbufferorbidistilledwater.

DNA staining with methylene blueOccasionally,ethidiumbromideisnotsuitableforstainingDNA.Analternativeismethyleneblue.Insuchcases,however,itmustbekeptinmindthatthemassofDNAusedmustbeincreasedbyabout30%andthatabout1.5hmoremustbeplannedfordestainingsteps.WithamethyleneblueconcentratesuppliedbyAppliChem,youachieveverygoodresults,evenforsmallfragmentsofabout200bp.

Mass calculations for single fragmentsUsingDNAmarkersofadefinedorigin,thecalculationofthemassofsinglefragmentsisrelativelyeasy.Theamountloadedperlane,e.g.1µg/10µl,isdividedbythenumberofbasepairsoftheDNAusedandismultipliedbythefragmentsize.Forexample:the267bpfragmentofthepBR322HaeIIImarkerwithaloadingamountof1µg:1µg:4361bp(pBR322)=0.229ng/basepairx267bpofthefragment=61.2ng/267fragment.Assuming comparable staining (saturationwithdye), themassofunknown fragments canbedetermined in thisway. Ifrequired,concentrationgradientscanbeloaded.

Less is often moreThedifferentgelformatsforagaroseandpolyacrylamidegelelectrophoresisandthevaryingsensitivityofstainingordetec-tionmeanthatitisonlypossibletogiveanapproximationoftherecommendedDNAamounttobeloaded.MostDNAmar-kersshowthebestseparationwithloadingamountsof0.5-1µgonagarosegels.Thegeneralruleis:thelowerthenumberofmarkerbands, the smaller the total amountneeded.A low loadingamount is alsoof advantagewith shortmigrationdistancesorverysensitivedetection.Whenusingpolyacrylamidegels,generallyonlyabout1/5-1/2theamountrequiredforagarosegelsisnecessary.ForDNAmarkerswithlargefragmentsandahighmass(e.g.equimolarmixturesorlambdaDNAmarkers),dependingontheagaroseconcentration,aminimumseparationdistanceisrequiredtoseparateorcompletelydistinguishbetweenthedominantupperbandsintermsofwidth.Thisseparationdistancecanoftenbereducedby10-15%byreducingthemarkeramount.Byusingequalizedmarkers,inadditiontoamarkedreductionofthemarkeramount,theseparationdistancecanbereducedbyafurther10-15%,frome.g.70to50mm,onastandardformatgel,toobtainthebestresults.

i n t r o d u c t i o n

1.1


How to achieve results quicklyEspeciallydeproteinisedDNAlengthmarkerscanbeseparatedverywellusinghighvoltages,e.g.within20minuteswithamigrationdistanceof70mmona1.2%agarosegelat120Volt.Thiscanonlyberealized,however,withahighqualitygelmigrationbuffer(thewaterqualitymustbe takenintoaccount!)andanadequateamountofbuffer(400-600ml).Poorbufferqualityresultsinoverheatingoftheagarosegelathighervoltagesandthisthereforecausesproblems.Ahighbuffervolume(thebuffercanusuallybereused4-6 times inoneweekwithoutproblems) leads theheatoff,provided thegelchamberforsubmersionisofadequatesize.

Staining front too intense?Insomecases,userswhoregularlyperformassayswithourproductssometimescommentthatthedyeconcentrationinthegelloadingbufferwesupplyistoohigh(onlylyophilisedmarkers).Ifthisisthecase,simplydissolvetheDNAlengthmarkerindoubleconcentrationinthegelloadingbufferandfurtherdiluteitwith1/2volumeTEbuffer.Evenwhenusinga50%solution,therearenoproblemswiththeresulting7%glycerolconcentrationtoincreasethesolutiondensity.

Plausibility of the results and ion concentrations:Non-plausible results (e.g.unexpected fragment runsat500bp insteadof350bp)mayoccurunder relatively extremeconditions(separationofrestrictionorPCRsampleswithaveryhighsaltcontent>150mM)oratveryhighvoltageswithrelativelyshortseparationdistances.Toidentifyerrors,1/10volumerestrictionbuffercanbeaddedtothealiquotoftheDNAmarker,forexample,ortherestrictionsamplecanbeappropriatelydilutedwithgelloadingbuffer(usually1/2volume).Inmanycases,loweringthevoltageduringseparationisalsohelpful.TheaccuratedeterminationofthesizeoffragmentsorPCRproductsmayalsobeimpaired,whensaltfrontscombinedwithlocalproblemsofleadingoffheatwithconductingawayheatandveryhighelectrophoresisvoltages,resultinpartialdenaturationoftheDNAfragments.TheseareconditionsthattheoreticallymayalsooccurwhenprocessingsamplesfromMaxam-Gilbertsequencingincombinationwithsamplebufferscontainingformamide.

i n t r o d u c t i o n


Prod. No. DNA Marker Bands Fragment Sizes [bp]

A3470 DNALadder100bp(lyophilised) 11 1000 900 800 700 600 500 400 300 200 150 100

A5191 DNALadder100bp 10 1000 900 800 700 600 500 400 300 200 100

A3302 DNALadder100bpequalized(lyophilised) 11 1000 900 800 700 600 500 400 300 200 150 100

A5216 DNALadder100bpplus 11 1500 1000 900 800 700 600 500 400 300 200 100

A3660 DNALadderMix100-5000(lyophilised) 17 5000 4000 3000 2500 2000 1500 1000 900 800 700 600 500 400 300 200 150 100

A3982 DNALadder250bp(lyophilised) 16 8000 6000 5000 4000 3000 2750 2500 2250 2000 1750 1500 1250 1000 750 500 250

A8640 DNALadder250bpplus(lyophilised) 15 12000 10000 8000 6000 5000 4000 3000 2000 1750 1500 1250 1000 750 500 250

A2667 DNALadder1kb(lyophilised) 11 10000 8000 6000 5000 4000 3000 2500 2000 1500 1000 500

A5207 DNALadder1kb 13 10000 8000 6000 5000 4000 3000 2500 2000 1500 1000 750 500 250

A6430 DNALadder1kbconcatamer(lyophlised) >25 1000 bpsteps(1000-approx.25000)

A7215 DNAMarkerquick-run(lyophylised) 5 2500 2000 1500 1000 500

A7222 DNAMarkerquick-runextended(lyophylised) 9 6000 4000 25000 2000 1500 1000 500 200 100

A4406 DNAMarkerpBR322-HaeIII(lyophilised) 22 587 540 502 458 434 267 234 213 192 184 124 123 104 89 80 64 57 51 21 18 11 8

A5229 DNAMarkerpBR322-HaeIII 22 587 540 502 458 434 267 234 213 192 184 124 123 104 89 80 64 57 51 21 18 11 8

A6927 DNAMarkerpBR328Mix(lyophilised) 12 2176 1766 1230 1033 653 517 453 394 298 234 220 154

A5194 DNAMarkerPhageLambdaStyI 11 19329 7743 6223 4254 3472 2690 1882 1489 925 421 74

A4412 DNAMarkerPhageLambdaBstEII(lyophilised) 14 8454 7242 6369 5686 4822 4324 3675 2323 1929 1371 1264 702 224 117 BstEII(lyophilised) 14 8454 7242 6369 5686 4822 4324 3675 2323 1929 1371 1264 702 224 117 Bst

A5220 DNAMarkerPhageLambdaBstEII 14 8454 7242 6369 5686 4822 4324 3675 2323 1929 1371 1264 702 224 117 BstEII 14 8454 7242 6369 5686 4822 4324 3675 2323 1929 1371 1264 702 224 117 Bst

A5589 DNAMarkerPhageLambdaHindIII(lyophilised) 8 23130 9416 6557 4361 2322 2027 564 125 HindIII(lyophilised) 8 23130 9416 6557 4361 2322 2027 564 125 Hind

A5223 DNAMarkerPhageLambdaHindIII 8 23130 9400 6557 4361 2322 2027 564 125 HindIII 8 23130 9400 6557 4361 2322 2027 564 125 Hind

A5235 DNAMarkerpUC19-MspI 12 501 489 404 331 242 190 147 111 110 67 34 26

A3996 DNAMarkerpUC19-MspI(lyophilised) 12 501 489 404 331 242 190 147 111 110 67 34 26

Lyophilised markers

Starting materialTheDNAinourlyophilisedlengthmarkersisamplifiedinlow-nucleasehostbacteria,restrictedwithqualityenzymes,andisdeproteinised.Thisensuresthattheycontainhigh-quality,low-proteinornuclease-freeproductswithverygoodmigrationpropertiesonagarose,agarandpolyacrylamidegels.

Quality controlNumerous gel separations of undigested plasmids and digested fragments formpart of themanufacturing process. Thefragmentmassisdeterminedusingtwocalibratedphotometersviaconversion(1OD260nm=50µgdsDNA)andisaliquotedat100%(+2%).EachbatchofDNAandgelloadingbufferissubjecttoafinalqualitycheckusinggelelectrophoresis.

StabilityLyophilisation of the DNA markers results in very stable products, without any significant impairment of quality atroom temperature after transport (even at the height of summer,> 35°C for several days) or after long storage times(>4yearsat-20°C).

ResuspensionBeforeuse,lyophilisedDNAmarkerscanbedissolvedingelloadingbufferoroptionalforend-labelinginsterile,bidistilledwater. The lyophilised form allows you the highest degree of flexibility (labeling, concentration, intensity of stainingbands).

Legal noteTheDNAladdersweremanufacturedbydigestionofplasmidswithEcoR I.Theplasmidsarelegallyprotected.Toproducecopiesapplyforapermissionfromthemanufacturer.

selection guide


selection guideselection guideselection guideselection guideselection guideselection guideselection guideselection guideselection guideselection guideselection guide

) 11 1000 900 800 700 600 500 400 300 200 150 100

A5191 DNALadder100bp 10 1000 900 800 700 600 500 400 300 200 100

) 11 1000 900 800 700 600 500 400 300 200 150 100

A5216 DNALadder100bpplus 11 1500 1000 900 800 700 600 500 400 300 200 100

) 17 5000 4000 3000 2500 2000 1500 1000 900 800 700 600 500 400 300 200 150 100

) 16 8000 6000 5000 4000 3000 2750 2500 2250 2000 1750 1500 1250 1000 750 500 250

A8640 DNALadder250bpplus(lyophilised) 15 12000 10000 8000 6000 5000 4000 3000 2000 1750 1500 1250 1000 750 500 250

) 11 10000 8000 6000 5000 4000 3000 2500 2000 1500 1000 500

A5207 DNALadder1kb 13 10000 8000 6000 5000 4000 3000 2500 2000 1500 1000 750 500 250

A7222 DNAMarkerquick-runextended(lyophylised) 9 6000 4000 25000 2000 1500 1000 500 200 100

III(lyophilised) 22 587 540 502 458 434 267 234 213 192 184 124 123 104 89 80 64 57 51 21 18 11 8

III 22 587 540 502 458 434 267 234 213 192 184 124 123 104 89 80 64 57 51 21 18 11 8

A6927 DNAMarkerpBR328Mix(lyophilised) 12 2176 1766 1230 1033 653 517 453 394 298 234 220 154

I 11 19329 7743 6223 4254 3472 2690 1882 1489 925 421 74

EII(lyophilised) 14 8454 7242 6369 5686 4822 4324 3675 2323 1929 1371 1264 702 224 117

selection guideEII(lyophilised) 14 8454 7242 6369 5686 4822 4324 3675 2323 1929 1371 1264 702 224 117

selection guideEII 14 8454 7242 6369 5686 4822 4324 3675 2323 1929 1371 1264 702 224 117 selection guideEII 14 8454 7242 6369 5686 4822 4324 3675 2323 1929 1371 1264 702 224 117 selection guideIII(lyophilised) 8 23130 9416 6557 4361 2322 2027 564 125 selection guideIII(lyophilised) 8 23130 9416 6557 4361 2322 2027 564 125 selection guideIII 8 23130 9400 6557 4361 2322 2027 564 125 selection guideIII 8 23130 9400 6557 4361 2322 2027 564 125 selection guideI 12 501 489 404 331 242 190 147 111 110 67 34 26selection guideI 12 501 489 404 331 242 190 147 111 110 67 34 26selection guideI(lyophilised) 12 501 489 404 331 242 190 147 111 110 67 34 26

selection guideI(lyophilised) 12 501 489 404 331 242 190 147 111 110 67 34 26

selection guideDNA marker

DNA Ladder 100 bp (lyophilised) A3470DNAsizestandardformedium-sizedfragmentsandPCRproducts

Description Thefragmentsofthissizemarkeroccurinequimolarproportions,i.e.alsoallpossiblemarkingpositionsontheterminalEcoRIgeneratedrecessedends.Thebandmassincreaseswithfragmentlength.TheDNA isdeproteinizedand lyophilized.Thismarker isparticularly suitable forcom-parisonswithmedium-sizedDNAfragmentsandPCRproducts.The500basepairbandmasshasbeendoubledforeasierorientationon1.5-2.0agarosegels.Thesizeandmassofplasmidin-ser-tions or PCR products can be determined precisely in the range of up to 1000 base pairs atintervals of 100basepairs. An additional bandwith150basepairs hasbeen included for thedeterminationofsmallerPCRproducts.Thebestresultsareachievedafteramigrationdistanceofapprox.70-80mmonagarose gels. Each laneof anormal sized agarose gel (approx.80ml)

shouldbeloadedwithapprox.0.4-0.8µgofmarker.Loadingbufferissuppliedseparately.

Number of bands 11

Fragment sizes (bp) 1000,900,800,700,600,500(x2),400,300,200,150,100

Supplied with 1 ml 10mMTris·HCl(pH7.5);5mMsodiumacetate;2mMEDTA;10%glycerol;loading buffer (1X) 0.02%bromophenolblue;0.015%xylenecyanol

Storage -20°C

Loading volume 0.4-0.8µgperlane

Package size A3470,0050 50µg

1.21.21.21.21.21.21.21.2) 11 1000 900 800 700 600 500 400 300 200 150 100 1.2) 11 1000 900 800 700 600 500 400 300 200 150 100

A5191 DNALadder100bp 10 1000 900 800 700 600 500 400 300 200 100 1.2 A5191 DNALadder100bp 10 1000 900 800 700 600 500 400 300 200 100

) 11 1000 900 800 700 600 500 400 300 200 150 100 1.2) 11 1000 900 800 700 600 500 400 300 200 150 100

1.3


DNA Ladder 100 bp A5191

Description 100bpDNAladderisidealfordeterminingthesizeofdouble-strandedDNAfrom100to1000basepairs.The100bpand500bpfragmentsarepresentatincreasedintensitytoalloweasy

identification.Allfragmentsareblunt-ended.

Number of bands 10

Fragment sizes (bp) 1000,900,800,700,600,500x2,400,300,200,100x2

Storage buffer 10mMTris·HCl(pH7.8),10mMNaCl,1mMEDTA

Concentration 0.2mg/ml

Storage -20°C

Loading volume 1µgperlane

Package sizes A5191,0005 0.05mg(=250µl)

A5191,0025 0.25mg(=1.25ml)

Assay conditions: 1.7 % agarose

DNA Ladder 100 bp equalized (lyophilised) A3302DNAsizestandardformedium-sizedfragmentsandPCRproducts

Description Withthismarker,thebandmassofthelargerfragmentshavebeenreducedandhavebeenreinforcedforthesmallerfragments(ascomparedtomarkerA3470)byuptoafactoror4.5.Theresultisanevenappearanceinintensityoftheindividualbands. Themolnumberofthefragmentsdecreasesfromthelargertothesmallerfragments,asdothepossiblemarkingpositionsontheterminalEcoRIgeneratedrecessedends. The500bpband,withanincreaseinmassofafactorof3,enablesrapidorientationon1.5-2.0%agarosegels.Thankstotheevendistribution,ashortermigrationdistanceisneededforthelargerfragments,inordertoachieveacleardistinctionbetweenthebands. Thebestresultsareachievedafteramigrationdistanceofapprox.60-80mmonagarosegels.Eachlaneofanormalsizedagarosegel(approx.80ml)shouldbeloadedwithapprox.0.2-0.5µgofmarker.Theequalized100bpladderisthereforeveryeconomicalinuse.

Loadingbufferissuppliedseparately.

Number of bands 11

Fragment sizes (bp) 1000,900,800,700,600,500(x3),400,300,200,150,100

Supplied with 1 ml 10mMTris·HCl(pH7.5);5mMsodiumacetate;2mMEDTA;loading buffer (1X) 10%glycerol;0.02%bromophenolblue;0.015%xylenecyanol


Storage -20°C


DNA marker

ThequalityoflyophilisedDNAsizingstandardsdiffersconsiderablyfromproductsofferedbymostothermanu-

facturers thanks to the elaboratemanufacturingmethod,which also fulfils the highest quality requirements!

ThedigestedDNAisnotsimplyprecipitatedandresuspended,butalsoextractedinphenol.Thisguaranteesthe

extremelylongshelf-lifewithoutanylossofquality.Notonlythepricesshouldthereforebecompared.


DNA Ladder 100 bp plus A5216

Description 100bp+1.5kbDNALadderissuitableforsizinglineardouble- strandedDNAfragmentsfrom0.1to1.5kb.Useincombinationwitha loadingbuffer(pleaseseeourpresentcatalogforalistofloading buffers).

Number of bands 11

Fragment sizes (bp) 1500,1000(x2),900,800,700,600,500(x2),400,300,200,100(x2). The500bpand1kbbandsarebrighterthantheotherbandsintheladder.

Concentration Themarkerissuppliedinaconcentrationof0.2mg/ml in10mMTris·HCl(pH8.0),1mMEDTA,10mMNaCl.

Storage Storeat-20°C.Preparesmallaliquotstopreventrepeated freeze&thawing!

Loading Werecommendloadingof0.4-0.6µg(2-3µl)perlane.


A5216,0025 0.25mg(=1.25ml) Assay conditions: 1.7 % agarose

DNA Ladder Mix 100-5000 (lyophilised) A3660DNAsizestandardforthedeterminationofthesizeofmedium-sizedfragmentsandplasmids

Description The 100 bp ladder (A3470) was extended with fragments in the size range of 1500-5000. The extension by 40 % mass in the larger fragments means easy

orientationfrom1500bpupwardson1.2-1.5%agarosegels.InadditiontosmallandlargeplasmidinsertionsandPCRproducts,thesizeandquantityofplasmidvectorscan also be determined. The best results are achieved after a migration distance of 80-90 mm on agarose gels with loading volumes of 0.5-0.8 µg per lane on

standardsizedgels.

Number of bands 17

Fragment sizes (bp) 5000,4000,3000,2500,2000,1500,1000,900,800,700,600, 500(x2),400,300,200,150,100.

Supplied with 1 ml 10mMTris·HCl(pH7.5);5mMsodiumacetate;2mMEDTA;loading buffer (1X) 10%glycerol;0.02%bromophenolblue;0.015%xylenecyanol

Loading volume 0.5-0.8µgperlane(forgoodstainingpropertieswithethidiumbromide)

Migration distance Bestresultsareachievedafteramigrationdistanceof70-90mm.

Storage -20°C. Avoid repeated thawing and refreezing. Portioning in small aliquots is recommended. The lyophilised product is stable for many weeks even at room temperature!


This sizing standard wasmanufactured using digestion of 7 plasmids withEcoR I. The plasmids are legallyprotected.Permissiontoproducecopiesmustbesoughtfromthemanufacturer.TheDNAwasdeproteinised,precipitatedandlyophilisedafterdigestionwithphenol/chloroform.Ifthefragmentsaretobelabeled,themarkershouldbedissolvedindistilledwater(10minutesatroomtemperature,shakingoccasionally).


DNA Ladder 250 bp (lyophilised) A3982UniversalDNAsizestandardforthedeterminationofthesizeofplasmidsandtheirDNAinsertions

Description With its twoclearly reinforcedbandsat2500and1500basepairs, the250bp ladderenables rapidorientationon1.0-1.2%agarosegels.Usinggelelectrophoresis,withintervalsof250base

pairsinarangeof250-3000basepairs, it ispossibleafterrestrictiontodetermineclearlyandprecisely,forexample,largeplasmidinsertions,andvectorplasmidsabove3000basepairswithintervalsof1-2kb.IncomparisontoDNAmarkerswithequimolarbanddistribution,themassofthesmallerbandsofthis250bpladderupto1000bphasbeenreinforcedandthelargerbandsabove3000bphavebeenreduced.Thismeans thatoptimumresultsarealreadyachievedafteramigrationdistanceof60-80mmonagarosegelsand thatsmallamountsofabout0.7µgper

lanecanbeusedwithstandardsizedagarosegels(80ml).

Number of bands 16

Fragment sizes (bp) 8000,6000,5000,4000,3000,2750,2500(x3),2250,2000,1750,1500(x3),1250,1000,750,500,250


Loading volume 0.4-0.8µgperlane(forgoodstainingpropertieswithethidiumbromide)

Migration distance Bestresultsareachievedafteramigrationdistanceof75-85mm.

Storage -20°C.Avoidrepeatedthawingandrefreezing.Portioninginsmallaliquotsisrecommended.Thelyophilisedproductisstableformanyweeksevenatroomtemperature!


DNA Ladder 250 bp plus (lyophilised) A8640DNAsizestandardforgenomicDNA,plasmidsandtheirinsertions

Description TheDNALadder250bpplusisintendedforuseinthesizingofDNAfragmentsduringcloningandofgenomicDNA.Bycombiningtwogroupsof fragmentsdifferinginthefragmentsizerange,smallerfragments(250-2000bp)aswellasplasmidvectorsandfragmentofgenomicDNA(3000-12000bp)mybesizedonthesamegel.Themassofthelargerfragmentsisreducedtoachieveabetterseparationonshortdistancesof70-90mmonly.TheterminalEcoRIrestrictionsitesallowforanefficientlabelingofthefragments.Bestresultsareachievedbyloading0.8-1.0µg/laneona1.2%agarosegel(standardminigelsize).TheDNAissupplieddeproteinizedandlyophilized.

Number of fragments 15bands

Fragments sizes (bp) 12000,10000,8000,6000,5000,4000,3000,2000,1750,1500,1250,1000,750,

500,250


Loading volume 0.4-0.8µg/lane(e.g.1.0-1.2%agarosegel)

Storage Storeat-20°C.Storeassmallaliquots,ifresuspended.


This sizing standard was manufactured using digestion of plasmids with EcoR I. The plasmids are legally protected.Permissiontoproducecopiesmustbesoughtfromthemanufacturer.TheDNAwasdeproteinised,precipitatedandlyophilisedafterdigestionwithphenol/chloroform.Ifthefragmentsaretobelabeled,themarkershouldbedissolvedindistilledwater(10minutesatroomtemperature,shakingoccasionally).


DNA Ladder 1 kb (lyophilised) A2667

Description Thesizeofmedium-sizedplasmidsandfragmentscanbedeterminedwith11DNAbands between10,000and500basepairs.Unliketheequimolardistributionofthebandsinother

DNAmarkers and the longmigrationdistancenecessarywith these for the separationof large fragments, the mass of the larger bands of the 1 kbp DNA ladder has beenreduced. This permits relatively short separation distances for a 1 kbp DNA ladder(approx.70mmon1.2%agarosegels) andmeans that it is very economical inuse(0.4-0.6 µg per lane, or 400 separations) on normal sized gels. Loading buffer issupplied separately. The fragments have terminal EcoR I generated recessed ends.

TheDNAisdeproteinizedandlyophilized.

Number of bands 11

Fragment sizes (bp): 10000,8000,6000,5000,4000,3000,2500,2000,1500,1000,500



Storage -20°C

Package sizes A2667,0050 50µg

A2667,0200 4x50µg

DNA Ladder 1 kb A5207

Description 1 kbDNA Ladder is a convenientmarker for determining the size of double-strandedDNA from 250 to 10,000 base pairs. The 1,000 and

3,000bpfragmentshaveincreasedintensityrelativetotheotherbandsonethidiumbromide-stainedagarosegels,andserveasreference

indicators.Allfragmentsareblunt-ended.

Number of bands 13

Fragment sizes (kb) 10.0,8.0,6.0,5.0,4.0,3.0(x2),2.5,2.0,1.5,1.0(x2),0.75, 0.5,0.25



Storage -20°C


A5207,0025 0.25mg(=1.25ml)

DNA marker


DNA Ladder 1 kb concatamer (lyophilised) A6430DNAsizemarkerasalternativetoirregularl-fragments(from1toapprox.25kb)Description Apartial ligationof1000bpHind III fragments results inaDNA ladder ranging from1000 to approx. 22000 bp in 0.8 to 1% gels using standard agarose (Prod.-No. A2114; separation

range up to approx. 25 kbp). The upper limit of the fragment ligation results in the mainmass of the ligation products in the separation range optimal for standard agaroses (agar). Forabetterorientation,the3000bpand10,000bpbandsarebrighter.Optimalresultswillbeachievedafteradistanceofapproximately80-100mminagarosegelsandaloadingvolumeofapproximately1µgperlaneinnormal-sizedgels.This1000bpconcatamerladdermaybeappliedeitherinreducedloadingvolumes(foraseperationrangee.g.<10000bp)orinincreasedvolumes(>15000bp). Evenunderoptimizedligationconditions,anintramolecularreligationmayoccur,especiallywithsmallerfragments.Therefore,athighgelloadingvolumes,ligationproducts(closedcircles)havebeen

observedbetweenthe1000and3000bpbands.Forabetterorientation,the3000bpbandisbrighter.

Number of bands >25Fragment sizes (bp) 1000bpsteps(1000-approx.25000)


Loading volume approx.1.0µgperlane

Migration distance Optimumresultsafteradistanceof15-25mmonly.Storage -20°C.


DNA Marker quick-run (lyophilised) A7215DNAsizestandardfordeterminationofDNAfragmentsaftershortgelruns(15-25mm)

Description Thismarkerisidealforusewithminigels,sinceitsbandsareseparatedverywellevenafterveryshortgelrunsasshortas15-25mminane.g.1.5-1.8%agarosegel.Thesinglebandshavebeenequalizedintermsoftheirmass.The1500bpfragmenthasanincreasedmassforbetterorientation.Optimumresultsareachievedina1.5-1.8%agarosegelwithaslittleas0.25µgofthemarkerperlane(normalsizedminigel).Pleasenote that theDNA fragments have to be saturated with ethidium bromide in

therunningbufferduringtheseparation.

Number of bands 5

Fragment sizes (bp) 2500,2000,1500*,1000,500 Themassofthemarkedfragment*hasbeenincreasedforbetterorientation.

Supplied with 1 ml 10mMTris·HCl(pH7.5);5mMsodiumacetate;2mMEDTA;10%glycerol;loading buffer (1X) 0.015%bromophenolblue

Loading volume 0.25µgperlane(foragooddetectionwithethidiumbromide)

Migration distance Optimumresultsafteradistanceof15-25mmonly.

Storage -20°C.Preventrepeatedfreezingandthawing(>20x).Werecommendtoprepare smallaliquots.Thelyophilisedformisstableevenforweeksatambienttemperature


ThismarkerhasbeendesignedasanalternativeforirregularλDNAfragmentmarkers,fortheseparationinstandardlowendoosmosisagarose(Prod.-No.A2114;separationrange70bptoapproximately25kbp).Incomparisontootherconcatamermarkers,ithasareducedmassinthenon-separatingrange(highmolecularweightrange),resultinginabetterresolution(goodreadingsupto24kbp).Forapplicationsrequiringagoodseparationinthehighmolecularweightrangepulsed-fieldelectrophoresisisrecommended.Duetotheproductionprocedure(ligationof1000bpHindIIIfragments),itisimpossibletodeterminethespecificmassofasingleband.TheDNAofthismarker(50µgsufficientforapproximately100loadings)isdeproteinised,Iyophilisedandissuppliedwith1mlofthe1xgelloadingbuffer.

Thismarker ismadeofplasmidswithspecificmutagenesissites.TheEcoRI-digestedDNAhasbeendeproteinizedwithphenol/chloroform,desalted,precipitatedandlyophilised.ResuspendtheDNAinthesterile-filteredgelloadingbuffer(suppliedwiththemarker)byincubationfor10minutesatroomtemperaturewithoccasionalshaking.


DNA Marker quick-run extended (lyophilised) A7222DNAsizestandardfordeterminationofDNAfragmentsaftershortgelruns(35mm)

Description Thismarker is ideal for usewithmini ormidi gels, since its bands are separatedverywellevenafterveryshortgelrunsasshortas35mminane.g.1.2%agarosegel. The single bands have been equalized in terms of their mass. The 1500 bpfragmenthasanincreasedmassforbetterorientation.Optimumresultsareachieved

withaslittleas0.3-0.5µgofthemarkerperlane(normal-sizedmini/midigel).

Number of bands 9

Fragment sizes (bp) 6000,4000,2500,2000,1500*,1000,500,200,100 Themassofthemarkedfragment*hasbeenincreasedforbetterorientation.

Supplied with 1 ml 10mMTris·HCl(pH7.5);5mMsodiumacetate;2mMEDTA;10%glycerol;loading buffer (1X) 0.015%bromophenolblue

Loading volume 0.3-0.5µgperlane (foragooddetectionwithethidiumbromideinaminiormidigel)

Storage -20°C.Prevent repeated freezingand thawing(>20x).Werecommend toprepare

smallaliquots.Thelyophilisedformisstableevenforweeksatambienttemperature!


Thismarkerismadeofplasmidswithspecificmutagenesissites.TheEcoRI-digestedDNAhasbeendeproteini-zedwithphenol/chloroform,desalted,precipitatedandlyophilised.ResuspendtheDNAinthesterile-filteredgelloadingbuffer(suppliedwiththemarker)byincubationfor10minutesatroomtemperaturewithoccasionalshaking.

DNA Marker pBR322 - Hae III (lyophilised) A4406DNAsizestandardforhigh-percentageagarosegelsandpolyacrylamidegels

Description Plasmid pBR322 was digested with Hae III, deproteinized and lyophilized. This marker is particularly suited to comparisons with small PCR fragments and DNA

from restriction samples on polyacrylamide gels or high-percentage agarose gels (> 2.2 %). The best results are achieved after a migration distance of approx. 70-80mmonagarosegelsor100mmon6-8%polyacrylamidegels.Eachlaneofanormalsizedgel(approx.80ml)shouldbeloadedwithapprox.1µgof marker

foragarosegelsor0.5µgforPAAgels.Loadingbufferissuppliedseparately.

Number of bands 22

Fragment sizes (bp) 587,540,502,458,434,267,234,213,192,184,124,123,104,89,80,64,57, 51,21,18,11,8



Storage -20°C

Package size A4406,0050 50µgPlease note! Extremelysmallfragmentscanonlybevisualizedonhigh-percentagePAAgels.

DNA marker


DNA Marker pBR328 Mix (lyophilised) A6927DNAsizestandardformiddle-sizedfragmentsandPCRproductsDescription PlasmidpBR328wasdigestedwithHinfIandBglI,respectively,deproteinisedand

lyophilised. The fragments have beenmixed in an equimolar ratio. Thismarker isparticularlysuitableforcomparisonwithPCRfragments(e.g.fooddiagnostics)andDNAfromrestrictionsamplesonagarosegelswithsizesbetween150and2000basepairs.Theconcisedistributionoffragmentsofthemarkermakesthisproductidealfor long and short gel runs with running distances of 70-80mm (1.5% agarosegel).Load1µgofthemarkerperlaneofanormalsizedagarosegel(approx.80ml

Loadingbufferissuppliedseparately.

Number of bands 12

Fragment sizes (bp) 2176;1766;1230;1033;653;517;453;394;298;234;220;154


Loading volume 1.0µgperlane

Storage -20°C.Prevent repeated freezingand thawing(>20x).Werecommend toprepare smallaliquots.Thelyophilisedformisstableevenforweeksatambienttemperature!

Package sizes A6927,0050 50µg

ResuspendtheDNAinthesterile-filteredgelloadingbuffer(suppliedwiththemarker)for10minutesatroom

temperaturebyoccasionalshaking.

DNA Marker pBR322 - Hae III A5229

Description ThismarkerisgeneratedbydigestionoftheplasmidpBR322withHaeIII.

Number of bands 22

Fragment sizes (bp) 587,540,502,458,434,267,234,213,192,184,124,123,104,89,80,64,57, 51,21,18,11,8



Storage -20°C

Package sizes A5229,0005 0.05mg(=250µL)

A5229,0025 0.25mg(=1.25ml)


DNA marker


DNA Marker Phage Lambda - Sty I A5194

Description Lambda DNA (cI857 Sam 7) Sty I markers are prepared by digesting Lambda DNA with Sty I, followed by inactivation of the enzyme. The DNA fragments arethenethanol-precipitatedandresuspendedinthestoragebuffer.

Number of bands 11

Fragment sizes (bp) 19329*;7743;6223;4254*;3472;2690;1882;1489;925;421;74

Storage buffer 10mMTris·HCl(pH8.0),1mMEDTA

Concentration 0.2-0.5µg/µl

Storage -20°C


A5194,0025 0.25mg(=1.25ml)Please note! Thecohesiveendsoffragments1and4(*)maycausetheformationofanextraband(23583bp). Thefragmentsmaybeseparatedbyheatingto65°Cfor3minutesbeforeloadingthesampleontothegel.

DNA Marker Phage Lambda - BstE II (lyophilised) A4412DNAsizestandardmadeofphagelDNAforthedeterminationoflargeDNAfragments(digestionofgenomicDNA)Description Natural lambdaDNAwasdigestedwithBstE II, deproteinizedand lyophilized.This markerissuitableforcomparisonswithfragmentsizesbetween8400and700base

pairs.Thebestresultsareachievedafteramigrationdistanceofapprox.80-90mmon1.0-1.2%agarosegels.Eachlaneofanormalsizedgel(approx.80ml)should

beloadedwithapprox.1µgofmarker.Loadingbufferissuppliedseparately.

Number of bands 14

Fragment sizes (bp) 8454*,7242,6369,5686*,4822,4324,3675,2323,1929,1371,1264,702, 224,117


Loading volume 1.0µgperlane

Storage -20°C

Package size A4412,0100 2x50µgPlease note!Thecohesiveendsoffragments1and4(*)mayformanadditionalband(14140bp).Beforeloadingontothegel,thefragmentscanbeseparatedbyheatingto65°Cfor5minuteswithsubsequentincubationonice.

DNA marker


DNA Marker Phage Lambda - BstE II A5220

Description Phage Lambda (cI857 Sam 7)DNA/BstE II markers are prepared by digesting Lambda DNA with BstE II, followed by inactivation of the enzyme. The DNA fragmentsarethenethanol-precipitatedandresuspendedinthestoragebuffer.

Number of fragments 14

Fragment sizes (bp) 8454*,7242,6369,5686*,4822,4324,3675,2323,1929,1371,1264,702, 224,117



Storage Storeat-20°C


A5220,0025 0.25mg(=1.25ml)Please note!Thecohesiveendsoffragments1and4(*)maycausetheformationofanextraband(14140bp). Thefragmentsmaybeseparatedbyheatingto65°Cfor3minutesbeforeloadingthesampleontothegel.

DNA Marker Phage Lambda - Hind III (lyophilised) A5589DNAsizestandardmadeofphagelDNAforthedeterminationoflargeDNAfragments(digestionofgenomicDNA)Description NaturallambdaDNAwasdigestedwithHindIII,deproteinizedandlyophilized.This marker is suitable for comparisonswith large tomedium-sized fragments between

23,000and600basepairs.Thebestresultsareachievedafteramigrationdistanceofapprox.80-90mmon1.0-1.2%agarosegels.Eachlaneofanormalsizedgel(approx.80 ml) should be loaded with approx. 0.5-0.8 µg of marker. Loading buffer is

suppliedseparately.

Number of bands 8

Fragment sizes (bp) 23130*;9416;6557;4361*;2322;2027;564;125



Storage -20°C

Package size A5589,0100 2x50µgPlease note! Thecohesiveendsoffragments1and4(*)mayformanadditionalband(27491bp).Beforeloadingontothegel,thefragmentscanbeseparatedbyheatingto65°Cfor5minuteswithsubsequentincubationonice.

DNA marker


DNA Marker Phage Lambda - Hind III A5223

Description LambdaDNA(cI857Sam7)/HindIIImarkersarepreparedbydigestingLambdaDNAwithHind III, followedbyheat-inactivationof theenzyme.TheDNAfragmentsare thenethanol-precipitatedandresuspendedinstoragebuffer.

Number of bands 8

Fragment sizes (bp) 23130*;9400;6557;4361*;2322;2027;564;125



Storage -20°C


A5223,0025 0.25mg(=1.25ml)Please note! Thecohesiveendsoffragments1and4(*)maycauseformationofanextraband(27491bp). Thefragmentsmaybeseparatedbyheatingto65°Cfor3minutesbeforeloadingthesampleontothegel.

DNA Marker pUC19 - Msp I A5235

Description DigestionofthepUC19plasmidyields12fragments.

Number of bands 12(9visible)

Fragment sizes (bp) 501,489,404,331,242,190,147,111,110,67,34x2,26



Storage -20°C


A5235,0025 0.25mg(=1.25ml)


DNA Marker pUC19 - Msp I (lyophilised) A3996DNAsizestandardforhigh-percentageagarosegelsandpolyacrylamidegels

Description PlasmidpUC19wasdigestedwithMspI,deproteinizedandlyophilized.Thismarkerisparticu-larlysuitableforcomparisonswithsmallPCRfragmentsandDNAfromrestrictionsamplesonpolyacrylamidegelsorhigh-percentageagarosegels.Thebandsat501/489bpand111/110bphaveapprox.doublemassandproviderapidorientationafterashortmigrationdistanceon2.0-2.2%agarosegels.Thebestresultsareachievedafteramigrationdistanceofapprox.50-70mmonagarosegelsor100mmon6-8%polyacrylamidegels.Eachlaneofanormalsizedagarosegel (approx.80ml) shouldbe loadedwith approx.0.5-1.0µgofmarker. Loadingbuffer issuppliedseparately.

Number of bands 12

Fragment sizes (bp) 501,489,404,331,242,190,147,111,110,67,34,26



Storage -20°C

Package size A3996,0050 50µgPlease note! Extremelysmallfragmentscanonlybevisualizedusinghigh-percentageandhigh-qualityagarosegels(>2.2%)withlargemassoffragments.


dyes for nucleic acidsProd.-No. Description DNA/RNA Dye Complex Excitation/Emission Complex Excitation/Emission Complex (Detection)

dyes for nucleic acids(Detection)

dyes for nucleic acids Sensitivity recommended working

A1398 Acridineorange dsDNA/RNAgreenfluorescence; 490nm/525nm 50ng 30µg/ml 1µg/mlinwater;2-8°CA1398 Acridineorange dsDNA/RNAgreenfluorescence; 490nm/525nm 50ng 30µg/ml 1µg/mlinwater;2-8°CA1398 Acridineorange dsDNA/RNAgreenfluorescence; 490nm/525nm 50ng 30µg/ml 1µg/mlinwater;2-8°C ssDNA/RNAredfluorescence

A0691 Crystalviolet DNA(purple-red) 590nm 10ng 1µg/ml

A1001 DAPI specificbindingtoAT-Basepairs; 365nm/450nm 70ng 0.1µg/mlinwaterA1001 DAPI specificbindingtoAT-Basepairs; 365nm/450nm 70ng 0.1µg/mlinwaterA1001 DAPI specificbindingtoAT-Basepairs; 365nm/450nm 70ng 0.1µg/mlinwater intercalationintoGC-Basepairs;white-bluefluorescence

A1151 Ethidiumbromide* DNAintercalation 260-360nmor546nm/590nm 0.5ng 0.2-0.5µg/ml 10mg/mlinwater;2-8°C

A2388 Malachitegreenoxalate DNA 626nm A2388 Malachitegreenoxalate DNA 626nm

A1402 Methyleneblue DNA,RNA-stainingatacidicpH 297nm/672nm 40ng 0.2%in0.4MNaOAc/0.4MAceticacid

A5595 DNA-DyeMethyleneblue DNA 297nm/672nm 40-80ng 200-foldconcentratedsolution;2-8°CA5595 DNA-DyeMethyleneblue DNA 297nm/672nm 40-80ng 200-foldconcentratedsolution;2-8°C

A1403 Methylgreen specificbindingtoAT-richsequences;DNA(green) 638nm 1µg/ml

A0581 Methylorange(C.I.13025) 10ng 0.0005% 0.25%inwaterA0581 Methylorange(C.I.13025) 10ng 0.0005% 0.25%inwater

A3918 Nileblue DNAintercalation(blue) 630nm/673nm 40ngonagarose;4ngondriedgel 1µg/ml 1µg/mlin0.25XTAEbuffer(pH8.0)

A2261 Propidiumiodide DNA-intercalator;nouptakeintolivingcells 530nm/625nm 1µg/ml 2-8°CorRTA2261 Propidiumiodide DNA-intercalator;nouptakeintolivingcells 530nm/625nm 1µg/ml 2-8°CorRT

A1406 PyroninY DNA/RNA(red) 488-530nm/565-574nm 0.05%or1µg/ml

A3944 Silvernitrate DNA(brown) 2.5ngdsDNAonagarose A3944 Silvernitrate DNA(brown) 2.5ngdsDNAonagarose

A1400 Stainsall RNA(blue-violet)

*Thereareseveralready-to-usesolutionsavailable! A1152Ethidiumbromide-Solution1%BioChemica DAPI(4',6-Diamidino-2-phenylindoledihydrochloride);PBS(Phosphate-bufferedsaline);NaOAc(Sodiumacetate)A2273Ethidiumbromide-Solution0.07%“dropper-bottle” PBS(Phosphate-bufferedsaline)

Protocol for staining with DNA-Dye Methylene blue:Approx.50ml1XDNA-DyeMethylenebluestainingsolutionarerequiredtostainanagarosegelwiththedimensionsofapprox.80x60mm(widthxlength)andathicknessof3-4mm.The gel should be trimmed down appropriately, with the edge of a ruler for example, toremoveareasofthegelthatwerenotusedforDNAseparation.Thestainingisperformedinanadequatelysizeddish,inwhichthegelisfloatedtoadepthofabout5mmwithdyesolution.To prepare theDNA-DyeMethylene blue staining solution for use, 250µl are dissolved in50mlwater.Thegelislefttostainforabout20minutesandthedishisshakenoccasionally.Torevealthebands(differentstainingintensities)thegelisdestainedwithtapwaterseveral

timesforabout5-10minutes.Theblue-stainedDNAbandsareclearlyvisibleintransmittedlightandcanbemeasuredwitharulerordocumentedphotographically.MethylenebluedoesnotstainDNApermanently:dependingonthethicknessofthegelandthestoragetempe-rature,theDNAbands(especiallysmallerfragments)maylosetheirstainingaftersometime.

DNA-Dye Methylene blue A5595Nontoxicsolutionforthestainingofnucleicacidsinagarosegels

Amount 10ml200-foldmethylenebluedyeconcentrate

Storage 2-8°C

Stability 18-24months(shakewellbeforeuse)

Package size A5595,0010 10ml

The DNA marker Phage Lambda-Hind III (A5589) was loaded on the 1st lane (*).

2.1

2.2

*


dyes for nucleic acids Sensitivity recommended working concentration Stock solution / Storage

A1398 Acridineorange dsDNA/RNAgreenfluorescence; 490nm/525nm 50ng 30µg/ml 1µg/mlinwater;2-8°CA1398 Acridineorange dsDNA/RNAgreenfluorescence; 490nm/525nm 50ng 30µg/ml 1µg/mlinwater;2-8°C

A0691 Crystalviolet DNA(purple-red) 590nm 10ng 1µg/ml

A1001 DAPI specificbindingtoAT-Basepairs; 365nm/450nm 70ng 0.1µg/mlinwaterA1001 DAPI specificbindingtoAT-Basepairs; 365nm/450nm 70ng 0.1µg/mlinwater

0.5ng 0.2-0.5µg/ml 10mg/mlinwater;2-8°C

A2388 Malachitegreenoxalate DNA 626nm

A1402 Methyleneblue DNA,RNA-stainingatacidicpH 297nm/672nm 40ng 0.2%in0.4MNaOAc/0.4MAceticacid

A5595 DNA-DyeMethyleneblue DNA 297nm/672nm 40-80ng 200-foldconcentratedsolution;2-8°C

A1403 Methylgreen specificbindingtoAT-richsequences;DNA(green) 638nm 1µg/ml

A0581 Methylorange(C.I.13025) 10ng 0.0005% 0.25%inwater

A3918 Nileblue DNAintercalation(blue) 630nm/673nm 40ngonagarose;4ngondriedgel 1µg/ml 1µg/mlin0.25XTAEbuffer(pH8.0)

A2261 Propidiumiodide DNA-intercalator;nouptakeintolivingcells 530nm/625nm 1µg/ml 2-8°CorRT

A1406 PyroninY DNA/RNA(red) 488-530nm/565-574nm 0.05%or1µg/ml

A3944 Silvernitrate DNA(brown) 2.5ngdsDNAonagarose

Abbreviations DAPI(4',6-Diamidino-2-phenylindoledihydrochloride);PBS(Phosphate-bufferedsaline);NaOAc(Sodiumacetate)

A2273Ethidiumbromide-Solution0.07%“dropper-bottle” PBS(Phosphate-bufferedsaline)

Repeated use of the 1X staining solutionThe prepared 1X methylene blue staining solution can be kept in a suitable brown-glass bottle at 2-8°C for4-6monthsandcanbeusedseveraltimes.Thestainingperiodshouldbelengthenedbyabout20%fortheseconduse,andbythesameagainforthethirduse.Afterthis,thestainingcapacityofthesolutionislargelyexhaustedandanewsolutionshouldbeprepared.

SensitivityThebandsina1kbladder(loadingamount1µg)canbestainedwithnoproblems.Smallfragments(150bp)canalsobestained.Thedecisivefactorsinsensitivityarethethicknessofthegel,thegel:stainingsolutionratio,andthedestainingtime.Byvaryingthestainingconditions,youcaneasilydeterminethebestconditionsforyoursystem.

HandlingAlaboratorycoatandglovesshouldbewornwhenhandlingthestainingsolution.

PrecautionOncontactwiththeskinorclothing,theareashouldbewashedwithlargeamountsofsoapandwater.

Short protocol1. DiluteDNA-DyeMethyleneblue200Xconcentratedownto1X(250µlDNA-DyeMethyleneblue+50mlwater)2.Trimdowntheagarosegeltothesmallestareapossible.3. Incubate the gel (dimensions approx. 80 x 60 mm) with 50 ml DNA-Dye Methylene blue for about 20 minutes;shakeoccasionally.Thestainingsolutionshouldcoverthegeltoadepthofabout5mm.

Please note:thestainingsolutioncanbeusedseveraltimes!4.Destainrepeatedlyinwaterfor5-10minutes.


Ethidium bromideisthemostcommonlyuseddyeforstainingDNAduringorafterpolycrylamideoragarosegelelectrophoresis.Excitationwithlightatawavelenghtof254nmisabsorbedbytheDNAandtransferredtoethidiumbromide.Excitationat366nmdirectlyexcitesthedye(3).Anaqueousstocksolutionispreparedataconcentrationof10mg/mlandemployedat0.2-0.5µg/ml(5).Stainingisperformedafterthegelrun(especiallyPAGE)orduringthegelrun(agarose).ThelatterallowstheobservationofthegelrunbycontrolunderUVlight.Ethidiumbromideisaddedtotheagarosesolution.Pleasenote,that'prestaining'mayleadtoartifacts(4).In another application, ethidium bromide may be used to sensitively quantify DNA by spectrofluorimetry.Excitationat250nmwithUVlightandanemissionat605nm,thesensitivityisevenbetterascomparedtothedyeHoechst33258.Ataconcentrationof0.5µg/mlEtBr,thedetectionlimitof10ng/mlDNAisreachedwithalinearrangeofmeasurementfrom20to1250ng/ml(6).Caution:Ethidiumbromideisapowerfulmutagenandismoderatelytoxic.Glovesshouldbewornwhenworkingwithsolutionsthatcontainthisdye,andamaskshouldbewornwhenweighingethidiumbromide.

dyes

for

nucl

eic

acid

s

Ethidium bromide - Solution 1 % BioChemica A1152Synonym 2,7-Diamino-10-ethyl-9-phenylphenanthridiumbromide-Solution

Composition Ethidiumbromide:10mg/ml filteredsolution

Storage Storethestocksolutionat2-8°Cprotectedfromlight.

Package sizes A1152,0025 25ml

A1152,0100 100ml

Ethidium bromide - Solution 0.07 % “dropper-bottle” A2273Synonym 2,7-Diamino-10-ethyl-9-phenylphenanthridiumbromide-Solution

Composition Ethidiumbromide:0.7mg/ml filteredsolution

Storage Storethestocksolutionat2-8°Cprotectedfromlight.

Description EthidiumbromideactsasaDNAintercalatorandhasaconsiderablemutagenicpotential.InordertoreducethepossiblecontacttoethidiumbromideAppliChemprovidesaready-to-usesolutionina'dropperbottle'.Theconcentrationofethidiumbromidesolution(0.7mg/ml)isadjustedsothattheadditionofonedrop(ofavolumeof50µl)ofthissolutionissufficienttostain50mlagarosegelsolution.

Package sizes A2273,0005 5ml A2273,0015 15ml

Literature(1) Waring, M. (1975) in Antibiotics Vol. III , 141-165 (J.W. Corcoran & F.E. Hahn eds.) Springer-Verlag: Review article about ethidium and propidium.(2) Lunn, G. & Sansone, E.B. (1987) Anal. Biochem. 162, 453-458 Ethidium bromide: Destruction and decontamination of solutions.(3) Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd Edition. Cold Spring Harbor Laboratory Press. Cold Spring

Harbor, New York.(4) Gärtner, U. et al. (1996) Anal. Biochem. 243, 194-196 Apparent degradation of cleaved genomic DNA may be an ethidium bromide prestaining artifact.(5) Lucey, M.J. et al. (1997) BioTechniques 23, 780-782 Reducing the ethidium bromide quantity in agarose gel electrophoresis.(6) Bonasera, V. et al. (2007) BioTechniques 43, 173-176 Protocol for high-sensitivity/long linear-range spectrofluorimetric DNA quantification using EtBr.

2.3


Protein Marker I (14-116) A5238Description Thebandsof3µlmarkerareeasily visualizedwithCoomassiestainingina

polyacrylamidegel.ProteinMarkerIisamixtureof7purifiedproteinssuppliedingelloadingbufferfordirectapplicationto

anSDSpolyacrylamidegel.

Number of bands 7

Fragment sizes (kDa) 116.0;97.4;66.2;37.6;28.5;18.4;14.0

Loading buffer 50mMTris·HCl(pH6.8), 100mMdithiothreitol,2%SDS, 0.1%bromophenolblue,10%glycerol

Assay conditions 3µl/12%PAGE

Package size A5235,0500 500µl

protein marker

precisionprecision

Prod. Protein Marker Bands Fragment Sizes [kDa] No.

A5238 ProteinMarkerI(14-116) 7 116 97.4 66.2 37.6 28.5 18.4 14

A5418 ProteinMarkerII(6.5-200)prestained 8 200 116 68 43 30 20 14.4 6.5

A4402 ProteinMarkerIII(6.5-200) 8 200 116 68 43 30 20 14.4 6.5

A3993 ProteinMarkerIV(10-150) 8 150 100 80 60 40 30 20 10

A8359 ProteinMarkerV(10-175)prestained 11 175 130 95 70 62 51 42 29 22 1410.5

A8889 ProteinMarkerVI(10-245)prestained 12 245 180 135 100 75 63 48 35 25 20 17 11

General ConsiderationsSizeestimation:Prestainedproteinmarkersarenotrecommendedforprecisedeterminationofthemolecularweightsizesincetheirbehaviorduringelectrophoresisstronglydependsonelectrophoresisconditions.Forexactdeterminationsofproteinsizesuseunlabeledmarkerproteins,i.e.non-prestainedmarkers.

Proteinblotting:Thetransferofproteinsduringblottingdependsontheirsize(e.g.lysozymeisfullytransfer-redafter30minutes,whilemyosinrequires2.5hours@1V/cm2).

3.13.13.13.13.1

3.2


Protein Marker IV (10-150) A3993Recombinantproteinsizemarkerforgelelectrophoresis

Description The bands of 2.5 µl marker are easily visualized with Coomassie staining in a polyacrylamidegel.

Number of bands 8

Protein sizes (kDa) 150;100;80;60;40;30;20;10

Assay conditions 10µl/4-20%SDS-PAGEgradientgel (Tris-Glycine);Coomassiestaining

Storage -20°C, if stored longer than 1 month. Pleasenotethatrepeatedfreezingand thawingwillreduceproductquality.

Preparationofsmallaliquotsisrecommended.Thawedaliquotsarestable

foroneweekat+4°C.


Thisisa ready-to-usemarkercontainingrecombinantproteins.Theproductmaycontainresidualiodoacetamide.

Protein Marker III (6.5-200) A4402Description Thebandsofthe2.5µlmarkerareeasily visualizedwithCoomassiestainingina polyacrylamidegel.

Number of bands 8

Protein sizes (kDa) 200.0;116.0;68.0;43.0;30.0;20.0; 14.4;6.5

Assay conditions 5µl/4-20%SDS-PAGEgradientgel; Coomassiestaining

Storage -20°C,ifstoredlongerthan1month. Pleasenotethatrepeatedfreezingandthawingwillreducepro-

ductquality.Preparationofsmallaliquotsisrecommended.

Package size A4402,0001 1.0ml

Thisisaready-to-usemarkercontainingacylatedproteins.Theproductmaycontainresidualiodoacetamide.

precision

Application ●Theproteinemarkershavetobe“preheated”toroomtemperature,toguaranteethatallcom-ponentsaredissolved.●Apply1-5µlofthemarkertoamini-gel(10x10cm,1-0.75mmthick,7mmslot).Use1XLaemmlibuffertodilutethismarker,ifnecessary.●Tominimizemyosinaggregation,thealiquottobeloadedontothegelmaybeheatedto95°Cfor1-2minutes.


Protein Marker II (6.5-200) prestained A5418Prestainedproteinsizemarkerforgelelectrophoresis

Description This is a ready-to-use marker containing covalent prestained proteins. The product containsformamide.

Number of bands 8

Protein sizes (kDa) 200.0;116.0;68.0;43.0;30.0;20.0; 14.4;6.5

Assay conditions 10µl/4-20%PAGEgradientgel Tris-Glycine;leftlaneprestainedbutnot

Coomassie-stained;rightlaneprestained andadditionalCoomassiestaining.

Storage -20°C,ifstoredlongerthan1month. Pleasenotethatrepeatedfreezingand

thawingwillreduceproductquality.Pre- parationofsmallaliquotsisrecommended.


Protein Marker V (10-175) prestained A8359PrestainedproteinmarkerforgelelectrophoresisandWesternblotting.MagentaMarker

Description Ready-to-useprestainedproteinsizemarker.Suppliedinloadingbuffer.

Number of bands 11

Protein sizes (kDa) 175;130;95;70;62;51;42;29;22;14;10.5

Threereferenceproteinscoupledwithabluedyeforeasyidentification: approx.10,40,and90kDa.

Storage At2-8°Cformax.3months;at-20°Cforlongtermstorage.


prestained

3.33.3


Protein Marker VI (10-245) prestained A8889PrestainedproteinmarkerforgelelectrophoresisandWesternblotting.Blue-Green-RedProteinMarker

Description ProteinMarkerVIprestainedisathree-colorproteinstandardwith12prestainedproteinscoveringawidemolecularweightrangefromapprox.10to245kDa.Proteinsarecovalentlycoupledwitheitherabluechromophoreoronegreen(25kDa)andonereddye(75kDa),respectively.GelrunmaybemonitoredduringproteinseparationonSDS-PAGE(Tris-glycinebuffer,“Laemmlisystem”).The main applications are the monitoring of protein migration duringSDS-polyacrylamide gel electrophoresis, the verification of Western transferefficiency onmembranes (PVDF, nylon, or nitrocellulose), and the sizing ofproteins on SDS-polyacrylamide gels and Western blots. The size marker is

suppliedready-to-useingelloadingbuffer.

Number of bands 12

Protein sizes (kDa) 245;180;135;100;75;63;48;35;25;20;17;11


RecommendationsforLoading1.Thawtheladdereitheratroomtemperatureorat37-40°Cforafewminutestodissolvepre-cipitatedsolids.Donotboil!

2.Mixthoroughlytoensurethesolutionishomogeneous.3.LoadthefollowingvolumesoftheladderonSDS-polyacrylamidegel:5µlperwellformini-gels,2.5µlperwellforblots;10µlperwellforlargegels,5µlperwellforblots.

prestained3.33.3

SizeMarker•©2010AppliChem

Sometimes, less is more!

For The Sake OfThe Environment

Manyreagentspreparedinbiomedicalresearchlabsareidealnutrientbrothsforunwantedgerms(bacteria,fungi).Topreventtheirgrowth,reagentsareeitherautoclaved,sterilefiltered,orantibiotics/antimycoticsortoxicsubstancesareadded.OneoftheseadditivesisThimerosal,amercury-containingmoleculewhichisdangerousfortheenvironment.Ouroriginalimmunoasssaybuffercontainedthischemicaltoo,butnowwehavereplaceditbythenontoxicProClin® 300.Forthesakeoftheenvironment.

For Your Safety

Wewouldliketokeepyouhealthysothatyoustayourcustomer.FYI:Thimerosalisclassifiedastoxic.Thelethaldose(rat,s.c.)is9mg/kg,comparedtoethidiumbromidewithalethaldose(mouse,s.c.)of110mg/kg.Insomecountries,Thimerosalisaforbiddenadditive.

For Better Results

Changingthecompositionhasnonegativeinfluenceontheperformanceoftheproducts.WithCrossDownandallotherimmunoassaybuffersyouareeveninabettermoodandyourimmunoassaysshowabetterquality.


Abbreviations:BSA(BovineSerumAlbumin);CBB(Coomassie®Brilliantblue);MeOH(Methanol);RuBPS(Ruthenium(II)tris(bathophenanthrolinedisulfonate));TCA(Trichloroaceticacid);&onlyincombinationwithCoomassie®!$detectionofphosphoproteins;formationofinsolublephosphatecomplexes(sensitivity:0.1nM)


dyes

for p

rote

in g

els Prod.-No. Description Comment Absorption maxima Sensitivity Recommended Concentration Stock solution

A2176 BismarckbrownR increasessensitivityofCoomassie® BrilliantBlueR-250staining

lmax.468nm 25ng& CoomassieBrilliantblueR-250(0.2%w/v)BismarckbrownR(0.05%w/v)

SolventMeOH:Aceticacid:Water(40:7:53);dissolvedyesseparately,thenmix1:0.75(v/v)

A3480 Coomassie®BrilliantBlueG-250 stainsampholytesinIEFgelstoo!* <0.5ng Mix80mlofthestocksolutionwith20mlofMeOHjustbeforeuse.

0.1%w/vCBBin2%w/vPhosphoricaicd,10%w/vAmmoniumsulfate(Donotfilter!)

A1092 Coomassie®BrilliantBlueR-250 stainsampholytesinIEFgelstoo!* lmax.withprotein549nm,w/oprotein555nm

10ng 0.1%in20%MeOH,10%Aceticacid

A0822 EosinY reversiblestaining similartoCBBR-250 0.25%(w/v)in0.1NNaOH preparestainingsolutionfreshdaily;maybeusedforseveralgels;

A1346 EriochromeblackT 560nm 10ng 0.01% 0.02%in40%MeOH/7%Aceticacid;incombinationwithRhodamineB

A1401 FastGreenFCF dyecomplexinacidicpHrange lmax.(pH8.3)615;(MeOH)620nmlmax.(withprotein)635nm

400ng 0.25%(w/v)in10%Aceticacidupto1%(w/v)in7%Aceticacid

A2385 Kongored stainingintheacidicpHrange lesssensitivethanCBB 0.1%(w/v)in0.2MAcetatebuffer(pH3.5) 1%(w/v)indistilledwaterA2388 Malachitegreenoxalate Phosphatasestainingingels Stainingsolution=

3Vol.Solution1+1Vol.Solution2(stablefor3weeks;filterpriortoeachuse)

Solution1:0.15gMalachitegreenoxalatein300mlWater;Solution2:4.2gAmmoniummolybdatetetrahydratein100ml5NHCl;

Nilered 5ngA1405 PonceauS acidicdiazodye lmax.(H2O)517-823nm 500ng 0.1-0.5%in3%TCAor1mlAceticacid

glacialad100mlWaterpreparefreshpriortouse

A7808 Proteo-DyeRuBPS lmax.617nm 2-5ng 1µM 1mMA3930 RhodamineB$ 560nm 10ng 0.01% 0.02%in40%MeOH/7%Aceticacid;incombinationwith

EriochromeblackTA3972 Silvernitrate brown 2-5ngA1400 Stainsall** stainsdifferenttypesofproteins

indifferentcolorslmax.forBSA515nm likeCBB 0.005% 0.1%inFormamideor5.6mg/10mlin50%Dioxane;

preparefresh

Zinc-Imidazole reversiblestaining 10-20ngSDS-PAGE40-80ngnativePAGE

Coomassie® Brilliant blue R-250 (C.I. 42660) A1092Synonym BrilliantblueR,Xylenebrilliantcyanine

Order No. Quantity Formula M CAS-No.

A1092,0010 10g C45H44N3NaO7S2 825.98g/mol 6104-59-2

A1092,0025 25g

A1092,0100 100g

®registeredtrademarkofImperialIndustriesPLC

Coomassie®BrilliantBlueR-250isoneof themostcommonlyusedstains forproteins,after theirseparationbypolyacrylamidegelelectro-phoresis. The protein-dye complex has an absorptionmaximum at 549 nm, the dyewithout protein at 555 nm (in 0.01M citrate buffer,pH3).The intensity in stainingofproteinsprobablydependson thebasicityofaprotein.Perpositivelychargedaminoacidapproximately1.5-3moleculesofCoomassie®willbebound.Thisvariationcomplicatetheexactproteindeterminationwithalbuminasastandard,sincethisproteincontainsmorebasicaminoacidsthanmanyotherproteins.TheredoexistmanyprotocolsforsensitivestainingprocedureswithCoomassie®(e.g.ref.3,4).Thesensitivityreachesalimitat25ngprotein.Werecommendthefollowingprotocol:I. Staining solution: 0.1 % Coomassie® Brilliant Blue R-250 (Prod.-No. A1092), 20 % methanol (or ethanol), 10 % acetic acid.TheSDSgel(without'stackinggel')isstainedfor1hourat60°Corfor2hoursat50°CorovernightatRT.

II. Destainingsolution:20%methanol(orethanol)and10%aceticacid.Destainthegel for3-4hoursat50-60°C.Addsomesponges.Subsequentlywashthegelfor15minutsinwateranddryundervacuumat60°Cfor2-3hours.

4.1

4.2

dyes

for p

rote

in g

els

4.2

dyes

for p

rote

in g

els

*Allen,R.E.et al.(1980)Anal. Biochem. 104,494-498.StainingProteinsinIsolelectricFocusingGelswithFastGreen.**stainse.g.sialoglycoproteinsandphosphoproteinsblue,almostallotherproteinsred.


Prod.-No. Description Comment Absorption maxima Sensitivity Recommended Concentration Stock solution

A2176 BismarckbrownR increasessensitivityofCoomassie® BrilliantBlueR-250staining

lmax.468nm 25ng& CoomassieBrilliantblueR-250(0.2%w/v)BismarckbrownR(0.05%w/v)

SolventMeOH:Aceticacid:Water(40:7:53); dissolvedyesseparately,thenmix1:0.75(v/v)

A3480 Coomassie®BrilliantBlueG-250 stainsampholytesinIEFgelstoo!* <0.5ng Mix80mlofthestocksolution with20mlofMeOHjustbeforeuse.

0.1%w/vCBBin2%w/vPhosphoricaicd, 10%w/vAmmoniumsulfate(Donotfilter!)

A1092 Coomassie®BrilliantBlueR-250 stainsampholytesinIEFgelstoo!* lmax.withprotein549nm, w/oprotein555nm

10ng 0.1%in20%MeOH,10%Aceticacid

A0822 EosinY reversiblestaining similartoCBBR-250 0.25%(w/v)in0.1NNaOH preparestainingsolutionfreshdaily; maybeusedforseveralgels;

A1346 EriochromeblackT 560nm 10ng 0.01% 0.02%in40%MeOH/7%Aceticacid; incombinationwithRhodamineB

A1401 FastGreenFCF dyecomplexinacidicpHrange lmax.(pH8.3)615;(MeOH)620nmlmax.(withprotein)635nm

400ng 0.25%(w/v)in10%Aceticacidupto1%(w/v)in7%Aceticacid

A2385 Kongored stainingintheacidicpHrange lesssensitivethanCBB 0.1%(w/v)in0.2MAcetatebuffer(pH3.5) 1%(w/v)indistilledwaterA2388 Malachitegreenoxalate

Phosphatasestainingingels

Stainingsolution= 3Vol.Solution1+1Vol.Solution2 (stablefor3weeks;filterpriortoeachuse)

Solution1:0.15gMalachitegreenoxalatein300mlWater;Solution2:4.2gAmmoniummolybdatetetrahydratein100ml5NHCl;

Nilered 5ngA1405 PonceauS acidicdiazodye lmax.(H2O)517-823nm 500ng 0.1-0.5%in3%TCAor1mlAceticacid

glacialad100mlWaterpreparefreshpriortouse

A7808 Proteo-DyeRuBPS lmax.617nm 2-5ng 1µM 1mMA3930 RhodamineB$ 560nm 10ng 0.01% 0.02%in40%MeOH/7%Aceticacid;incombinationwith

EriochromeblackTA3972 Silvernitrate brown 2-5ngA1400 Stainsall** stainsdifferenttypesofproteins

indifferentcolorslmax.forBSA515nm likeCBB 0.005% 0.1%inFormamideor5.6mg/10mlin50%Dioxane;

preparefresh

Zinc-Imidazole reversiblestaining 10-20ngSDS-PAGE 40-80ngnativePAGE

Coomassie® Brilliant blue G-250 (C.I. 42655) A3480Synonym BrilliantblueG

Order No. Quantity Solubility (20°C) Formula M CAS-No.

A3480,0010 10g ~10g/L(H2O) C47H48N3NaO7S2 854.04g/mol 6104-58-1

A3480,0025 25g

A3480,0100 100g

®registeredtrademarkofImperialIndustriesPLC

TheassaysofNeuhoffetal.(Neuhoff,V.etal.(1988)Electrophoresis9,255-262)haveshownthatstainngwithCoomassie®G-250ismoresensitivethanwithR-250.FormoreinformationsseeA1092.


dyes

Protocol for staining of gels with Proteo-Dye Blue-Vis: SDS-PAGEminigels (1mm thickness, 10% acrylamide, Tris-glycine buffer system) aretreatedfor1hourwith30%v/vmethanolafterelectrophoresis toremovetheexcessofSDSandtofixtheproteinsinthegelmatrix.Thegelsarestainedfor2hoursinavolumeof100mlProteo-DyeBlue-Vis.Afterthis,thegelsarewashedwithacetatebuffer(0.2M;pH4.5),containing20%v/vmethanolfor90-120minutesuntilthedarkredbackgroundisreducedtoaweakpinkbackgroundincontrasttotheblueproteinbands.Thegelsaredocumentedwiththehelpofavideosystem(transilluminator312nm,whitelightplate)oronascannerinthetransmissionmode.

Proteo-Dye Blue-Vis A6810

Synomym Proteindyeinthevisiblerange

Storage 2-8°Cprotectedfromlight

HS-No. 38220000

Package size A6810,1000 1L

1Lissufficientforapprox.30minigels,since3xreusable detectionlimit3-5ng/mm² reversiblestainingofproteins

Protocol for staining of gels with Proteo-Dye Red-Fluo: SDS-PAGEminigels(5x8cm,1mmthickness,10%acrylamide,Tris-glycinebuffersystem)arefixedfor30minutesinasolutionof7.5%v/vaceticacid,20%v/vethanol.Afterthis,thegelsarestainedwithProteo-DyeRed-Fluo(100ml)for2-3hours.Thebackgroundfluorescence canbe removedby repeatedwashingwith the fixation solution (7.5% v/vaceticacid,20%v/vethanol;3-4washingsteps,30minuteseach).Thedocumentationof the gels is carried out with a video system (transilluminator 312 nm, adapting filter590nm).

Proteo-Dye Red-Fluo A6803

Synomym Proteindyewithredfluorescence


HS-No. 38220000

Specification Emissionmaximum:630nm

Exicitationwavelenght:312nm


1Lissufficientforapprox.30minigels,since3xreusable detectionlimit1-3ng/mm² reversiblestainingofproteins moresensitivethanmostotherproductsavailableinthemarket

4.3


for protein gels

Proteo-DyeGreen-Fluoisafluorescenceproteindye,basedonametal-chelatecomplexinconjunctionwithadetergentinsubmicellarconcentrationinanaqueoussolution.Thisdyesolutionenablestodetectverysmallamountsofproteinsandthestainingisfullyreversible.Usethedyesolutionuptothreetimes!

Protocol for staining of gels with Proteo-Dye Green-Fluo: SDS-PAGEminigels(5x8cm;1mmthickness,10%acrylamide,Tris-glycinebuffersystem)arepostelectrophoretically treated for30minuteswithamethanol solution(30%, v/v)whilegentlyshaking(100rpm).Afterthis,thegelsarestainedfor2hoursinavolumeof100mlProteo-DyeGreen-Fluo.Reductionofthebackgroundfluorescenceisachievedbyrepeatedwashingsteps(3-4 times,30min.each)with30%v/vmethanol.Thegelsaredocumentedwithavideosystem(transilluminator365nm,adaptingfilter520nm).

Protocol for staining of blots with Proteo-Dye Green-Fluo: BystainingwithCoomassieorsilvertheproteinsinthegelbecomeirreversiblydenaturatedand are difficult to transfer to blottingmembranes.Metal chelate stainingmethods arereversible, resulting in good preconditions for the following blot transfer. Blots wereperformedbyatypicalsemi-drymethod.Aftertheblottingprocess,theblotswerewashedindistilledwaterfor1houranddriedatroomtemperature.BlotswerethenstainedwithProteo-DyeGreen-Fluo for2hours,brieflywashed in30%v/vethanolanddriedagain.Only in thedried state, intenselygreen fluorescentproteinbandsbecomevisible inUVtoplight(365nmUVlight)andaredocumentedwithavideosystem(adaptingfilter420nm).Thestainmaybeeasilyandfastlyremovedjustbyrinsingin30%v/vethanol.

Proteo-Dye Green-Fluo A6794

Synomym Proteindyewithgreenfluorescence


HS-No. 38220000

Specification Emissionmaximum:520nm

Excitationwavelenght:365nm


1Lissufficientforapprox.30minigels,since3xreusable detectionlimit3-5ng/mm² reversiblestainingofproteins suitableforgelsandforblots


CommentRuBPSisafluorescencedyeforproteindetectionine.g.SDS-and2-D-gels.Itprovidesthehighsensitivity of silver staining without its drawbacks. The excellent contrast, good linearity andhomogeneitymadethisdyethestainingreagentofchoiceinproteomics.AminimalinterferencewithMALDI-TOFanalysisisobservedandcompatibilitywithMS/MSanalysisisgiven.Thephoto-chemicallystabledyeisexcitedwithUV-lightofthewavelength473or488nmbluelaser.A532nmlasermaybeusedaswell,albeitwithreducedefficiency.ItisofadvantagethattheexcitationwavelengthofRuBPSislongerthantoseofthearomaticaminoacids.The information given in terms of formula, molecular weight and CAS number refer to theraw material! The concentrate supplied is diluted 1 : 1000 (1 ml ad 1 L) resulting in an 1µMworkingsolution.

Special features of Proteo-Dye RuBPS• Providesthesensitivityofsilver-stainingwithoutitsdrawbacks (limiteddynamicrange,inhomogeneityandinterferencewith MSanalysis)• Carefullypurifiedproductoffersmuchimprovedsignal-to- backgroundratio• Excellentcontrastingelimagesenablesproteinspotstobe detectedmoreeasilybyimageprocessingsoftware• Highsensitivity• Goodlinearityandhomogeneity• MinimalinterferencewithMALDI-TOFanalysis• CompatiblewithMS/MSanalysis,providedthatcleaningofthe samplewithreversephaseadsorptioniscarriedout

Protocol for gel staining / destaining(accordingtoRef.2)1. Prepare a 1 µM Proteo-Dye RuBPS solution by diluting the concentrate 1000-fold (e.g.,1mlto1L)

2. Fixthegelin30%ethanol,10%v/vaceticacidovernight.3. Rinsethegelin20%ethanolv/vfor30minandrepeat3times.4. Incubatethegelin1µMProteo-DyeRuBPSsolutionfor6h.5. Equilibrate the gel in water for 10 min and repeat once (a first scan is possible at thisstage).

6. Destainthegelwith40%ethanol/10%aceticacidv/vfor15h.N.B. for low protein concentration the destaining time might be too long. In such cases, shor-ter destaining times may optimize the procedure resulting in greater sensitivity.7. Equilibratethegelinwaterfor10min,repeatonceandscan.

Proteo-Dye RuBPS A7808

Synomym Ruthenium(II)tris(bathophenanthrolinedisulfonate)tetrasodiumsalt

Formula C72H42N6Na4O18RuS6

Molecularweight 1664.58g/mol

CAS-No. [301206-84-8]

Concentration(inH2O) 1mM

lmax.Emission(inH2O) 617nm

lmax.Exitation 277,437,460nm

Storage 2-8°C,protectedfromlight

recommendedworkingsolution 1:1000dilution(1µMfinalconcenrtation)

Stability 2years

Packagesize A7808,0001 1ml

2-D gels of E. coli proteins stained with (1) SYPRO Ruby© and (2) with RuBPS. For details see Reference 2.

Literature

[1] AcomparisonbetweenSyproRuby©and

ruthenium(II)tris(bathophenanthrolinedisulfonate)

asfluorescentstainsforproteindetectioningels.

(Rabilloud,T.etal.(2001)Proteomics1,699-704)

[2] Improvedruthenium(II)

tris(bathophenanthrolinedisulfonate)staining

anddestainingprotocolforabettersignal-to-

backgroundratioandimprovedresolution.

(Lamanda,A.etal.(2004)Proteomics4,599-608)

©2010AppliChem•SizeMarker

Electrophoresis Reagents Prod. No.AcrylamideMolecularbiologygrade A3812

BisacrylamideMolecularbiologygrade A3636

AgaroseBasic A8963

AgaroselowEEO(AgaroseStandard) A2114

AgaroseMP A1091

LoadingbufferDNAI A3144

LoadingbufferDNAII A2571

LoadingbufferDNAIV(forAgarosegels) A3481

TAEbuffer(50X) A1691

TAEbuffer(10X) A1416

TBEbuffer(10X) A0972

Transfer membranes Prod. No.ReprobeNitrocellulosesupported0.22µmTransfermembrane A5237

ReprobeNitrocellulosesupported0.45µmTransfermembrane A5242

PureNitrocelluloseunsupported0.22µmTransfermembrane A5250

PureNitrocelluloseunsupported0.45µmTransfermembrane A5239

PureNylonNeutralTransfermembrane0.22µm(30cmx3m) A4399

PureNylonNeutralTransfermembrane0.45µm A5248

ReprobeNylonPositivelychargedTransfermembrane0.45µm(30cmx3m) A5255

PVDF-StarTransfermembrane0.45µm A5243

Other Related Products Prod. No.ChemiluminescenceDetectionKitsforHorseradishPeroxidase

Cheluminate-HRPPicoDetect A3417

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Transfer Membranes

AppliChemsuppliesarangeoftransfermembranesdesigned andtestedspecificallyforRNA,DNAandproteinanalysis.

AppliChemalsoprovidesourcustomerswithtried andprovenprotocolsdevelopedtoobtainconsistentreproducibleanddependableresultswhenusedwithAppliChemmembranes. 22protocolsandalltypesofmembranes–allfromonesource.

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Documents

Gel Electrophoresis Size Marker - AppliChem · The different gel formats for agarose and polyacrylamide gel electrophoresis and the varying sensitivity of staining or detec-tion mean