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Protein(s)

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unit = 1 Dalton(Da)

1 kilo Dalton (kDa) = 103 Da

1 Dalton = approx. Mw of Hydrogen atom

(1,66 x 10 -24 g = 1.66 yoctograms)

= 1/16 of Oxygen Mw

Molecular weight of proteins

Amino acid (average) = 110 Da

1 pair of bases (average) = 649 Da

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Proteinsconsistof amino acids

H2N

C

H

R

COOH

amino group

carboxyl group

variable groupor

side chain

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Proteinsconsistof amino acidsconected by peptide bond

H2O

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Proteinshaveseveral levelsofstructure

Van der Wals

Hydrogen bridge

Cystein Bridge

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Proteinstability andhandling- extremely heterogeneous class of biological macromolecules.- they are often unstable when not in their native environments, which can vary considerably amongcell compartments and extracellular fluids. If certain buffer conditions are not maintained,extracted proteins may not function properly or remain soluble.

- proteins can lose activity as a result of proteolysis, aggregation and suboptimal buffer conditions.

- purified proteins often need to be stored for an extended period of time while retaining theiroriginal structural integrity and/or activity.

- the extent of storage shelf life can vary from a few days to more than a year and is dependenton the nature of the protein and the storage conditions used.

- optimal conditions for storage are distinctive to each protein; nevertheless, it is possible to

suggest some general guidelines for protein storage and stability.

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Proteinstability andhandlingGeneral considerations for protein storage

Temperature:

Protein Concentration:

- diluted protein solutions (< 1 mg/ml) more prone to inactivation and loss as a result of low-level binding to thestorage vessel. Therefore, it is common practice to add carrier or filler protein, such as purified bovineserum albumin (BSA) to 1-5 mg/ml (0.1-0.5%), to dilute protein solutions to protect against such degradation

and loss.

- generally, proteins are best stored at 4C in clean, autoclaved glassware or polypropylene tubes.

- storage at room temperature often leads to protein degradation and/or inactivity, commonly as a result of microbialgrowth.

- shortterm storage(1 day to a few weeks), many proteins may be stored in simple buffers at 4C. Protein stabilizingcocktail helps to extend the shelf-life of most proteins for storage at 4 C or -20C compared to storage in simplephosphate or Tris buffers.

- longterm storagefor 1 month to 1 year, some researchers choose to bead single-use aliquots of the protein in liquidnitrogen for storage in clean plastic containers under liquid nitrogen. This method involves adding the protein solutiondropwise (about 100 l each) into a pool of liquid nitrogen, then collecting the drop-sized frozen beads and storingthem in cryovials under liquid nitrogen.

-frozen at -20C or -80C is the more common form of cold protein storage

-freeze-thaw cycles decrease protein stability, samples for frozen storage are best dispensed and prepared in single-use aliquots so that, once thawed, the protein solution will not have to be refrozen

- alternatively, addition of 50% glycerol or ethylene glycol (see Additives section below) will prevent solutions fromfreezing at -20C, enabling repeated use from a single stock without warming (i.e., thawing).

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Additives:

Many compounds may be added to protein solutions to lengthen shelf life: Protein Stabilizing Cocktail is a solution that helps to extend the shelf-life of most proteins for storage at 4C

or -20C.

Cryoprotectants such as glycerol or ethylene glycol to a final concentration of 25-50% help to stabilize proteins bypreventing the formation of ice crystals at -20C that destroy protein structure.

Protease inhibitors prevent proteolytic cleavage of proteins.

Anti-microbial agents such as sodium azide (NaN3) at a final concentration of 0.02-0.05% (w/v) or thimerosal at afinal concentration of 0.01 % (w/v) inhibit microbial growth.

Metal chelators such as EDTA at a final concentration of 1-5 mM avoid metal-induced oxidation of SH groups andhelps to maintain the protein in a reduced state.

Reducing agents such a dithiothreitol (DTT) and 2-mercaptoethanol (2-ME) at final concentrations of 1-5 mM alsohelp to maintain the protein in the reduced state by preventing oxidation of cysteines.

Proteinstability andhandling

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Basicproteincharacteristics

Molecular weight

Amino acid composition

pI- The pH of a dispersion medium of a colloidal suspension or an ampholyteat which the solute does not move in an electrophoretic field.

Hydrophobicity-hydrophilicity index

Immunogenic index - : lysine, argenine, glutamic acid, aspartic acid,glutamine, asparganine

Signal sequence

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MRTSVVVLAVVALIGAVIADERCSSACTLEYNPICGADALNHYETFGNPCAFNYYNCEHPFSPMRLVRAGECTAAETDEE

Analysis Whole ProteinMolecular Weight 8692.84 Da

Length 80 AA101 microgram = 115.037 pMoles

Molar Extinction coefficient 67105%

1 A(280) = 1.30 mg/ml 0.73 mg/mlIsoelectric Point 4.34Charge at pH 7 -6.92

lysine, arginine, glutamic acid, aspartic acid, glutamine, asparganine

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Sequence length = 70Measure Position Value Cutoff signal peptide?

max. C 20 0.825 0.32 YES Cleavage sitemax. Y 20 0.815 0.33 YES Y= C+ Smax. S 14 0.985 0.87 YES Signal peptide

Most likely cleavage site between pos. 19 and 20: VIA-DE

Sequence Prediction: Signal peptideSignal peptide probability: 0.999Signal anchor probability: 0.000

Max cleavage site probability: 0.979 between pos. 19 and 20

Signal peptideMRTSVVVLAVVALIGAVIADERCSSACTLEYNPICGADALNHYETFGNPCAFNYYNCEHPFSPMRLVRAGECTAAETDEE

SignalP 3.0 Server:http://www.cbs.dtu.dk/services/SignalP/

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acetylation, the addition of an acetyl group, either at the N-terminus [2] of the protein or at

lysine residues [3] (also see histone acetylation) [4][5]

deacetylation

alkylation, the addition of an alkyl group (e.g. methyl, ethyl)

methylation the addition of a methyl group, usually at lysine orarginine residues.

(This is a type of alkylation.)

demethylation

amidation at C-terminus

biotinylation, acylation of conserved lysine residues with a biotin appendage

formylation

gamma-carboxylation dependent on Vitamin K[6]

glutamylation, covalent linkage ofglutamic acid residues to tubulin and some otherproteins.[7] (See tubulin polyglutamylase)

glycosylation, the addition of a glycosyl group to eitherasparagine, hydroxylysine, serine, or

threonine, resulting in a glycoprotein. Distinct from glycation, which is regarded as a

nonenzymatic attachment of sugars.

glycation, the addition of a sugar molecule to a protein without the controlling action of an

enzyme.

glycylation, covalent linkage of one to more than 40 glycine residues to the tubulin C-

terminal tail

heme moiety may be covalently attached

hydroxylation

iodination (e.g. of thyroid hormones)

isoprenylation, the addition of an isoprenoid group (e.g. farnesol and geranylgeraniol)

lipoylation, attachment of a lipoate functionality

prenylation

GPI anchor formation

myristoylationfarnesylation

geranylgeranylation

multiple reactions: diphthamide

nucleotides or derivatives thereof may be covalently attached

ADP-ribosylation

flavin attachment

nitrosylation

oxidation

palmitoylation

pegylation

phosphatidylinositol may be covalently attached

phosphopantetheinylation, the addition of a 4'-phosphopantetheinyl moiety from coenzyme A, as

in fatty acid, polyketide, non-ribosomal peptide and leucine biosynthesis

phosphorylation , the addition of a phosphate group, usually to serine, tyrosine, threonine or

histidine

polysialylation, addition ofpolysialic acid, PSA to NCAM

pyroglutamate formation

tRNA-mediation addition of amino acids such as arginylation

sulfation, the addition of a sulfate group to a tyrosine.

selenoylation (co-translational incorporation ofselenium in selenoproteins )

Posttranslational modificationscanchangepropertiesofproteins

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MRTSVVVLAVVALIGAVIADERCSSACTLEYNPICGADALNHYETFGNPCAFN

YYNCEHPFSPMRLVRAGECTAAETDEE___________________.....................................................T...T...Name S/T Pos G-score I-score Y/N Comment

----------------------------------------------------------------------------Sequence T 3 0.491 0.048 . Sequence S 4 0.382 0.031 .

Sequence S 24 0.255 0.070 . Sequence S 25 0.236 0.051 . Sequence T 28 0.285 0.055 . Sequence T 45 0.231 0.059 .

Sequence S 62 0.236 0.046 . Sequence T 73 0.575 0.027 T Sequence T 77 0.605 0.035 T

Glycosylation

NetOGlyc 3.1 Server : http://www.cbs.dtu.dk/services/NetNGlyc/

- enzymatic process that links saccharides to produce glycans attached to proteins, lipids, or other organic molecules

- form of co-translational and post-translational modification

- enzyme-directed site-specific process, as opposed to the non-enzymatic chemical reaction of glycation

- glypiation which is the addition of a GPI anchor which links proteins to lipids through glycan linkages(Glycosylphosphatidylinositol is a glycolipid that can be attached to the C-terminus of a protein during posttranslational modification)

Five classes of glycans are produced:

- N-linked glycans attached to a nitrogen of asparagineor arginine side chains

-O-linked glycans attached to the hydroxy oxygen of serine,threonine,tyrosine,hydroxylysine,orhydroxyproline side chains, or to oxygens on lipids such as ceramide

- phospho-glycans linked through the phosphate of a phospho-serine

- C-linked glycans, a rare form of glycosylation where a sugar is added to a carbon on a tryptophan side chain

NetNGlyc 1.0 Server : http://www.cbs.dtu.dk/services/NetNGlyc/

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SequenceMRTSVVVLAVVALIGAVIADERCSSACTLEYNPICGADALNHYETFGNPCAFNYYNCEHPFSPMRLVRAGECTAAETDEE 80..T....................SS.................................... S..........T....... 80

Phosphorylation sites predicted: Ser: 3 Thr: 2 Tyr: 0

Serine predictions

Name Pos Context Score Pred_________________________v_________________Sequence 4 -MRTSVVVL 0.038 .

Sequence 24 DERCSSACT 0.848 *S*Sequence 25 ERCSSACTL 0.844 *S*

Sequence 62 EHPFSPMRL 0.952 *S*_________________________ _________________

Threonine predictions

Name Pos Context Score Pred_________________________v_________________

Sequence 3 --MRTSVVV 0.506 *T*Sequence 28 SSACTLEYN 0.022 .

Sequence 45 NHYETFGNP 0.050 .Sequence 73 AGECTAAET 0.675 *T*Sequence 77 TAAETDEE- 0.341 .

_________________________ _________________Tyrosine predictionsName Pos Context Score Pred

_________________________v_________________Sequence 31 CTLEYNPIC 0.208 .

Sequence 43 ALNHYETFG 0.411 .Sequence 54 CAFNYYNCE 0.160 .Sequence 55 AFNYYNCEH 0.039 .

_________________________^_________________

Sequence- andstructure-basedpredictionofeukaryoticproteinphosphorylationsites. Blom, N., Gammeltoft, S., and Brunak, S. Journal ofMolecularBiology: 294(5): 1351-1362, 1999.

Phosphorylation

NetPhos2.0 Server :http://www.cbs.dtu.dk/services/NetPhos/

- addition of a phosphate (PO4) group to a protein or other organic molecule- activates or deactivates many protein enzymes, causing or preventing the mechanisms of diseases such ascancer and diabetes.

- kinases (phosphorylation) and phosphatases (dephosphorylation)

- serine > threonine >> tyrosine (rare) [tyrosine phosphorylated proteins easy to purify using antibodies well studied]

- Histidine and Aspartarte sometimes phosphorylated in signalling pathways

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Histonecode

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Proteinquantification

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Proteinquantification- UV

- the most simple and quick method, but also the most likely to produce inaccurate results

- usually at 280 nm -> depends on the presence of aromatic amino acids in proteins.

Tyrosine and tryptophan absorb at 280 nmPhenylalanine has a peak of absorbance at 260 nm.

Higher orders of protein structure may also modify the molar absorptivities of tyrosine and tryptophan andthus the UV detection is sensitive to pH and ionic strength.

Unless the protein sample is pure and its extinction coefficient is known, UV determination of proteinconcentration will invariably be significantly in error.

A useful equation (Layne, 1957) for approximate concentration determination using UV absorbance is asfollows:

[protein] (mg/ml) = (1.55 A280) (0.76 A260)

This equation is not sufficiently accurate for routine biochemical calculations.

Glass and polystyrene cuvettes absorb UV light

Quartz cuvettes should be used expensive!!!Cuvettes

Methacrylate plastic cuvettes

- transmit60% of280-nm light and it is possible to zero spectrophotometers with them- significantly less expensive than quartz cuvettes (e.g., teaching laboratories)

If using a pure protein with a known extinction coefficient (), it is possible to directly determine the

concentration from the measured A280. The extinction coefficients for many proteins have beenpublished (Fasman, 1989). Because of the different mole fractions of tyrosine and tryptophan residues indifferent proteins, their extinction coefficients can be very different.

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Unknownproteinsorprotein mixtures- Path length for most spectrometers is 1 cm.

Concentration (mg/ml) = Absorbance at 280 nm divided by path length (cm.)

Pureproteinofknown absorbancecoefficient- concentration is in mg/ml, %, or molarity depending on which type coefficient is used.

Concentration = Absorbance at 280 nm divided by absorbance coefficient

To convert units, use these relationships:

Mg protein/ml = % protein divided by 10 = molarity divided by protein molecular weight

Absorbance coefficients of some common proteinstandards:

Bovine serum albumin (BSA): 63Bovine, human, or rabbit IgG: 138Chicken ovalbumin: 70

Unknownswithpossiblenucleic acidcontamination

Concentration (mg/ml) = (1.55 x A280) - 0.76 x A260)

Proteinquantification- UV

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Proteinquantification- Bradford

Highlights:Ready-to-use dye-binding reagent formulation

Fast (almost immediate) color development; measure at 595 nm

Compatible with reducing sugars, reducing substances and thiols

Refrigerated reagent is stable for 1 year

Useful for determining protein concentration from 100-1,500 g/ml

Micro method determines protein concentration in the range of 1-25 g/ml

- Dr. Marion Bradford in 1976.- acidic environment of the reagent, protein binds to the coomassie dye.This results in a spectral shift from the reddish/brown form of the dye(Amax at 465 nm) to the blue form of the dye (Amax at 610 nm).

difference between the two forms of the dye is greatest at 595 nm,

- Development of color in coomassie dye-based (Bradford) associated withthe presence of arginine, lysine and histidine in the protein.

- mass of a peptide or protein must be at least 3,000 daltons- ssay is performed at room temperature- compatible with most salts, solvents, buffers, thiols, reducing substances

and metal chelating agents encountered in protein samples.

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Proteinquantification- BCA

Highlights:

Colorimetric method; read at 562 nm

Compatible with most ionic and nonionic detergentsFaster and easier than the Lowry method

Working reagent stable for 24 hours

Linear working range for BSA from 20-2,000 g/ml

Minimum detection level of 5 g/ml with the enhanced protocol

Less protein-to-protein variation than dye-binding methods

-combines the well-known reduction of Cu2+ to Cu1+ by protein in an alkalinemedium with the highly sensitive and selective colorimetric detection of

the cuprous cation (Cu1+) by bicinchoninic acid.

- first step is the chelation of copper to form a light blue complex(biuret)- second step - (BCA) reacts with the reduced (cuprous) cation that wasformed in step one.

- intense purple-color chelation of two molecules of BCA with one Cu.- BCA/copper complex is water-soluble

and exhibits a strong linear absorbance at 562 nm

2,2'-BicinchoninicAcid or 4,4'-Dicarboxy-2,2'-biquinoline

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Proteinquantification- Lowry

Highlights:

The most widely cited colorimetric method; read at 750 nm

Preformulated cupric sulfate-tartrate reagent stable for 1 year

at room temperature

Linear results from 1-1,500 g/ml for BSA

Convenient microplate or cuvette format

Less protein-to-protein variation than dye-binding methods

Sensitivity : requires 10-200 ug of protein only

1. Formation of a complex between Cu2+ and protein amide (peptide) bonds in an alkaline solution causing a reduction of copper

to Cu+

(B

IURETreaction)

Cu2+ + protein > [Cu2+-proteincomplex]

Cu2+ + (polar amino acids, Trp, Tyr)red > Cu+ + (amino acids)ox

2. Cu+ and radical groups of tryptophan, tyrosine, and cysteine reduce a yellow phosphomolybdate-phosphotungstatecomplex(Folin-Ciocalteu reagent: Na2MoO4 + Na2WoO4 + H3PO4) to a deep blue color

Cu+ + (F-C)ox

> Cu2+ + (F-C)red

(F-C) = phospho-Mo-Tungstate acid

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Antibodies

Avidity - strength of interactions between a multivalent antibodyand a multivalent antigen

Most produced Ig. Found in mucosal areas, such as the gut, respiratory andurogenital tract, and prevents their colonization by pathogens. Resistant todigestion and is secreted in milk.

Function unclear. Works with IgM in B-cell development; mostly B cell bound

Binds to allergens and triggers histamine release from mast cells and isinvolved in allergy. Also protects against parasitic worms.

Major Ig in serum. Provides the majority of antibody based immunityagainst invading pathogens. Moderate complement fixer (IgG3) can crossplacenta.

First response antibody. Expressed on the surface of B cells and in asecreted form with very high avidity. Eliminates pathogens in the early

stages of B cell mediated immunity before there is sufficient IgG.

Ab

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There are several advantages to choosing chickens, rather than rabbit or goats to produce your polyclonal antibodies.1. Chickens are not mammals and therefore are more able to make high-avidity antibodies to mammalian antigens (especially highly conservedmammalian proteins).

2. To our knowledge, it is the most ethical way to produce polyclonal antibodies. There is no need to bleed the chicken. Simply collect the eggs.Our hens are kept in flocks of six in coops that have outside runs. Our unique egg identification system allows our chickens to live freely.

3. A single chicken can produce an enormous amountof antibody,upto 3 gofIgYper month, which is 10-20 times the amount of a rabbit.

Furthermore, compared to rabbits, chickensproduce antibody muchquicker, high-titre antibody is available from eggs as early asday 25.

4. Fc region of chicken IgY is sufficiently different from mammalian IgG: Does not bind to mammalian Fc receptors

5. By having the immunoglobulin Y (IgY) packaged conveniently in eggs, one can collect and store eggs over a long period of time andretroactively purify the IgY from the eggs of desired titre/avidity.

6. It is cheaper to feed and house chickens than rabbits.

7. IgY is a stable antibody sharing the following characteristics with mammalian IgG: Divalent degraded by papain to yield divalent Fabfragment

May be enzyme-labeled, biotinylated and goldlabeled by standard procedures

ChickenIgYAb

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Tissue culturesupernatant

AscitesWholeantiserum

Purifiedantibody

WB / dot blot 1/100 1/1000 1/500 1 ug/ml

IHC / ICC neat to 1/10 1/100 1/50 to 1/100 5 ug/ml

EIA / ELISA 1/1000 1/10000 1/500 0.1 ug/ml

FACS / FlowCytometry

1/100 1/1000 1/500 1 ug/ml

IP ? 1/100 1/50-1/100 1 to 10 ug/ml

Concentration

estimate

1 to 3 mg/ml 5 to 10 mg/ml 1 to 10

mg/ml

AntibodiesAbAntiserumPolyclonal antibodies are often available in relatively unpurified formats, described as serumor antiserum. Antiserum refers to the blood from an immunized host from which clotting

proteins and red blood cells have been removed. The antiserum will containantibodies/immunoglobulins of all classes as well as other serum proteins.TissueculturesupernatantMonoclonal antibodies may be grown as hybridoma cell cultures (cells secreting cytokines) andharvested as hybridoma tissue culture supernatantsAscitesfluidMonoclonal antibodies can be produced by growing hybridoma cells within the peritoneal cavityof a mouse (or rat). When injected into a mouse, the hybridoma cells multiply and produce fluid

(ascites) in its abdomen. This fluid contains a high concentration of antibody which can beharvested. This usually provides higher antibody yields than hybridoma cell culture.Unpurified antibody concentrationsUnpurified antibody preparations vary significantly in specific antibody concentration. If thespecific antibody concentration of a given unpurified antibody preparation is unknown, one mayrefer to the following "typical ranges" as a guideline for estimation:

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Polyclonal antibodies- Recognise multiple epitopes on any one antigen.-Serum obtained will contain a heterogeneous complex mixture of antibodies of different

affinity- Polyclonals are made up mainly of IgG subclass

General advantages:

- Polyclonals will recognize multiple epitopes on any one antigen which has the followingadvantages:

- More tolerant of minor changes in the antigen, e.g., polymorphism, heterogeneity ofglycosylation, or slight denaturation, than monoclonal (homogenous) antibodies.

- Polyclonal antibodies are often the preferred choice for detection of denatured proteins.

Disadvantages:

- Prone to batch to batch variability.-They produce large amounts of non-specific antibodies which can sometimes give backgroundsignal in some applications.

-Multiple epitopes make it important to check immunogen sequence for any cross-reactivity. Drosophila-rabbit Ab

Ab

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Monoclonal antibodies

- Detect only one epitope on the antigen.-They will consist of only one antibody subtype.

- Where a secondary antibody is required fordetection, an antibody against the correct subclassshould be chosen..

Advantages:Once hybridomas are made it is a constant andrenewable source and all batches will be identicalMonoclonals detect one epitope only on any oneantigen which has the following advantages:- less background from staining of sections and

cells.- less likely to cross-react with other proteins.

-specificity excellent as the primary antibody in anassay, or for detecting antigens in tissue, and willoften give significantly less background stainingthan polyclonal antibodies.

-Disadvantages:They can produce large amounts of specificantibodies but may be too specific (e.g. less likelyto detect in across a range of species)More vulnerable to the loss of epitope throughchemical treatment of the antigen than arepolyclonal antibodies. This can be offset by pooling

two or more monoclonal antibodies to the sameantigen.

Ab

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Inexpensive to produce Expensive to produce

Technology required is low High technology required

Skills required are lowTraining is required for the technologyuse

Time scale is short Time scale is long for hybridomas

Produces large amounts of non specificantibodies

Can produce large amounts of specificantibodies but may be too specific

Recognizes multiple epitopes on any oneantigen

Recognizes only one epitope on anantigen

Can be batchto batch variabilityOnce a hybridoma is made it is aconstant and renewable source and allbatches will be identical

Polyclonal v. MonoclonalAb

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Principle of ELISA/RIA

1 2

3 4

ELISA - Enzyme-Linked ImmunoSorbent Assay

1. Antigen coating wells

2. Add serum

3. Add enzymeconjugated Ab(anti-human IgG)

4. Add substrate

5. Read color reaction

Ab

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Denaturing ConditionsElectrophoresis is performed under denaturing conditions using an anionicdetergent such as sodium dodecylsulfate (SDS).

Non-Denaturing(Native) ConditionsElectrophoresis is performed under non-denaturing (native) conditions usingbuffer systems that maintain the native protein confirmation, subunitinteraction, and biological activity. During native electrophoresis, proteins areseparated based on their charge to mass ratios.

Reducing ConditionsElectrophoresis is performed under reducing conditions using reducing agentssuch as dithiothreitol (DTT) or -mercaptoethanol (-ME). The reducing agentscompletely unfold the denatured proteins into their subunits by cleaving thedisulfide bonds between cysteine residues.

Proteinelectrophoresis

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SDSPAGE

- proteins are denatured by heating in the sample buffer which contains SDS

-> all proteins retain only their primary structure-> all proteins are fibrous and have an equal negative charge per length unit given

by binding to SDS => migration dependends on their molecular weight only!

SDS (Sodium Dodecyl Sulfate) anion detergent

solubilizes and denatures proteins

it gives protein negative charge

Most proteins binds SDS in this ratio : 1,4gSDS/gproteinu

Increasedtemperaturedenaturures proteins

OS

O

O

O

-

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH3

+SDS

+heat

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SDSPAGE

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SDSPAGE - Gels

Tris-glycine -Tris-acetate

Tricine

Bis-Tris

Zymogram

It has a lowernegativechargethan glycine allowing it to migratefaster. In addition its high ionic strengthcauses more ion movement and less protein movement. This allows for low molecularweightproteinsto beseparatedin lowerpercentacrylamide gels. Tricine has been documented in the separation of proteins inthe range of 1 to 100 kDa by electrophoresis.

-includes a substrate copolymerised with the polyacrylamide detection of enzymatic activity (gelatin,casein)

- samples are prepared without denaturing the active enzymes present in the samples

- following electrophoresis, the gel is placed in an enzyme activation buffer which allows the enzymespresent in the sample to become active and digest the substrates copolymerised in the gel

- the zymogram is subsequently stained and the areas of enzyme activy and digestion become visible

High-resolution pre-cast gel system for SDS-PAGE applications, fast run times (25-minute quick check), 12-month shelf life at room temperature, and efficient transfer.

High-performance pre-cast gels for large protein separation

Laemmli-based Tris-glycine gels

Precasted: 1-D2-D

Gradient gels : 4-20%

Shelf life: weeks to 18 month

Thickness: 0.75 - 1 1.5 mm

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PrepareSamplesPrepareRunningBufferPrepare 1X Tris-Glycine SDS Running Buffer by adding 100 ml of 10XNovex Tris-Glycine SDS Running Buffer to 900 ml of deionized water.LoadSampleLoad the appropriate concentration and volume of your protein sample

on the gel.LoadBufferFill the Upper Buffer Chamber with 200 ml and the Lower BufferChamber with 600 ml of 1X Tris-Glycine SDS Running Buffer.Run ConditionsVoltage: 125 V constantRun Time: 90 minutes (dependent on gel percentage)Expected Current: 30-40 mA/gel (start); 8-12 mA/gel (end)

Sensitivity Level10% Zymogram (Gelatin) Gel: 10-6 units of collagenase12% Zymogram (Casein) Gel: 7 x 10-4 units of trypsin4-16% Zymogram (Blue Casein) Gel: 1.5 x 10-3 units of trypsin

StainingtheGelZymogram (Blue Casein) 4-16% gels do not require staining. For nonprestained Zymogram gels, stain the gels withColloidal Blue Staining Kit or the SimplyBlue Safestain available from Invitrogen. Areas of protease activity willappear as clear bands against a dark background

DevelopingtheGel1. Dilute Novex Zymogram Renaturing Buffer (10X) and Novex Zymogram Developing Buffer (10X), 1:9 withdeionized water. You will need 100 ml of each buffer per one or two mini-gels.2. After electrophoresis, remove the gel and incubate the gel in 1X Zymogram Renaturing Buffer from Step 1for 30 minutes at room temperature with gentle agitation.3. Decant the Zymogram Renaturing Buffer and add 1X Zymogram DevelopingBufferfrom Step 1 to the gel.4. Equilibrate the gel for 30 minutes at room temperature with gentle agitation.5. Decant the buffer and addfresh 1X Zymogram DevelopingBufferfrom Step 1 to the gel.6. Incubate the gel at 37C for at least 4 hours or overnight for maximum sensitivity. The optimal result is

determined empirically by varying the sample load or incubation time.

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SDSPAGE

Voltage 5-7 V/cm

Coomassie Brilliant Blue R stains canusually detect a 50-ng protein band Ponceau-SSilver

Loading capacity: up to 50ug/lane

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SDSPAGE

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2-D gel elfo

Data Analysis

Sample preparation for Mass Spectrometry

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2-D gel elfo

200

90

60

25

kDa

IEF

SDSPAGE

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2-D gel elfo

IPGstrips (store at-20C)- pH gradient is generated by a number (6-8) of well-defined chemicals (immobilines) which are co-polymerized with the acrylamide matrix

Advantages:- better reproducibility- easy manipulation !!!

Disadvantages:- high price- hydrophobic proteins can bind to plastic

holder

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IPGstriprehydration

A minimum of 10 h is required for rehydration; overnight is recommended

Rehydration solution - 8 M urea, 0.5% (w/v) CHAPS, 0.2% (w/v) DTT, 0.5% (v/v) IPG Buffer orPharmalyte, 0.002% bromophenol blue

Urea solubilizes anddenaturesproteins,unfoldingthem toexposeinternal ionizable aminoacids. Commonly 8M urea is used, but the concentration can be increased to 9 or 9.8 M if necessary for complete sample

solubilization. Thiourea,in addition to urea, can be used to further improve protein solubilization

Detergentsolubilizeshydrophobicproteins and minimizesprotein aggregation. Thedetergentmust have zeronet chargeuse only non-ionic and zwitterionic detergents. CHAPS, Triton X-100, or NP-40 in the range of 0.5 to4% are most commonly used.

Reductantcleavesdisulfide bondsto allowproteinstounfoldcompletely. DTTor DTE(20 to 100 mM) arecommonly used. 2-Mercaptoethanol is not recommended, because higher concentrations are required, andimpurities may result in artifacts. Tributyl phosphine (TBP) is not recommended as reductant for IEF due to its

low solubility and poor stability in rehydration solution. Reductants should be added directly before use.

IPGBufferorPharmalyte(carrier ampholyte mixtures) improve separations, particularly with high sample loads.Carrier ampholyte mixtures enhance protein solubility and produce more uniform conductivity across the pHgradient without disturbing IEF or affecting the shape of the gradient.

IPG Buffers are carrier ampholyte mixtures specially formulated not to interfere with silver staining following 2-D electrophoresis. Select an IPG buffer with the sample pH interval as the Immobiline DryStrip to berehydrated. Use IPG Buffer 3.55.0 for Immobiline DryStrip 3.54.5 and 4.05.0. Use IPG Buffer 611 for

Immobiline DryStrip 69 and 611. Pharmalyte 310 may be used for separations on Immobiline DryStrip pH 310and 310 NL. Pharmalyte 58 may be used for separations on Immobiline DryStrip pH 47.

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After IEF proceed to the second-dimension separation immediately or store the IPG strips at -70 C

in screw-cap tubes.

Isolelectricfocusing

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Protein(s)

Increase in resolving power in the first dimension with overlapping pH range IPG strips. E colilysate (40 l per gel) was run on 11 cm

overlapping narrow pH range ReadyStrip IPG strips and focused for 20,000 V-hr. The strips were then transferred to 816% Tris-HCI Criterion

precast gels for the second dimension run. The gels were strained with colloidal Coomassie Blue stain. More proteins were detected on the three

overlapping gels than on the single 310 pH range gel. Note the improved resolution of proteins in the circled areas.

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ImageAnalysis

Commonly Used software:

ImageMasterMelanie IIIPDQuest

Spot picker

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WESTERNBLOTTING- analytical technique used to detect specific proteins in a givensample of tissue homogenate or extract.

SDS PAGE

transfer to a membrane

Detection

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WESTERNBLOTTING

- Run the transfer for 6-8 hours to overnight at 30 volts (or 30-60 mA).

- to 100 V 150V (about 200 mA) and carrying out the transfer to 1-2 h (longer durations willcause severe heating). Even when using high voltage, it is recommended that the initialtransfer (first 20-30 minutes) be done at low voltage to allow low molecular weight proteins to

bind.

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PVDF - much more sensitive to SDS - keep it

limited to less than 0.05%.

PVDF is a naturally hydrophobic, unsupportedpolyvinylidene fluoride transfer membrane - western transfer- protein sequencing

Amido Black, Colloidal Gold, Coomassie Blue, IndiaInk and Ponceau-S.

- 0.22 and 0.45 um

- from 5k Daltons to 700k Daltons- binding capacity averages 125 g/cm for larger

globular proteins such as immunoglobulins andhigher binding capacities for small peptides.

PVDF (polyvinylidene fluoride)

Binding Capacity:- 209 g/cm2 of proteins (0.2 m)- 80 g/cm2 of proteins (0.45 m)

- 0.2 m proteins (20 kDa)

-Compatible with commonly used transferconditions and detection methods such asstaining, immunodetection, fluorescence, orradiolabeling

- Provides high-sensitivity with low background

Nitrocellulose

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WESTERNBLOTTING

E

primary antibody

conjugated secondary antibody

substrate

protein

membrane

3,3,5,5-Tetramethylbenzidine (TMB)

3,3'-Diaminobenzidine (DAB)

Horse redish peroxidase (HRP)

Enzyme Substrate

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Proteinexpression

NEXTProteinpurification

Protein assays

Protein modelling