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Department of Medical Biochemistry and Microbiology

Responsible teacher: Jin-ping Li/DS Version: 2

Date: 2015-08-19 Number of pages: 13

Biomedicinprogrammet T1

Biokemi

Teachers’ instructions/Amanuensinstruktion

PROTEIN SEPARATION

Department of Medical Biochemistry and Microbiology Responsible teacher: Jin-ping Li/DS

Version: 2 Date: 2015-08-19

Teachers’ instructions – complement to students’ instructions

Observe: Read through the students’ lab instructions part (pp. 6-) so that you are aware of the background of the lab (as available for the students). Additional hints and tips are given in this teachers’ manual including expected answers in lab reports.

Time lineFour weeks before Check the list of materials and reagents that are needed, anything that

needs to be ordered?

Two weeks before Perform a prerun. Make sure all reagents are indeed working, and that there is enough for all groups of students. Do columns need repacking? Evaluate the results of the prerun carefully and if needed discuss with the responsible teacher.

Two days before Set up the lab with all necessary materials, plan and make sure things are in a good order for the students. Make sure columns are not dry. Prepare aliquots of reagents, if needed.Plan this with Eva Gottfridsson.Ask your head teacher for a printed attendance list.

15 minutes before Take out reagents from freezerSet up for introduction (projector available, ask Eva G)

Lab starts!

Wear a clean lab coat with name tag, stay focused – no cell phones during the lab! Be on time (i.e. at least 15 minutes before scheduled course time!). Be prepared!

Critical steps during the practical

DEAE-SepharoseMake sure that the students know how to manage the pump and reinforce the importance of checking the direction of pumping as well as not letting air come into the column. Should the gel dry out after adding the sample but before starting to collect fractions (and opening the gradient mixer) it is possible to repack the column (make sure that the sample has reached the column so it is not lost in the tubing!). If the column dries while collecting the fractions it is advisable to leave it as it is and to repack it before washing since you will otherwise disturb the gradient.

Make sure that the gradient mixer is open and mixing (no air bubbles trapped in the channel) when the experiment starts (especially if students leave during the elution). It is advisable to let the students stay for at least until the two first fractions have been collected to insure that the collector is turning as it should. Also ensure that there is no fluid leakage at any connection point.

Gel castingMake sure that the students are only wearing gloves when handling the non-polymerized acrylamide, which shall be present and handled only in the fume hoods. Gloves are not necessary during any other part of the lab except for when handling the Coomassie staining solution.

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To minimize the stress of the students casting gels (and thus potentially keeping the mistake rate low) have only one group working at a time in a hood (but use as many hoods as you have available) and make sure that the ones queuing do not stand too close to the others working. The best is to keep the queuing groups at their benches until there is a free place for them to work. This also allows the amanuenses to monitor the work in the hood without having to squeeze through a crowd to do so.

In order to avoid air bubbles in the stacking gel resulting in loss of wells, fill the stacking gel to the brim of the lower plate and then push down the comb. This will result in overflowing but excess gel can be removed after polymerization, just watch out for splashing when pushing the comb down.

Gel runningAlways remove non-polymerized gel remnants from the wells after removing the comb by rinsing using a syringe with a 20-gauge needle (while assembled in the gasket and after addition of running buffer).

Make sure that the gaskets are not leaking running buffer after assembly. If the level becomes too low the current will not be sustained and electrophoresis will stop.

Coomassie stainingIntensely staining (though easily removed from surfaces by 70 % ethanol), avoid splashing as much as possible; use bench paper on surfaces where solution is handled and use gloves.

Unused gels can be used to practise handling gels before taking apart the gels from the electrophoresis equipment.

Note: Coomassie staining solution is not to be poured down the sink! Collect the solution after staining; it may be reused several times. De-staining can be speeded up by folding a piece of tissue paper (or rubber foam) and leave it in the de-staining solution, it will absorb excess stain from the solution and reduce the number of solution changes.

Common sources of error

DEAE-Sepharose: Air getting trapped in the system when disassembling and reassembling of tubing Sample has not been loaded onto column Pump has not been started Pump going in the wrong direction Fraction collector has not been started. Fraction collector going past all tubes at the switch or not switching, check tubing length at sensor Fraction collector does not rotate – Make sure that the tray matches the stand (holder with versus one without ruffles):

Ruffles No ruffles

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SDS-PAGE: Adding of APS and TEMED to the gel-solution before assembling glass plates in the casting frames (-> gel polymerized already) Adding too much or too little (or no) APS and/or TEMED resulting in very fast or slow polymerization Trying to pipette semi-polymerized gel-solution (gelatinous) resulting in uneven top or cavities in the gel Forgetting to layer water (or isopropanol) on top of the separation gel Forgetting grey rubber pads in casting frames – solution runs through Assembling plates with the spacers facing away from short plate resulting in no space to cast gel Adding TEMED and APS to both stacking gel and separation gel at the same time, sometimes casting the gels in different plates (one with separation and the other with all the stacking)

Make sure that that the timer on the power supply is turned off or set to a sufficient time

Staining: Getting the gel stuck to the green wedge resulting in tearing of the gel, easily avoided by wetting the wedge Destruction of the gel by microwaving too long, make sure to stop once the solutions start to boil (if using the microwave)

Lab report

Unless otherwise specified all reports should conform to the following points.Numbered figures with legend underneath Numbered tables with legend on topAll figures and tables need to be referred to in the textResult section should not only be figures and tables but connected by text so that it is easy to follow what has been doneIf the report is in English, use the decimal point (not decimal comma as in Swedish)To pass the practical the students should hand in a report, no later than 5 working days after the lab (observe – will be controlled for plagiarism – students have to upload document at the SP).

The report should have the following parts:1) Background/theory

A brief description of the concepts of the lab.Describing the principles of ion-exchange chromatography and SDS-PAGE, paying special attention to what the basis of separation is in the different methods.

2) MethodsDescribing how the work was preformed in general, and more in-depth descriptions of crucial steps.

3) ResultsFocus on the questions in the handout, but still retaining a cohesive and understandable report.

Pictures or scans of the gel and Western blot should be included (otherwise it is virtually impossible to interpret the results and the reasonability of the results). Results are not to be discussed in the result part.

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All results should be included and presented in an understandable way, with paragraphs of text not just diagrams.

4) DiscussionDescribe in short how the experiments went, both practically and also the main results. Are the results reasonable? Expected? If not, is there an explanation? What went wrong? What conclusions may be drawn? Sources of error should be presented where deemed relevant but not every minor thing should be listed.

General note: The list of proteins included in the handout is that of plasma proteins, since the sample used is serum some proteins (mainly fibrinogen and coagulation factors) will not be present on the gel.

Note! The responsible teacher sets the criteria for the lab report – communicate to ensure everybody knows what to expect/demand!

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Safety and rules

General rules for the course lab (there may be further specific points for individual labs).No eating or drinking in the lab (includes water, coffee, etc.).No usage of cell phones (exceptions for usage in calculations can be made, teachers decide).No outdoor clothes (e.g. jackets and hats) should be worn in the lab. There are hangers at the entrance to the lab, keep bags at the same place.A clean lab coat is to be worn while in the lab.Use extra safety equipment when needed (e.g. glasses when handling strong acids/bases).If gloves are used, take them off when they are not needed. Do not go around contaminating other areas, such as door handles, with dirty gloves! Note: Gloves are thrown in the regular garbage bin, not in “Riskavfall” unless you are specifically instructed to do so. Please do not put regular waste, e.g. tissues, in the boxes for pipette tips, these are for sharp or contaminated waste.

Safety Emergency exits are found in both ends of the corridorFire extinguisher, eye showers, bandages etc. can be found at the entrance to the corridorYou will be instructed about additional precautions during the lab, for instance when/if you need gloves or work in the fume hoods.

SEPARATION OF SERUM PROTEINS BY ION EXCHANGE CHROMATOGRAPHY

PURPOSEThe isolation of specific proteins from sources such as muscle or serum is a central part of practical biochemistry. In this exercise you will use of one of the most frequently employed separation techniques: ion exchange chromatography. With this technique you will separate proteins in bovine serum. To visualize the separated proteins you will use polyacrylamide gel electrophoresis.

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LEARNING OUTCOMESHaving done this lab, you should be able to describe:

how ion exchange chromatography can be used to separate proteins how spectrophotometry can be used to determine protein concentrations how SDS/PAGE can be used to assess the composition of protein samples and to

estimate the molecular mass of proteinsFurthermore, you should be able to:

prepare an SDS/PAGE gel and use it for the analysis of protein-containing samples

THEORYIn ion exchange chromatography the protein sample is pumped through a column containing gel beads with fixed negative or positive charges; proteins with a charge opposite to that of the beads will then bind to the gel. The salt concentration in the buffer running through the column is then increased, which causes the proteins to detach, those with little charge coming out first and vice versa. The experimental setup is shown in Figure 1. You will use a positively charged gel and increase the salt concentration gradually through the use of a gradient mixer; this device consists of two connected vessels and a magnetic stirrer. The eluted proteins will be collected in test tubes (fractions) and the protein concentration determined by UV-measurement. Finally, you will use polyacrylamide gel electrophoresis to visualize individual proteins in the obtained fractions.

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Figure 1. Set-up for ion exchange chromatography. Individual parts are labeled by name.



1. ION EXCHANGE CHROMATOGRAPHY

MATERIAL Tris/HCl, 25 mM, pH 7.5 (Buffer A); NaCl (solid)Magnet, magnetic stirrerColumn with DEAE-Sepharose Cl 6B (1.0 x 5.0 cm)Fraction collector, peristaltic pump with 1 mm diameter tubing, gradient mixer, clampsProtein sample (1.0 ml) containing bovine serum diluted 10X in 25 mM Tris/HCl (Buffer A).Test tubes: Eppendorf; 3-ml E-tube and 15-ml conical tubes and stoppers; 50-ml centrifuge tubesAnalytical balance, regular balance, spatulaConductivity meterPasteur pipettes, silicone tubing, felt pensMeasuring cylinder 100 ml, beaker 100 mlGraduated pipetteBovine serum albuminUV-cuvette, spectrophotometers

PROCEDURE

A. Adjustment of chromatography system1) Set the pump rate to 3 (1X), check that you have buffer (buffer A) in a beaker (or similar vessel) and that the ingoing tubing is well beneath the surface. Start the pump. Collect the outgoing solution in a pre-weighed test tube for a determined time, e.g. 3 min.

2) Weigh the test tube again and calculate the flow rate (1 g equals 1 ml; flow rate unit: ml/h). With the assumption that the flow rate is linearly proportional to the scale on the pump, adjust the flow rate to 12-15 ml/h. Check that the conductivities of the incoming buffer (Buffer A) and outgoing solution are the same (±25%): Add 20 µl from incoming and outcoming solution to 2 ml deionized water each, and measure the conductivity.

3) Calculate the time (in min) needed for collecting a 0.90-1.0 ml fraction. Put three empty tubes into the fraction collector and collect outcoming solution to check if the fraction volume is correct:

switch on fraction collector (at the back), press ‘Run/End’ to ‘End’ press “Collection mode” to “min”, press “Fraction size” and enter your calculated

time, and then press “Store” press “Run” and start the pump.

Fill in the table below.

Gel typeSize of the gel (length and diameter, cm)Adjusted flow rate (ml/h)Fraction volume (ml)

B. Preparation of elution buffer (Buffer B)1) Prepare 20 ml of Buffer B: Add solid NaCl to 20 ml of Buffer A, so that you get a final concentration of 0.5 M NaCl in the solution. Molecular weight of NaCl: 58.5 g/mol (calculate how many grams of NaCl you need to weigh in).

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C. Chromatography1) Make sure that the pump is off, then take the tubing out of the buffer A beaker and put the end of the tubing into the tube with the protein sample. Start the pump and suck the protein sample into the gel - then switch off the pump. OBSERVE: Air must not come into the tubing during this procedure. Put back the tubing into the buffer A-beaker and start the pump, let it run for about 5 minutes. Do not collect the outgoing fluid at this point. Meanwhile, number 20 fraction tubes with a pen and put them counterclockwise from 1-20 into the fraction collector (starting from the outermost slot in the fraction collector).

2) Measure 10 ml buffer A and add it to vessel A in the gradient mixer (with the valve vertically closed). Turn the valve slowly to the open position (horizontal position). When you see a drop running backwards into vessel B, close the valve. This will give you an idea when the valve is in the open position as well as removing air in the channel between the vessels. Then add 10 ml buffer B to vessel B. Stop the pump and connect the tubing with the fraction collector.

3) Align the arm of the fraction collector with the first tube (fraction 1). There is a stirring bar in the vessel A. Start the stirrer, pump and fraction collector. Open the valve (horizontal position is open).

4) Collect fractions till the height of the liquid in the gradient mixer is 2-3 mm. Then stop the pump.

D. UV-spectrum of protein The teaching assistants have prepared a protein solution of bovine serum albumin (BSA) at about 0.8 mg/ml. Determine the absorption spectrum from 400 to 230 nm of this solution according to separate instruction provided at the spectrophotometer.

Use these data to calculate the specific extinction coefficient (, unit ml/(mg•cm)) for albumin at 280 nm from the formula A=·c·l, where A is the absorbance, c is the concentration (in mg/ml) and l is the optical path length (1.0 cm).

E. Detection of proteinMeasure A280 for all 20 fractions according to separate instruction at the spectrophotometer.

Use the BSA sample to be able to determine the coefficient for later. Take out the BSA cuvette, rinse it with deionized water, then transfer the first of the

fraction samples to the cuvette and proceed as instructed. Repeat for all fractions, each value will be added to the list. N.B. Remember to save

the samples (pour them back into the tubes after measuring), you will use them later!

From these data draw an elution diagram of the ion exchange chromatography run.Consult the teaching assistants which 8 fractions should be saved for later analysis with SDS/PAGE.

F. Determination of salt concentrationWhen you are using ion exchange chromatography it is usually important to know at what salt concentration different proteins are eluted from the column. The salt concentration in the different fractions can easily be determined through measurement of the conductivity, since this value is proportional to the salt concentration.

For this purpose, add 20 µl from every second fraction to 2 ml deionized water each, and measure the conductivity. Do the same with Buffers A and B (which contain 0 and 0.5 M NaCl, respectively). Based on the conductivity and the known salt concentrations of buffer A

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and B, create a standard curve and calculate the NaCl concentration (in M) in the different fractions.

Close your tubes with lid and give the whole rack to an assistant teacher.

2. POLYACRYLAMIDE GEL ELECTROPHORESIS IN SDS (SDS/PAGE)In the type of electrophoresis used in this lab (the Laemmli system), the proteins first enter a relatively loose gel (the stacking gel), the purpose of which is to make the proteins pre-separate and therefore separate as sharper bands in the following separation gel which has a denser network to achieve the final separation. The buffer contains sodium dodecyl sulphate (SDS), which denatures all proteins and gives them a strong negative charge, so that they will migrate according to their molecular mass in the separation gel.

A. MaterialBio-Rad Miniprotean II electrophoresis apparatusFour 1.5 mm spacers and two 15-well combs, 1.5 mm thickSeparation gel solution: 97 g/l acrylamide, 2.5 g/l bisacrylamide, 0.4 M Tris/HCl, pH 8.8, 0.5 g/l SDSStacking gel solution: 49 g/l acrylamide, 1.3 g/l bisacrylamide, 125 mM Tris/HCl, pH 6.8, 0.5 g/l SDSNNN'N'-tetramethylene ethylendiamine (TEMED)Electrode buffer: 0.19 M glycine, 25 mM Tris (pH 8.3), 1.0 g/l SDSAmmonium persulphate, 100 g/l (APS)Filter paperSample buffer: 63 mM Tris/HCl,pH 6.8, 20 g/l SDS, 100 g/l glycerol, 0,10 g/l bromphenol blue, 8 mM dithiothreitol (DTT)Injection syringe 5 mlPasteur pipetteEppendorf tubes 1.5 ml, centrifuge tube 50 mlHeating block (95 °C)Molecular mass markers (high range, Biorad)Capillary tips, waterproof pens, plastic glovesStain solution: 0.5 g/l CBB in methanol/ acetic acid/ water (3:1:6)Gel shaker, Eppendorf centrifuge

B. Casting of gel 1) Assemble the glass plates of the electrophoresis apparatus. (You will prepare two gels although you need only one).

2) Put a mark with a waterproof pen 17 mm from the upper edge of the smaller plate.

3) From this step on work in the fume hood! Gloves must be worn when acryamide solutions are handled! You will get 10 ml separation gel solution in a 15 ml plastic centrifuge tube. Add 40 µl APS and 8 µl TEMED and mix immediately by turning the tube upside down twice.

4) Use a Pasteur pipette to fill the gel solution between the glass plates up to the marks you made. Layer water carefully over the acrylamide solution with a Pasteur pipette. Leave the remaining gel solution in the centrifuge tube with the lid open in the hood, so that you can easily check if polymerization occurs.The gel takes 30-40 min to polymerize. While you wait, prepare your samples (see C).

5) When the gels have polymerized, the water is poured out on a paper towel.

6) You will get 5 ml stacking gel solution in a 15-ml centrifuge tube. Add 20 µl APS and 5 µl TEMED and mix as above. Fill the gel chambers up to the rim of the smaller glass plate with the gel solution and push down the combs. No air bubbles! Make sure that the combs are

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centered between the spacers. Leave the remaining gel solution in the centrifuge tube with lid open in the hood, so that you can easily check if polymerization occurs (30 min).

C. Preparation of sample1) Prepare eight Eppendorf tubes with 10 µl of sample buffer. Add 10 µl of one of the 8 fractions (chosen from the ion exchange chromatography experiment) into one of the tubes with sample buffer. Repeat for the other seven fractions.

2) Prepare one more tube and mix 5 µl of the original protein sample (which you used the day before, teachers will provide it) with 15 µl sample buffer.

3) Close the lids and heat the Eppendorf tubes for 3 min at 95 °C. Centrifuge the tubes for 5-10 s at maximum speed in the centrifuge.

D. Electrophoresis run1) When your gels have polymerized, take out the glass plates from the green holder and assemble the buffer chamber. Fill up the inner buffer chamber with electrophoresis running buffer and fill half of the outer buffer chamber.

2) Pull out the combs carefully and rinse the wells to remove all un-polymerized gel remnants. 3) Note down, in which order you want to load your 8 fractions, the original protein sample and the molecular mass marker. Apply 15 µl of the samples you prepared with a pipette with a tip; change the tip between each application. Apply 5 µl of the molecular mass marker (you will get it from the teaching assistants).

4) Attach the electrode leads to the power supply – red to red and black to black. Adjust the number of voltage on the display to 95 (V). After about 15 min, adjust the voltage to 200 V.

5) Turn off the power supply when the blue dye has reached the lower edge of the gel - takes 60-70 min.

E. Visualization of protein bands1) Disassemble the electrophoresis apparatus. Use the green wedge-formed tool to loosen the glass plates from each other.

2) Put the gel into a petri dish. Pour staining solution into it – do not use more than what just will cover a gel. Then put the petri dish on a shaker.

3) After 20 min, pour back the staining solution into the staining solution bottle and rinse the gel two times with deionized water to get rid of any remaining staining solution. Fill up the petri dish about half-way with deionized water. Heat the petri dish with the gel in microwave, until it has boiled for 15-20 s, to destain more effectively. After destaining, the assistant will put the gel into a plastic bag for later analysis.

3. ASSIGNMENTS1) Measure the distances from the upper edge of the separation gel to the bands of the molecular mass markers. Plot these values (x-axis) against the logarithms of the masses. Fit the points with a smooth line by hand or a straight line by computer.

2) Measure the corresponding distances to the three major bands in the fraction samples. Use the graph to estimate the molecular masses of these proteins.

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3) Below, in Table 1, you have a list with information on the various plasma proteins. Try to identify the major proteins in your gel by using these data.

4) Assume that albumin is the only protein in the mixture with the highest amount of protein. Estimate how much albumin was applied on the electrophoresis gel from that fraction using the specific absorbance coefficient you have calculated.

5) Estimate at what salt concentration the major protein was eluted.

6) Have you obtained a pure protein in any of the fractions? If not, what could be changed in this experiment to improve the separation?

4. LAB REPORT

Your report should contain the following information:

Short description of the principle of ion exchange chromatography Gel type, size of gel, flow rate, fraction volume Elution diagram with A280 and NaCl concentration (in the same graph if possible) Short description of the principle of SDS/PAGE Photocopy of the electrophoresis gel UV-spectrum of albumin solution and calculated specific absorbance coefficient Answers to assignments

Submittal:

1. Write your report in Word in English and save the document in the doc (not docx) format. Name the file “enzyme_kinetics_groupX.doc”, with X replaced with your group number.

2. Write the names of all group members at the top of the report.

3. Upload the report to Studentportalen under Inlämningar->Proteinseparation.

Table 1. Concentration, molecular weight and half-life data for several plasma proteins.

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50 + 25

63 + 55

38 + 9

300

185

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Uppsala Universitet - Välkommen till Web viewCapillary tips, waterproof pens, ... Write your report in Word in English and save the document in the doc (not docx)