Sds-polyacrylamide Gel Electrophoresis Introduction

SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS Introduction The separation of proteins by polyacrylamide gel electrophoresis (PAGE) is an important and widely used biochemical technique. Used under denaturing conditions, specifically in the presence of the anionic detergent sodium dodecylsulphate (SDS), it permits separation at very high resolution of polypeptides from one another on the basis of their relative molecular masses (the smallest polypeptides migrate fastest) and allows estimates to be made of their relative molecular masses.

Overview of what you will do You should now prepare samples of proteins for electrophoresis (SDS-PAGE). For each of the samples from the column. You should take an appropriate volume and mix with sample buffer. Sample buffer is a denaturing solution that contains a detergent, sodium dodecyl sulphate (SDS) and a reducing agent (either 2-mercaptoethanol or dithiothreitol). • • • •

Complete the gel by pouring a stacking gel Prepare samples for electrophoresis Load gel; start electrophoresis Return and stain gel

Making an SDS-PAGE slab gel

 WARNING - Acrylamide is toxic by cumulative skin absorption so ALWAYS wear gloves when preparing or handling gels.

 NOTE: Treat all solutions in the SDS-PAGE part of this experiment as TOXIC by mouth and skin contact. USE GLOVES. Note: the acrylamide concentration may be increased or decreased, to suit the molecular weights of the proteins being separated, but the acrylamide: bisacrylamide ratio is maintained at 37.5:1. THE GEL BACK PLATES AND SPACERS COST ALMOST £20 EACH! TAKE CARE OF THEM PLEASE. CLEAN THEM AFTER USE AND RETURN TO THE STORAGE DRAWER

Protocol:– SDS-PAGE resolving gel preparation For a 10% resolving gel with a 5% stacking gel: Stock Solutions • 30% (w/v) acrylamide stock (29.2 g acrylamide, 0.8 g methyl [bis]acrylamide) • 1 M Tris-HCl, pH 8.8 • 1 M Tris-HCl, pH 6.8 • 10% (w/v) Sodium dodecylsulphate (lauryl sulphate, SDS) • TEMED (N, N, N’, N’ –tetramethylene-diamine) • 10% (w/v) Ammonium persulphate • Water saturated n-butanol (50 ml water + 10 ml n-butanol, shake and use the upper phase for the overlay) • Storage buffer: 10 ml 1 M Tris-HCl, pH 8.8 + 20 ml distilled water Note : TEMED and APS can be increased for faster polymerisation. Resolving gels may be

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stored for up to 1 week overlaid with storage buffer and water saturated n-butanol

Protocol:– Preparation, loading and running of the samples [the resolving gel has now been poured and allowed to polymerise]

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Complete setting up the gel and start it running as instructed. The gel will take approximately 1 hour to run. Come back after 45 min to begin the staining of part of your gel.

For 10 ml of 5% stacking gel • 30% Acrylamide stock: 1 ml • 1 M Tris-HCl, pH 6.8: 1.25 ml • Distilled water: 7.7 ml • 10% SDS: 0.1 ml • TEMED: 10 µ l • 10% APS: 50 µ l Note: TEMED and APS can be increased for faster polymerisation

Procedure Note: Pour off overlay and rinse with water before pouring stacking gel

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• Mix the stacking gel solution by swirling together. Pour on top of resolving gel until the level is almost full and insert a Teflon comb, being careful not to introduce and trap air bubbles. Leave until the gel has set. Allow 15-30 minutes for polymerisation. Once the stacking gel has polymerised the gel should be used within 60 minutes. Fit the cassette into the base reservoir, put a well guide onto the front so that you will be able to see where the wells are and fill the back reservoir to the top with tank buffer. Carefully remove the comb. Fill the base reservoir with tank/running buffer until it covers the platinum wire. Make sure that the sample wells in the top of the gel are neat, unclogged and visible. Using a Gilson P20 and the special long-nosed gel loading tips, load 30µl of each sample and standards into the bottom of appropriate wells. Fit the cover and insert the leads into the power pack (UNPLUGGED FROM THE MAINS!) so that the red is +ve and the black is -ve. If the power pack has a polarity switch, check that it is set to POLARITY NORMAL.

10 x (10 times) running buffer, for 1L: • 30.3 g Tris-HCl (0.25 M) • 144.2 g Glycine (1.92 M) • 10 g SDS (1% w/v) Samples should be prepared in SDS loading buffer 4 x loading buffer: 4 ml glycerol 2 ml 2-mercaptoethanol 2 ml 10% SDS 2.5 ml 1 M Tris-HCl, pH 6.8 0.001% (w/v) bromophenol blue – vortex until completely mixed – store in 1 ml aliquots at -20°C

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Load 30 µ l of your concentrated protein sample, including a molecular weight marker in a separate well (100-200 ng in 30 µ l respectively).

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Make note of your loading scheme, keeping in mind the position of the molecular weight marker which should be loaded asymmetrically, so that you may identify the lanes after staining. Turn on the power, select CONSTANT VOLTAGE and adjust to 200V (or current to 20 mA) per gel. Run gel at 200 V for about 45 minutes. Leave until the blue tracker dye is below the top of the buffer in the base. Turn off, unplug and strip down the cassette, discarding the buffer down the sink. Carefully dismantle the cassette by SLIDING the spacers out of the top or bottom - DO NOT PULL THEM SIDEWAYS. Use a thin spatula or razor blade to GENTLY lever the glass plates apart AT THE SIDE OR BOTTOM CORNER – NOT AT THE TOP LUGS. Cut off the bottom left corner of the gel. Transfer the gel to a polythene box. Please make sure that your gel box is labelled. After removal of gel from cassette, separate portions of the gel to be used in blotting and Coomassie staining by gently cutting it with a razor blade. Stain one part with Coomassie blue. The other part will be retained for Western blotting.

Coomassie stain solution, for 1 L • 2.50 g Coomassie Brilliant blue R-250 (CBB) (not blue G) • 100 ml glacial acetic acid • 400 ml methanol • fill to 1 L with distilled water De-stain solution, for 1 L • 100 ml glacial acetic acid • 200 ml methanol • fill to 1 L with distilled water

Coomassie stain •

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Allow that part of the gel to stain for an hour at room temperature. The destaining will be continued for you for an additional few hours until an appropriate signal to noise ration is obtained. This is when protein bands are clearly visible against a relatively transparent background. Proceed to mass spectrometry preparation.

Protocol: Part 4 – Examination of the gel Plot a graph of distance of migration versus log [relative molecular mass (Mr)] for the marker proteins, which may be as follows (other proteins may be used): MBP-β-galactosidase (175,000), MBP-paramyosin (83,000), Glutamic dehydrogenase (62,000), Aldolase (47,500), Triosephosphate isomerase (32,500), β-Lactoglobulin A, (25,000), Lysozyme (16,500), Aprotinin (6,500 – will probably have run off the bottom of the gel). Note that this is simply a collection of convenient markers – they have no logical connection with your samples.

Aims and objectives After performing this experiment and writing your report you should be able to: •

Prepare a polyacrylamide gel for electrophoretic separation of proteins in the presence of SDS, and prepare samples of proteins for electrophoresis in such a gel.

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Explain how a separation is achieved, and why it may be used for determining the subunit

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composition of proteins and the molecular weights of their constituent polypeptides.

A. Western Blotting; semi-dry transfer NB:

Wearing gloves is crucial for the next steps; otherwise the membrane will be ruined. It is essential that the Whatman paper and membrane be cut into squares of approximate size to the gel. If these are too large then you will get “short circuits” and the gel will take longer to transfer, if they are too small you will not be able to transfer the whole gel.

1. Wet 2 pieces of Whatman paper in transfer buffer. Place them on the transfer apparatus 2. Next wet the blotting paper, in this case nitrocellulose, in transfer buffer and place squarely on top of the Whatman paper 3. On top of this you will place the unstained part of your gel from the practical on SDSpolyacrylamide gel electrophoresis. Make note of the orientation so that you will be able to identify your lanes later 4. Lastly, wet 2 more pieces of Whatman paper and place them on top of the gel 5. The transfer sandwich is complete. Put the top on the apparatus and allow the transfer to take place at 150 mA to 200 mA for 1h

6. After transfer, remove the blot and allow it to soak in blocking solution overnight at 4°C. The proteins in the blocking solution occupy remaining binding/adsorption sites on the membrane to prevent antibody binding non-specifically, hence reducing background signal 7. Wash twice with the buffer, PBST, 5 min each 8. Add the horseradish peroxidase (HRP) conjugated PAP antibody at 1:5000 dilution in PBS for 1 hr at RT 9. Wash twice with PBST, 10 min each 10. Wash twice with PBST, 5 min each 11. Perform Enhanced Chemi-Luminescence (ECL) reaction and develop film – to be demonstrated

B. Visualising the proteins by ECL 1.

Pour off the blocking solution

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Wash the blot twice with PBST on shaker for 5 min

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Add PAP antibody @ 1:5000 dilution in PBS for 1 hr at RT

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Wash twice with PBST on shaker for 10 minutes

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Prepare a piece of plastic wrap for the blot. About the size of half an A4

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Wash once with PBS and pour off

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Holding the membrane by the corner with gloves, allow the residual PBS to drain off. Do not allow the blot to dry completely, just allow the excess liquid to drip away 8. Perform ECL reaction in a dry container. Allow this to proceed for 1 minute and then pour off the residual ECL in the same fashion as last time

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9. After the excess liquid has run off the blot place it face down on the plastic wrap and fold the wrap over enclosing the blot thoroughly so no residual liquid can leak out. Ensure that the plastic wrapping is done smoothly with minimal creasing. 10.

develop the film in one of the department’s automated developers

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