SDS PAGE PROTOCOL SOLUTIONS

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1 SDS PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) Is a technique widely used to separate proteins according to their electrophoretic mobility (a function of length of polypeptide chain or molecular weight) SDS gel electrophoresis of samples having identical charge per unit mass due to binding of SDS results in fractionation by size. This protocol applies to PAGE in Biorad EQUIPMENT CHEMICAL INGREDIENTS AND THEIR ROLES Tris (tris (hydroxy methyl) aminomethane) Innocuous substance to most proteins. Glycine (Amino Acetic Acid) Used as slow ion because its value below that of the slowest known proteins of net negative charge in the ph range. The minimum ph of this range is approximately 8.0. Acrylamide is used to generate polyacrylamides Bisacrylamide (N,N' Methylenebisacrylamide) The most frequently used cross linking agent for polyacrylamide gels. Sodium Dodecyl Sulfate (SDS) SDS is the most common dissociating agent used to denature native proteins to individual polypeptides (giving a near uniform negative charge along the length of the polypeptide) Ammonium persulfate (APS) Initiator for gel formation. TEMED (N, N, N', N' tetramethylethylenediamine) Catalysor. Chemical polymerisation of acrylamide gel is used for SDS PAGE. The rate of polymerisation and the properties of the resulting gel depends on the concentration of APS and TEMED (normally it is used approximately in equimolar concentrations in the range of 1 to 10mM) SOLUTIONS Prepare Stock Solutions: AAbisAA and TEMED are commercial solutions (4 o C). Sample buffer is at room temperature (RT). Prepare fresh: Mix with β mercaptoethanol (950µl SB + 50µl β m) Prepare APS 10% and dispense it in 1.5ml eppendorf. Store at 20 o C (use new tubes each time) 1

2 Buffers (electrophoresis, running and stacking) can be prepared and stored at room temperature. Staining solution: Coomassie Brilliant Blue G 250 (Sigma B 0770). Covered with aluminium foil, RT Destaining solution. RT Electrophoresis buffer ph g Tris 30g Tris 14.4 g glycine Better prepare 10X concentrated 144 g glycine 10 ml SDS 10% 100 ml SDS 10% Set to ph with HCl, Make up to 1 liter of demi water Make up to 1 liter Running buffer 1.5M Tris.HCl ( g/l) ph 8.8 (with HCl) Stacking buffer 0.5M Tris.HCl (60.57 g/l) ph 6.8 (with HCl) Acryl/bisacrylamide (AAbisAA) 40% Bio Rad (fridge) (acryl:bisacryl = 29:1) Ammonium persulfate (APS) 10% 10g/100ml water (freezer, in small aliquouts of 1 ml) TEMED Sample buffer (already prepared) 250 mm Tris (30.28 g/l) ph8 2.5mM EDTA (0.73 g/l) 5% SDS 50% glycerol 0.002% bromophenolblue 25 mm DTT (3.85 g/l) Staining solution 1g Coomasie R 250 (0.1%) 400 ml methanol 100 ml acetic acid 2

3 500 ml demi water Destaining solution 400 ml methanol 100 ml acetic acid 500 ml demi water Prepare Fresh!!!: For small gel (10 ml): STACKING GEL ~2.5ml RUNNING GEL ~10ml Demi water 1.59 ml 4.35 ml Buffer 0.63 ml stacking buffer 2.50 ml running buffer AAbisAA 0.25 ml 3 ml SDS 10% 25 µl 100 µl TEMED 6 µl 20 µl APS 18 µl 50 µl For large gel (28 ml): STACKING GEL ~9ml RUNNING GEL ~28ml Demi water 4.77 ml 12.2 ml Buffer 1.89 ml stacking buffer 7ml running buffer AAbisAA 0.75 ml 8.4 ml SDS 10% 75 µl 280 µl TEMED 18 µl 56 µl APS 54 µl 140 µl 3

4 Pouring the gel To decide which system to use (mini (10 ml volume) or large gels (28 ml volume) ) first is better to start with small gels. If the protein bands are very busy and you cannot see the profile clearly I suggest to try with big volumes, Use gloves from this stage Clean the glass plates with water Place the large plate down first, place the 1 mm spacers and on top the short plate. Loosen the screw of the sandwich clamp and place each clamp to the appropriate side of the gel Place the sandwich on the gray sponge into the casting stand and lock the assembly in place Leakage often occurs. Test first, using 1ml of demiwater. Wait for a minute, and see if it is leaking. If everything is fine, remove the water turning down the assembly over a tissue. If it leaks, re assemble and re test. If the problem continues, consider to pour a small bottom layer using 0.5 (small gell) 1 ml (large gel) running gel (with 2 µl TEMED, 5 µl APS per ml) and wait 30 minutes before continuing. Now you can add the compounds to make your gel. RUNNING GEL Mix the compounds in a Falcon tube (R) in the order given above (note that polymerization will go fast after adding the APS ) and pipette between the glass plates. Carefully add water saturated butanol (±200µl mini, ±1ml large gels) on the running gel. You may now also store the gels overnight before continuing. When the running gel is polymerized (30 min., you can check whether in the bottom of the Falcon tube the remaining liquid has already polymerized), start with the second part: pouring the stacking gel. STACKINGGEL Figure 1 Mix the solutions for the stacking gel in Falcon tube (S) and pipet on the running gel. Put the comb in. 4

5 Wait around minutes (polymerization is finished) Running the gel When you mix the solutions, add APS in the last step (polymerization will start at that moment) so be relatively fast! Fill the electrophoresis tank with electrophoresis buffer ph 8.0 (prepared freshly from 10X electrophoresis buffer) After polymerization, remove the comb from the gel by pulling it straight up, slowly and gently Release the gel from the casting stand With the short plate facing the core, slide the sandwich in place, push the gel onto the core until you hear a click. Place a sandwich to the other side or use a set off glass plates without spacers to complete the upper chamber (do not forget). Place the attached gel assemblies into the buffer chamber When you are running large gels, leakage will often occur. Pour 300 ml buffer into the upper chamber to check if it is leaking. If so, re insert the gels in the sandwich. Check if the system is well connected by supplying power to the gel (let it run for 1 min, to check for possible errors, which at this stage you can still easily fix). Prepare the samples (see protocol for preparation of PAGE samples) o In mini gels normally you should run the gel with around µg of protein per well. Measure your protein concentration first! (see Protocol: Whole cell protein determination according to BCA (Pierce)) o Appropriate volume of sample is mixed with 2 volumes of Sample Buffer (or less if the total volume is more than the well volume) o Marker: 5µl per well for mini gel 10 µl for large gel (see below) o Load the gel by using the special gel saver tips. Mark the gel by not using one or more wells. o Run the gel at 100V 150V until Coomassie blue runs off from the gel o After running, turn off the power supply and take out the core. Remove the sandwich and the clamps and remove one glass plate. o Cut off an edge of the gel in order to remember its position. 5

6 Kaleidoscope (Bio rad) Prestained standards Markers Broad Range Protein Ladder (Fermentas) Page Ruler Unstained Bl Da 250KDa Pink Green Viol Oran Red Bl Staining the gel Put the gel on the glass plate in staining solution (Coomassie Brilliant Blue G 250) Stain for 60 minutes Remove the staining solution in the container (Coomassie Blue waste). Put the destaining solution (in order to remove staining solution in excess) and wash the gel. Remove it into the container and add new distaining solution covering completely the SDS page gel Distain for several hours or overnight Optional: if you are in a hurry, for distaining step you can put demi water instead of distaining solution. Add a handful of glass bead and boil the gel into de microwave for 1 2 minutes (max W). Be careful, don t boil too much! Make a picture and store your image for gel analysis Optionally: seal your gel into plastic, add a few drops of water before closing the seal. 6