SDS-PAGE: gel electrophoresis of proteins
TECHNIQUE is to set up gel plates before you mix the gel mixes. Use the THIN spacers and choose a comb--number of wells varies. Make both resolving gel and stack (NO APS or TEMED)--then add APS and TEMED to resolving gel, mix and pour about 3-3.5ml per gel. Before it polymerises, now add APS and TEMED to your stack mix and pour it gently on top of the resolv. gel (using a pasteur pipette works well). Put in the comb and let solidify. Should take 15-20 minutes. You can keep gels overnight at 4°C if well wrapped to prevent drying out and if you keep the comb in. Note: just before you want to load gels, wash out wells with distilled water to remove unpolymerised acrylamide.
GEL RECEPIES (enough for 2 thin mini-gels)
RESOLVING GEL (NOTE pH 8.8)
|
Percentage of gel |
8% |
10% |
12.5% |
|
30: 0.8% w/v acrylamide:bisacrylamide |
2ml |
2.5ml |
3.1ml |
|
1.0M Tris-Cl pH 8.8 |
3ml |
3ml |
3ml |
|
20% SDS |
38ul |
38ul |
38ul |
|
dH2O |
2.43ml |
1.9ml |
1.3ml |
|
Mix together. Add APS and TEMED just before pouring |
|
|
|
|
10% APS |
36ul |
36ul |
36ul |
|
TEMED |
5ul |
5ul |
5ul |
|
|
7.5ml |
7.5ml |
7.5ml |
STACKING GEL (NOTE pH 6.8)--use 4% stack for <10% res. gel and 6% stack (easier to handle) for >10% resolving gels.
|
Percentage of stack |
4% |
6% |
|
|
30:0.8% w/v acryl:bisacryl |
660 ul |
1ml |
|
|
1M Tris-Cl pH6.8 |
630 ul |
630 ul |
|
|
20% SDS |
25 ul |
25 ul |
|
|
dH2O |
3.6 ml |
3.6 ml |
|
|
Mix together. Add APSand TEMED just before pouring. |
|
|
|
|
10% APS |
25ul |
25ul |
|
|
TEMED |
5ul |
5ul |
|
|
|
5ml |
5ml |
|
Preparations of sample: Protein samples are in 1X sample loading buffer (also called Laemmli dye)...ie to 6 ul protein sample, add 3 ul 3X Laemmli dye stock. Boil 3 minutes before loading gel.
Sample loading buffer (Laemmli loading dye) 3X stock:
1M Tris-Cl pH 6.8 2.4 ml
20% SDS 3 ml
Glycerol (100%) 3 ml
B-mercaptoethanol 1.6 ml
Bromophenol blue 0.006g
10 ml (store 4°C)
10X Running buffer (also called Laemmli buffer):
Tris base 30.3 g
Glycine 144 g
SDS 10 g
make to 1L with dH2O
For BioRad apparatus: need 500 ml of 1X buffer so dilute 50 ml 10X stock + 450 ml dH20.
Running gels: Using the BioRad apparatus, run gels at 200V (constant voltage) until the bromophenol blue dye is just off. Takes 50-60 minutes. Gels are run at room temperature.
