SDS-PAGE--聚丙烯凝胶电泳
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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)
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.
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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 |
| dH2 O | 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 | |
| dH2 O | 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.
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