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℃ 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 |
38μl |
38μl |
38μl |
|
dH2O |
2.43ml |
1.9ml |
1.3ml |
|
Mix together.Add APS and TEMED just before pouring |
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10% APS |
36μl |
36μl |
36μl |
|
TEMED |
5μl |
5μl |
5μl |
|
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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% |
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|
30:0.8% w/v acryl:bisacryl |
660μl |
1ml |
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1M>Tris-Cl pH6.8 |
630μl |
630μl |
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|
20% SDS |
25μl |
25μl |
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|
dH2O |
3.6 ml |
3.6 ml |
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Mix together.Add APSand TEMED just before pouring. |
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10% APS |
25μl |
25μl |
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TEMED |
5μl |
5μl |
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5ml |
5ml |
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Preparations of sample: Protein samples are in 1X sample loading buffer (also called Laemmli dye)...ie to 6μl protein sample,add 3μl 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℃)
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 dH2O.
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.
