You will need the following reagents:
5x Sample Buffer
|
10% w/v |
SDS |
|
10 mM |
Dithiothreitol, or beta-mercapto-ethanol |
|
20 % v/v |
Glycerol |
|
0.2 M |
Tris-HCl, pH 6.8 |
|
0.05% w/v |
Bromophenolblue |
Should add up to 8M urea for really hydrophobic proteins
1x Running Buffer:
|
25 mM |
Tris-HCl |
|
200 mM |
Glycine |
|
0.1% (w/v) |
SDS |
1x Running Gel Solution
For different applications increase your desired percentage acrylamide, make up thirty ml of running gel by selecting one of the following percentages and mixing the ingredients shown below. After adding TEMED and APS your gel will polymerize fairly quickly, so do not add these until you are sure you are ready to pour.
|
7% |
10% |
12% |
15% | |
|
H2O |
15.3 ml |
12.3 ml |
10.2 ml |
7.2 ml |
|
1.5 M Tris-HCl, pH 8.8 |
7.5 ml |
7.5 ml |
7.5 ml |
7.5 ml |
|
20% (w/v) SDS |
0.15 ml |
0.15 ml |
0.15 ml |
0.15 ml |
|
Acrylamide/Bis-acrylamide |
6.9 ml |
9.9 ml |
12.0 ml |
15.0 ml |
|
10% (w/v) ammonium persulfate (APS) |
0.15 ml |
0.15 ml |
0.15 ml |
0.15 ml |
|
TEMED |
0.02 ml |
0.02 ml |
0.02 ml |
0.02 ml |
Stacking Gel Solution (4% Acrylamide):
|
H2O |
3.075 ml |
|
0.5 M Tris-HCl, pH 6.8 |
1.25 ml |
|
20% (w/v) SDS |
0.025 ml |
|
Acrylamide/Bis-acrylamide |
0.67 ml |
|
10% (w/v) ammonium persulfate (APS) |
0.025 ml |
|
TEMED |
0.005 ml |
Pouring the Gels:
Choose a percentage acrylamide based on the molecular weight range of proteins you wish to separate:
|
% Gel |
M.W. Range |
|
7 |
50 kDa - 500 kDa |
|
10 |
20 kDa - 300 kDa |
|
12 |
10 kDa - 200 kDa |
|
15 |
3 kDa - 100 kDa |
Gradient gels are somewhat more difficult to pour and it may be worthwhile to spend the money on precast gels if the need for a gradient gel arises. This may be the case if you want to resolve a very broad range of molecular weights or for some reason the proteins you are interested in do not separate well using a straight percentage of acrylamide.
Now mix the ingredients needed for the chosen percentage and pour the solution quickly into your gel casting form - be sure to leave a some room for the stacking gel - I usually leave about 2 centimeters below the bottom of the comb for the stacking gel. You can do this by inserting the comb into the dry form, and marking a region below the comb for the height of the stacker you want. Look for bubbles and remove them, then layer the top of the gel with water saturated butanol or, very carefully, with water. This will remove bubbles at the top of the gel and will ensure this part does not dry out. Wait for about 30 minutes for the gel to polymerize completely. (If you always use fresh ammonium persulfate, you're gel may polymerize more quickly and reliably.)
While waiting mix the reagents for the stacking gel, but LEAVE OUT the APS and TEMED until you are ready to pour this gel; stacking gels will polymerize more quickly than desired sometimes while one is trying to add combs to make wells.
When the running gel is polymerized wash out the butanol completely or your stacker may separate from the gel and you will get ugly running artifacts. Mix in the polymerizing reagents and pour the stacking gel on top of the running gel. Insert your combs trying not to get bubbles stuck underneath and allow another 30 min - 1 hour for complete polymerization. Your gels are ready!
Preparing your Sample:
Mix your protein 4:1 with the sample buffer. Heat your sample by either:
a) Boiling for 5-10 minutes (Works for most proteins)
b) 65 degrees C for 10 minutes (If you have smearing using the above procedure)
c) 37 degrees for 30 minutes (Membrane proteins or others that do not enter the gel otherwise may benefit from this type of sample preparation)
Running your gel:
Clamp in your gel and fill both buffer chambers with gel running buffer according to the instructions for your specific apparatus. Pipet your sample into the gel adjusting the volume according to the amount of protein in your sample. If you are going to stain using Coomassie, don't use much more than 5ug of your protein of interest to get a nicely defined band. Be sure to include a lane with molecular weight standards. Now attach your power leads and run the gel until the blue dye front reaches the bottom. I prefer to run at 250 V constant which in a four to twenty percent mini gel needs about 30 minutes total run time, but adjust to the thickness of your gel, the power supply used and the resolution desired. Remove the gel for the power supply and process further - Visualize your proteins using Coomassie Brilliant Blue, Silver stain, or any of the other protein stains. Use a carbohydrate stain for glycoproteins, or blot your gel for N-terminal sequencing or Western blotting.
Reference:
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.