Denaturing protein gel run and staining¶
Solutions required¶
20x MES-SDS running buffer stock solution: Suitable for separating proteins with a molecular weight less than 75 kDa.
It is also generally cheaper to order this as a pre-mixed 20x stock solution. If you need to make it yourself, the recipe is:
Component
Final concentration
g/L to final concentration
MES
1 M
195.2 g
Tris
1 M
121.13 g
EDTA
20 mM
5.845 g
SDS
2%
N/A
200x running buffer reductant: Ensures that the gel remains under reducing conditions when run. Add directly to 1x running buffer before filling the gel tank.
Component
Final concentration
g/L to final concentration
Sodium bisulfite
1 M
104.061 g
200 mM Tris-HCl stock:
Component
Concentration
g/L to final concentration
Tris-HCl
200 mM
31.52 g
NaOH
Add to pH 6.8
10% SDS stock: At low temperatures, the SDS may fall out of solution, but will redissolve when mixed with the other ingredients. Mix well before transferring.
0.1% bromophenol blue
Component
amount/50 mL to final concentration
bromophenol blue
50 mg
DI water
50 mL
Note
The bromophenol blue solution will not be blue. It is more brown at this concentration. It will be blue when you add it into other things.
2x sample buffer: Used to denature and solubilize protein samples. Can be stored
Component
Final concentration
Volume / 10 mL
200 mM Tris-HCl stock
100 mM
5 mL
Glycerol
20%
2 mL
10% SDS stock
2%
2 mL
0.1% bromophenol blue stock
0.01%
1 mL
Coomassie staining dye: When preparing this dye, pour the 10% methanol first, using it to dissolve the R-250. Then, add water. Add the glacial acetic acid last to prevent aggregation.
Component
Final concentration
Coomassie R-250
0.2% (2g/L)
Methanol
10%
Acetic acid
10%
Water
80%
Coomassie destain solution
Component
Final concentration
Acetic acid
10%
Water
90%
Running procedure¶
Dilute enough 20x MES-SDS running buffer to fill the gel tank, adding fresh 200x running buffer reductant if a gel has not been recently run.
Add 2x sample buffer to the protein sample of interest. Add 2-mercaptoethanol to a final concentration of 1%.
Warning
2-mercaptoethanol smells awful; always add it inside a fume hood.
Briefly heat the protein sample to above 80C for one minute, to ensure full protein denaturation.
Place a prepared bis-Tris protein gel in the gel-runner, and fill both chambers with the prepared 1% MES-SDS running buffer. The combs (with the shorter glass side) should be facing towards the center. Fill the center chamber first, and wait for a minute to check for leaks before filling the outer chamber. If there are leaks, reseat the gels in the runner and repeat.
Carefully load samples, including a protein standard.
Run the gels at constant current, about 30 mA per mini-gel. The dye band runs around 3-5 kDa, so it is typically ok to run the dye band to the bottom of the gel unless very small proteins are of interest.
Note
If the gel runner cannot reach 30 mA per mini-gel, double check that you oriented the gel properly! The loading side should be facing inward to ensure a full liquid seal.
Staining (Coomassie)¶
Fill a small dish (the top of a pipette tip box works great!) with DI water.
Carefully separate the gel plates from each other, without ripping the delicate gel. Invert the gel over the dish of water, and gently move the gel into the dish.
Place the gel dish on a rocker for 5 minutes to remove any unbound proteins.
Drain the water, and replace it with a roughly half-inch layer of Coomassie stain.
Either gently shake, while covered, for several hours, or microwave until the solution just reaches boiling (for accelerated diffusion). If microwaving, cover with Kimwipes to prevent spattering!
Drain the Coomassie stain off, replace with DI water, and rock for 5 minutes.
Drain the DI water, and replace with destaining solution.
Cover the gel with Kimwipes, and either shake, while covered, for several hours or microwave as before.
When the destain solution seems substantially blue or purple, replace it with fresh destain solution.