Protein gel casting¶
The recipes and procedures on this page were originally posted here.
Required stock solutions¶
40% Acrylamide stock solution: Solution of monomers for gel polymerization.
We find it cheaper to buy premixed 40% stock solution, with a acrylamide:bis-acrylamide ratio of 29:1 (3.3%). Stocks with a 37.5:1 ratio also work, and are typically used for resolving larger proteins.
3x bis-Tris gel buffer: Ion buffer used in gel casting.
Component
Concentration
g/L to final concentration
bis-Tris
1 M
209.242
HCl
Add to pH 6.5-6.8
10% APS: One of the polymerization initiators. Only a small quantity needs to be prepared; each gel only requires 25 uL.
Component
Concentration
g/L to final concentration
Ammonium persulfate
10%
100
Casting protocol¶
Warning
The acrylamide monomers used here are toxic. Read the SDS.
Perform polymerization steps with a lab coat in a fume hood, and collect rinse waste in a waste container.
Prepare 1X resolving and stacking buffers. These buffers can be stored in the refrigerator for several weeks. Recipes given here for enough for 2 0.75mm gels.
Resolving buffer: ~3 mL per gel (6.5 mL total):
Component
Volume
Final concentration
3x bis-Tris gel buffer
2.2 mL
1x
40% Acrylamide stock
1.3 mL
8%
DI water
3 mL
Stacking buffer: ~1.2 mL per gel (2.5 mL total):
Component
Volume
Final concentration
3x bis-Tris gel buffer
0.83 mL
1x
40% Acrylamide stock
0.32 mL
5%
DI water
1.36 mL
Bromophenol blue
50 uL
(enough to give color)
Gel casting setup¶
In-lab, we have the ability to cast two gels simultaneous; this is recommended even if you only need one, so that you have a backup in case of pouring mishaps. Our gel runner also requires two poured gels to properly seal.
Gels can be stored in zip-lock bags in the fridge for at least several days.
Locate two 0.75mm spacer plates and two short glass plates.
Use ethanol and a Kimwipe to clean both glass surfaces.
Assemble them in the green alignment device.
Lock the two gels into the transparent gel pouring device.
Resolving gel¶
Measure 6.5 mL of 1x resolving buffer per gel to pour.
Add 50 uL of 10% APS per gel, mix well.
Add 20 uL TEMED, mixing quickly. Pour both gels to the resolving gel height (3 mL per gel). Lightly tap and tilt the gel to remove any bubbles.
Once done pouring, quickly but carefully fill the remaining height with water, making sure the gel-water interface stays undisturbed.
Wait for the polymerization reaction to finish (noticeable by a change in refractive index).
Drain the water by tilting the gel past 90 degrees, and wicking away with a Kimwipe.
Stacking gel¶
Measure 2.5 mL of 1x stacking buffer to pour.
Add 20 uL of 10% APS, mix well.
Add 10 uL TEMED, mixing quickly. Fill the top of the gels until just before overflowing. Insert the comb into the top, letting it rest on the spacers.
Wait for the stacking gel to polymerize.
Rinse with water to remove unpolymerized acrylamide.
If removing the combs prior to storage, slowly remove the comb, ensuring that wells are not broken.