SDS-PAGE gels (Conlon lab)

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Protocol submitted by VGP from Conlon lab protocols [1]

link to protocol document [2]


CASTING A SDS-PAGE (DENATURING) GEL


IMPORTANT Clean glass spacers and plates prior to and IMMEDIATELY after use! Failure to do so will result in uneven gel preparation, cracking of glass plates, and leaking of gel before polymerization.

1. Clean sufficient amount of glass spacers and plates for your experiment (If you are running two gels, clean two spacers and two plates). Clean first with 95% ethanol and wipe clean with kimwipe, then clean with distilled water, wipe clean. Allow to air-dry sufficiently on paper towel until ready to cast gel.

2. Prepare stacking and resolving gel solutions, minus the APS and TEMED, which polymerize the gel. For protein gel applications, we use Bio-Rad bisacrylamide #161-0148, pore size 37.5:1. BE SURE TO CHECK % of stock solution before using. The % resolving gel you use should be determined by the sizes of proteins you are attempting to resolve. 7.5% is better for viewing larger proteins, 120kDa and up. 15% is better for viewing smaller proteins ( i.e. <50 kDa). The stacking gel is fixed at 5% acrylamide. This allows all the proteins in your sample to run evenly into the resolving gel.

CAUTION: Bisacrylamide in liquid form is really bad for you. Use extreme caution to make sure you don’t spill it, especially on yourself. Once polymerized to solid state, it is no longer a danger.


Recipe for gels (adjust water and acrylamide for 40% stock):

Resolving Gels (15 ml)

7.50% 10% 12% 15%
1.5M Tris pH 8.8 5 ml 5 ml 5 ml 5 ml
30% acrylamide 3.75 ml 4 ml 6 ml 6 ml
water 6.15 ml 5.9 ml 3.9 ml 2.4 ml
20% SDS 75 ul 75 ul 75 ul 75 ul
10% APS 75 ul 75 ul 75 ul 75 ul
TEMED 25 ul 25 ul 25 ul 25 ul


Stacking Gels (5 ml)

0.5M Tris pH 6.8 .62 ml
30% acrylamide .833 ml
0.5M Tris pH 6.8 .62 ml
water 3.817 ml
20% SDS 25 ul
10% APS 50 ul
TEMED 5 ul


These recipes can make up to 4 gels – making this much for 2-3 gels is not a bad idea. This way you ensure you have enough solution in case you have leakage issues.

3. Assemble gel spacers and holders in mini-gel casting apparatus.

4. Add 10% APS and TEMED to Resolving Gel Solution and mix well. Using P1000, add 1 ml solution at a time in between assembled spacer and plate. Leave about ¾ inch space from top of the plate (or just fill to top green horizontal bar on gel holder).

5. Immediately add 100% EtOH to overlay the resolving gel. This will remove any bubbles in the gel. EtOH does not mix with the acrylamide, so as the acrylamide polymerizes, the EtOH will remain atop the gel. Allow to polymerize at least 10 minutes.

6. Rinse off EtOH will copius amounts of distilled water. Ensure there is no water left on the resolving gel.

7. Add 10% APS and TEMED to Stacking Gel Solution and mix well. Using P1000, add 1 ml solution at a time until gel is completely full. AVOID BUBBLES! Add appropriate gel combs to stacking gel immediately. Again, wait at least 10 minutes for gel to polymerize.

8. Assemble gel in electrophoresis chamber. Fill inner chamber with running buffer. Remove gel combs, and fill inner chamber again. There should be no need to clear out wells, there should be no acrylamide or bubbles in them. Load gel and run. Recommended: Run 70V for about 20-30 min until sample has run through the stacking gel. At that point, voltage can be increased to 150V to 200V if needed. Better resolution will occur at lower voltages!

9. Once gel has finished running, gently pop glass plate off the spacer using the PLASTIC green spatula. Gently remove gel and transfer or Coomassie stain as normal.

10. Make sure to clean glass plates well after use! A common practice is to soak them in HOT water while tending to immediate needs, such as transfer or staining. Do not leave them for long after you have run the gel, as residual acrylamide on plates will affect casting of subsequent gels. Be sure to clean off with hot water and then DI water to remove any salts. Place in drying rack.


Notes:

10% APS (Ammonium persulfate) should be made fresh and stored at 40C for up to ~2 weeks. Aliquots can be stored at -200C indefinitely.

TEMED smells bad (or so Erin says). Open and close quickly. Close tightly.

Gels can be cast and left overnight at 4 degrees. To do so, wrap tightly with wet paper towels and again with plastic wrap to keep gel from drying. Keep in mind, this should be done minimally as the number of plates are limited.