King lab SDS PAGE using criterion system

updated: Sept 2001 CHP

We pour our own because the precast have the stacker and separator together ( the sharp pH interface that facilitates good stacking has diffused so the bands are not as well stacked and thus not well resolved ). I use the criterion system from Biorad because it does not leak, the large upper reservoir allows a number of separators can be poured at a time and store without drying out and they are consistent. The cassettes are #165-6007 $5.00 each

1. Mix the Lower or Separating gel see recipes (note they will make exactly two lower gels).

• Mix the buffer, water, and acrylamide (in the best of all world degas, since acrylamide polymerase is inhibited by O2).
• Add 10 % ammonium persulfate (APS) [found in the desiccator in the weight room] (made with in the week) and TEMED Swirl.
• Pour 10 mls which is 1 cm below the comb. . - Try not to trap any bubbles.
• Prop flask at an angle, this helps to determine when the gel has polymerized.
• Gentle layer top with water or n-butanol on top. Using a pastur pipet
• Acrylamide Polymerization is inhibited by O2 this layering ensures a flat interface.
• Polymerization should occur with in 30 min.
--This can be determined by harden of the gel in the flask or when a defined line is observed between the water and lower gel.
I pour a number of separators at a time after polymerization I storedwith 1x lower buffer on top. And keep at 4ºC.

2. Pouring the upper / stacking gel.

Pour off water blot the area between the glass plates with filter paper. try not to touch the gel.
• Mix Upper /Stacking gel .
• Mix the water, buffer and acrylamide then add 10% APS and TEMED, swirl.
• Pour the stacking gel and put in the comb.
• Prop flask at an angle to help determine when the gel has polymerized
• Polymerization should occur in 30 min,
-- Look for defined line around the wells.
Riboflavin can be used to polymerizer the stacker still being worked at 10 mg/ml

3. Setting up the Gel

• Takeoff the bottom tape.
• Before taking out the comb you may consider marking the well positions. In the criterion system the well are marked, however if using a different comb, marking is advisable.
• Take out comb by gently pulling straight, to get out any unpolymerized acrylamide wash wells with running buffer, using a syringe with a 38 gauge needle.
• Put the gel in to the criterion box with buffer just below the line.
• Pour buffer in top.

4. Loading gels

• We use a 100 or 50 ul Hamilton syringe, rinse well between samples (6x in the bottom reservoir). 12 wells 40 ul, 18wells 30ul, 26 wells 20 ul
• Put point of the needle to the bottom of the well, slowly load sample, as one is pulling the needle out.

5. Running gels

• Attach leads the SDS coated positively charged protein will run to the negative red lead put the lid only one way
• Settings: SDS gels - 20 mAmps/gel