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Western Protocol

Making the Gels

Gather materials: big electric, 1000, 200, and 20 pipettes

Notes: recipe all in mL, the 10% APS, TEMED, & 30% Acryl are in the fridge

1. Use 2 glass pieces (1st drawer on right), 1 Big 1 Little

2. Clamp in green thing, make sure it is straight
3. Put green clamp in clear holder press down hard against sponge
4. Put all ingredients (EXCEPT APS AND TEMED) into 15 mL tube
a. Try to put ingredients for both resolving and stacking @ the same time to
save pipettes
5. Add TEMED and APS into resolving only
6. Swirl
7. Fill glass up until top of green clamp line
a. Place the pipette tip against the back glass, let it drip in
8. Drip in 500 uL of isopropyl alcohol on top
9. 20 mins polymerization
10. pour out alcohol
11. rinse 3x with distilled H2O squirt bottle
12. add TEMED and APS into stacking solution
13. fill all the way to the top
14. insert comb
15. 20 mins polymerization
16. remove comb carefully pull straight out
17. Rinse through with distilled H20
a. Wrap w/ 3 paper towels soaked thoroughly w/ distilled H2O
b. Wrap with cling wrap
c. Label
d. Place in fridge

Running Samples through Gels

Note: Take proteins out to thaw, ladder is in outside freezer (by ice box, green circle on
cap), RIPA buffer & SDS in first fridge

Max Cap for 10 well is 35 uL, for 15 well is 15-20 uL

Recipe for Diluting Ladder

5 uL ladder, 7uL 5X SDS, 23 uL RIPA buffer

Make Running Buffer

Dilute 10X SDS to 1X SDS (100 mL 10x SDS + 900 mL water)