Summary: 40
IEF and NEPHGE 2-D PAGE
Basic tube gel recipe, 5 ml volume:
Urea 2.75 g (ultrapure only)
ddH2O 1.65 ml
Acrylamide 0.67 ml 30% stock solution
TX-100 0.25 ml 20% stock solution (can substitute NP-40)
Combine and add ampholines to 2%
NEPHGE: use 250 µl of Ampholines 3.5-10
IEF: use Ampholines as desired:
standard = 50 µl of 3.5-10 + 200 µl of 4-6
alternative = same, but use 100 µl each of 4-6, 5-7.
Makes enough for ten 10.5 cm X 1.5 mm tube gels.
Reserve a small portion to make a 75% overlay solution.
Add 5 µl of TEMED and 3-5 µl of 25% APS, filter (0.45 mm), and pour gels.
Glass tubes must be cleaned overnight with 5% Chem-Solv prior to pouring gels.
Lysis buffer recipe, 1 ml volume:
Urea 0.55 g (ultrapure only)
ddH2O 0.35 ml
TX-100 50 µl 20% stock solution (can substitute NP-40)

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

Collections: Biology and Medicine