TAU Gels

Useful for the identification of states of modification to proteins such as acetylation and phosphorylation.

Best if run on large gels (does not work on our mini-gels). A 17cm long gel that was poured with a 1mm spacer worked fine.

Solutions:

Gel:
12% Acrylamide (20 ml 30% Acrylamide)
0.2% Bis-acrylamide (2 ml 2% Bis)
8 M Urea (24 g)
0.37 % Triton X-100 (200 ul Triton X-100)
5% Acetic Acid (2.6 ml Glacial AcOH)
APS (1000 ul of 10% APS)
Temed (100 ul)
H2O to 25 mls.

2X Loading Buffer:
8M Urea
10% glycerol
2.5% Acetic Acid

Running Buffer:
0.25 % Methyl Green
5% Acetic Acid

--After polymerization pre-run gel at 150 V for ~ 6 hours until amperage is steady.

--If using anything other than purified histones clean out wells with a syringe and place 50 ul preload buffer (1X Loading Buffer) in each well and run for an additional hour.

--Change running buffer before loading samples.

-- Boil samples in Loading buffer and when cool load on gel. Electrophorese at 90V overnight (18 hours) until blue component of dye is at the bottom of the gel. Make sure to reverse electrodes (runs to black).

-- Stain gel with Commassie Brilliant Blue and Destain as usual.

-- Infuse with a fluor if necessary.

(Make certain to reverse electrodes!)

Notes on drying!!!!

These gels are tough to dry without cracking especially after staining and fluor.

To help after staining wash away all acid in 5% glycerol/water. Then Infuse with Amersham amplify for 20-30minute.

Dry overnight with vacuum dryer on 3MM paper (no other paper works consistently even S&S) since it sticks hard to this paper.

Do not use any heat on drying or the gel will likely crack.

Exposure times are enhanced by preflashing the film.

Exposure time on Hyperfilm MP (preflashed as recommended by Kodak) for 50,000 cpm per Lane is about 2-4 weeks (sorry).