Western Blot Incubations - what solution is best for overnight incubations? (May/21/2007 )

Greetings, ALL!

I am trying to figure out what is the best way to do overnight incubation of primary antibody in my western blots.
Here is where I'm at:
First I tried using 5% milk / TBST, but it came out more faint than my one hour incubation . ( could it be the tween that is making it give less signal by stripping proteins off the PVDF?)
Then I tried TBS , but I only got the dreaded blank blot...
Now, I'm thinking of trying PBS instead of TBS.

Does anyone have much success with overnight incubations that they will share?

thanks in advance!

monkeybaji

-monkeybaji-

QUOTE (monkeybaji @ May 21 2007, 03:49 PM)

Tween-20 is indispensable in Western blot, including blocking, in my opinion, the problem may attribute to ur primary Ab or sample, and has none business with your blocking condition. by the way, anyway overnight is a better way for blocking.

-sciencect-

QUOTE (monkeybaji @ May 22 2007, 12:49 AM)

we have good experience with TTBS (tween-20 containing TBS) O.N.; increase the conc of primary Ab; to save Ab, use sealed bags; check the pH of TBS carefully, freshly prepared TBS changes pH O.N. and must be re-titrated; correct pH is very important for Ab-antigen binding!

-The Bearer-

I use 5% milk solution supplemented with 1X rotiblock(i think its tween) from carl-roth for 1 hr for blocking(BLOCK I) and subsequent 1-2hr with Pr-AB, then washed in PBS(ph 7.0) thrice for 1omins each. followed by incubation with 5% milk solution(BLOCK II) with Sec-AB for 1hr at room temperature wash in PBS(ph 7.0) thrice for 1omins each and develop.

for overnight i use the above block I together with Pr-AB and incubate at 4'C overnight with constant shaking.

results in nice clean blots

-rajgene-