Hi everyone,

I'm working WB with phospho-beta catenin antibody from tissue. I've a problem that I have no bands on my films. I prepared tissues with modified RIPA buffer and protease (PMSF: 1mM, Aprotinin and Leupeptin: 5g/ml) and phosphatase (okadaic acid:100nM and beta-glycerophosphate:10mM) inhibitors. I worked in ice all the steps and then for the protein concentration determination I use Lowry method. I load 70g protein on gel with 1X SDS smple buffer and use BSA insted of milk. I have no bands so I tried use another antibody different from the phospho form. I use bax antibody but the results were the same. I wrote my steps after the blotting. Besides my blots looks very well, I used presatined protein marker and I stained my gels after the electrophoresis. I used Chemikon WB detection kit.

It is difficult to identify where things are going wrong with my handling.
brief steps:

1) 1-1.5 hr blocking with Chemi-Block solution (that is supplied with the det. kit and has no milk) and 1X TBST(1X TBS+Tween-20 0.05%) at room temp.
----after this, wash 3 times with 1X TBS+Tween-20 0.05%.
2) O/N +4oC primary ab(1:1000) prepared in 1X TBS
3) wash 3 times with 5 min duration each with 1X TBST
4) 1 hr Sec ab (1:1000) prepared in 1X TBST+Chemi-Block (1:1).
5) wash 3 times with 5 min duration each, with 1X TBST
6) Chemicon WB det. kit and developing (expose time to film 30sec, 1min, 5 min, 15 min, 2 hours).

I've seen not so dark film, membran view but no bands or nothing.

I need to write this up in my project and i have no time.

Could anyone help? Thanks for answers.

Wilma

Hi,
We will try to help you.
First of all did you try before WB to use ponceau red to see if the transfer went ok?

Hi,

I used CBB instead of ponceau S, saw good bads, so the transfer is good. Thanks.

you also add phosphatase inhibitor in your blocking solutions and antibody solutions .
refer:
Sharma & Carew(2002) Anal Biochem. 307(1) 187-9