No bands in WB - (Sep/26/2006 )
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: 5µg/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 70µg 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.
1) 1-1.5 hr blocking with Chemi-Block solution (that is supplied with the det. kit and has no milk) and 1X TBS+Tween-20 0.05%) at room temp.
----after this, wash 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 TBS
4) 1 hr Sec ab (1:1000) prepared in 1X TBS
5) wash 3 times with 10 min duration each, with 1X TBS
6) Chemicon WB det. kit and developing (expose time to film approx. 2 hours).
I've seen not so dark film, membran view but no bands.
Could anyone help? Thanks for answers.
I don"t see any mistake in your protocol, or am I already tired?
Does your secondary antibody work and the substrate work? did you or someone else in the lab had good results with these products?
Maybe you could try to dot blot the first antibody, and try to detect it with your secondary antibody and substrate? Just to be sure.
I'm with Missele. dot-blots are key if you're not sure where the problem lives
a few things you might consider as parameters to test with your dot blot (you probably already know this, please bear with me)
1. different Ab concentrations
2. can you do a known dot blot as a positive control?
3. can you check just the reagents as a positive control?