help, :-( Actin completely blank on western blot - (Feb/14/2008 )

I am having some problems with my actin. Everytime I do an actin, it comes out blank and I have tried 3 different actin antibodies. 2 from santacruz and 1 from R&D. None of them work. I'm use mouse proteins and I can detect HIF-1a just fine with little to no background. Then after I strip the membrane the actin does not work. I've used goat anti-actin, rabbit-antiactin, and sheep-anti actin... none of them worked. I even blotted with HIF-1a again after stripping to see if the proteins dissapeared, and they didn't. Has anyone else had problems like this? I've remade all my wash buffers, transfer buffers, and tried different secondaries and nothing works.

If anyone has any ideas what is going on that would be great. In the mean time I'm going to do actin first before HIF-1a and see if that makes a difference.

-rubyeye

That's a good idea to try the actin before stripping, because the sensitivity may be lower than the HIF-1a and some of the protein may be lost during stripping.

Using mouse protein, I had good luck with the B actin antibody from cell signaling technology. It worked after stripping and had very low background. I used it at 1:1000 dilution.

Best thing is not to strip at all

But as you said, trying to blot actin first will tell you if your antobodies are good.

I've had some luck using antibodies from Cell Signaling Technologies and from Rockland immunochemicals.
But definitely try the actin first...that might help.

Thanks for the suggestions. I tried the actin first before stripping on a fresh transfer, but still nothing. The strange thing is that the western blot that is considered more technically challenging which is the HIF-1a blot works every time even after stripping 5 times. where as actin is supposed to be super easy... Now i'm trying 4 blots at once using 4 different actin antibodies, with different secondaries. Also have anyone tried Novus biological's actin antibody? so far I;m having no luck with R&D and santa cruz biotech.

Thanks for the suggestions. I tried the actin first before stripping on a fresh transfer, but still nothing. The strange thing is that the western blot that is considered more technically challenging which is the HIF-1a blot works every time even after stripping 5 times. where as actin is supposed to be super easy... Now i'm trying 4 blots at once using 4 different actin antibodies, with different secondaries. Also have anyone tried Novus biological's actin antibody? so far I;m having no luck with R&D and santa cruz biotech.

I HAVE ACTIN!!!

I now have actin bands showing up. The problem was the washing, blocking, and incubating buffer and not the antibodies. Normally our lab uses TBS-T with 0.1% tween and I cut tween down to 0.05% and increased Tris concentration to 25mM instead of 12.5mM. These changes helped a lot and now actin is showing up nice and bright. I also tried using PBS-T with 0.05% tween and the actin band was even darker and more well defined. Since we only use HRP conjugated secondaries PBS is compatible. However this washing formulation doesn't work for other antibodies as it increases background too much. So we still use TBS-T with 12.5mM tris and 0.1% tween. Looks like we're going to have to modify our washing buffers.

We now use TBS-T 12.5mM tris at ph7.5 with 0.3% tween 20 for other types of antibodies now to reduce background even more.

So it looks like in addition to adjusting antibody concentration, blocking time and formulation, and washing time for each antibody, changing washing buffer formulation might be needed if all else fails.

:sigh: western blots are really a PITA