problematic beta actin... - (Oct/17/2005 )

hiya.

wonder who can help me in this.

I am having problems detecting my beta actin.

and nothing is wrong with the primary and secondary antibodies.

would really appreciate it if anyone can point me in the right direction.

cheers.

What application are you using?
What dilution of ab are you using?
Are your abs tested for your application?
Is your beta actin being used as a reporter protein and if so are you getting your protein of interest coming up?
How much total protein are you using?
Are you running the protein in a gel?
If so what percentage gel do you use?

More people should be able to help if you answer these questions!

Rosie

Hi
Yes a few more details would help like why an antibody assay. You could use PCR and assuming you are performing ELISA what are these antibodies?
Regs
SParky

I am assuming that you are talking about a western blot.

I use B-actin from Sigma (5 ul in 14 ml 5% non-fat milk in 1X TTBS) and the secondary is from santa cruz.

First... How much protein are u loading on the gel??
Secondly, is ur processor working fine..I had a very similar problme yesterday and I realized that it was the processor to blame..

Another rough troubleshooting tip is:(u might think I am crazy but it works for me)
If you have saved ur PVDF at 4C just check if u see a band on the PVDF (a slight greyish band) If yes then the B-actin is present. Ur antibody conc may be too high and you might have to reduce the primary and secondary.

Good luck

dear pria, what do you mean when you said that your processor is not working?

i loaded 50microgram of prot in 10%SDS. Actually i have not had problems before, it's only lately i am getting this beta actin on an MIA mission.

i used my anti mouse from pierce at a 1:100k dillution. I have tried 1 in 30k as well, but not working.

the huge dilution is to minimise on back ground, by the way. and it has always worked before.