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Last month I order Anti-Actin antibody - Loading Control (ab1801) from your company.
I ran the western blot assays with this antibody (rabbit, 1:1000) and used anti-rabbit secondary antibody (1:1000), but I get the white bands of actin (light black background)and dark black bands of molecular markersin the X-tay films. At the same time, I use the anti-tubulin (cell signaling) with the same procedures andget the normal balck bands oftubulinin the X-ray films.
Could you tell me what is the problem?
Asked on Aug 17 2012
Thank you for taking time to contact us. I am sorry to hear that this antibody is not providing satisfactory results.
It sounds like you're seeing "inverse banding" which is white bands on a black background. Usually this means that you're using too much primary and/or secondary antibody. I'd recommend diluting the primary to 1:2000 and incubating this overnight at 4C in the blocking buffer (5% BSA) and using the secondary at 1:5000 - 10,000 for 1 hr at RT in blocking buffer. A 1:1000 dilution for a secondary antibody is quite high.
Are you blocking in 5% BSA?
How much protein are you loading and what are your samples (species, cell line, etc.)?
Do you have an image that you can send?
I would strongly recommend that you try diluting the primary and secondary more. Should the suggestions not improve the results, please do let me know.
In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.
I hope this information is helpful, and I thank you for your cooperation.
Answered on Aug 17 2012