I have ordered one antibody-osteopontin for my experiment. I tested it for western blot, but it was not working. however, some articles have shown that this antibody from this company can work well in the same samples, so I do not really know why it could not work.
I check the difference of their protocols and mine. only it is that they use 5% nonfat milk solution for blocking and I use 10% nonfat milk solution for blocking.
In addition, I will 12% SDS gel for my work, I do not know if it is ok?
Could anyone do me a favor?

Thank you!

Docqian

First of all are your positive and negative controls working? What are they for this blot? This always the best place to start for Westerns.

If all controls are working, then you must consider that the antibody sent to you by the company is no good. Ask them for postive control ligand or another aliquot of the antibody. Alternatively your protein prep procedure may not be working correctly, do you have control antibody such as B-actin to test your protein preps?

Rb

docqian said:

I have ordered one antibody-osteopontin for my experiment. I tested it for western blot, but it was not working. however, some articles have shown that this antibody from this company can work well in the same samples, so I do not really know why it could not work. I check the difference of their protocols and mine. only it is that they use 5% nonfat milk solution for blocking and I use 10% nonfat milk solution for blocking. In addition, I will 12% SDS gel for my work, I do not know if it is ok? Could anyone do me a favor? Thank you! Docqian

Thank you for your nice reply.
I have used other antibodies(such as beta-actin and Pex14p)to check these proteins, they are working very well. This means that my protein preps are ok.
For this osteopontin antibody, I only can get some unspecific bands when the membrane was exposed for a long time.

Thank you once more