Dear Naseer, 

Would you like to provide more details about the protocol that you used?

or , the link below show the protocol for western blot , hope it is helpful

http://www.abcam.com/protocols/general-western-blot-protocol

Receptors work as dimers or more. Do you had some reducing agent ?

Do you try some different lysis buffer in order to increase denaturation and disrupt some protein-protein interaction ?

Dear Dr. Roche,

Yes, we use RIPA buffer and Sonicator. But we do not heat our sample as for receptors recommendations are not to heat sample.

Dear Wai Chin,

Thanks alot, we are checking sphingosine receptors expression.

We are following protocol mentioned above.

Best regards,

Hello, Naseer, did you use any other antibodies to check quality of samples? I guess you are working with cardiac tissue, right? Try cnx43 antibody - this is the best and always works.

Dear Marina, Thanks. Yes I tried beta actin and today i run Akt also. Lets see.

Thanks for recommendation. I will try it.