Problem with control samples in cell IC
Posted 24 September 2010 - 05:28 AM
I'm trying to stain for Troponin I in a cardiac cell line. I've done this by blocking in 4% normal goat serum, use primary antibody from origene raised in rabbit and used anti-rabbit secondary raised in goat and coupled to a fluorophor. Problem was that I got a lot of staining in my control with cells that certainly do not express my target protein. However, no staining in the negative where I omitted primary antibody. I did some optimization, blocked with 10% instead of 4% NGS and used different concentrations of primary antibody (1:500, 1:1000, 1:5000).
Now, I don't think the secondary antibody is the problem as there is no staining when I omit the primary antibody, but perhaps it has something to do with blocking. Does anyone have have suggestions how to solve this problem? I thought about perhaps changing the composition of the blocking buffer to include BSA or milk.
Thank you in advance!
Posted 24 September 2010 - 07:16 AM
Posted 24 September 2010 - 07:20 AM
Posted 24 September 2010 - 10:22 AM
You know, I thought that might be as well but the western blot definitely says 1:1,000,000 which is why I think the IHC means 1:250,500. But the most important is to find the highest dilution possible that still works in the positive control. This way you are reducing the potential for background staining in the negative control.
Thank you for your reply. I'll certainly look into your remarks in details. About the dilutions recommended by the company, I thought it meant 1:250 OR 500. If indeed it means 1:250,500 I'm way off! I'll check with the company. Thanks again.