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