ELISA with undiluted cell lysate samples - (Feb/17/2011 )
Hello all. I'm trying to detect very low levels (pM) of my protein of interest in cell lysate. I estimate the concentration of my protein of interest to be on the picomolar range in 1x undiluted cell lysate. I'm lysing the cells using Pierce's Nuclear Cytoplasmic extraction kit. When I do a standard sandwich ELISA, I get a lot of non-specific signal (A450=~1.0) when I add in negative control 1x cell lysate (does not contain my protein of interest). It's not capture Ab-detection Ab cross-reactivity because I don't get this high non-specific when I omit the neg. control cell lysate. It's not the blocking agent either because I get the same 1.0 A450 regardless of whether I use 1%BSA, 1%casein, 5%nonfat milk or Pierce Starting Block. ELISA experts, have any of you successfully performed ELISA using undiluted cell lysate? Thanks in advance, please let me know if you need some clarifying information!
You get high background with the negative control?