I am trying to isolate glycophorin a (GYPA) from erythrocyte performing a crosslinking between GYPA and antibody at cell surface (crosslinking agent: DSP). After crosslinking reaction cells were lysed (Tris buffer containing 1% NP-40) and the lysate was added to a Pierce protein A/G agarose (overnight) for immunoprecipitation. After washing agarose beads, I resuspended the pellet into Laemmli buffer, boiled for 5 min at 95 ºC and applied it to the gel, following blotting. As a control I applied just protein a/g agarose resin into the gel, and the blot shows many bands related to it. Could someone help me?
Can you elaborate on your cross-linking protocol?
After the antibody is added and incubated for a time, do you add crosslink at that point?
Also, do you preclear your lysate to remove non-specific binding?
Magnetic beads are preferable as generally pre-clearing isn't needed and gives a cleaner prep.
Thank you Stanley for your answer!
In this condition I incubated antibody with cells for 2 hours at 4 ºC. After that I washed with PBS and resuspended the pellet in 0,5 mL of crosslinker solution (1 mM DSP in crosslinking buffer (PBS, MgCl2, NaN3 pH 8,0)) and incubated at room temperature with mixing for 1 hour. Thereafter pellet was washed with quenching buffer and resuspended with 0,1 mL of Pierce protein A/G agarose suspension following incubation at 4 ºC with mixing overnight. After incubation I washed the pellet according to data sheet instructions and resuspended it into Laemmli buffer.