1 reply to this topic

[Barbora]

Hi,
I perform immunoprecipitation on protein G agarose beads and I have a problem with high background (nonspecific binding). Preclearing doesn´t help much. I´d like to try BSA-blocking of beads, could anyone help me to clarify the right sequence of blocking protocol steps? Incubate the beads with primary antibody, then add BSA, wash it and finally add the lysate? Or BSA first and then antibody? Or, if using lysate+antibody step first, may pre-blocking have any effect?
Thanks for sharing your experiences!
Barbora

[laurequillo]

Barbora, on 14 April 2011 - 04:23 AM, said:

Hi,
I perform immunoprecipitation on protein G agarose beads and I have a problem with high background (nonspecific binding). Preclearing doesn´t help much. I´d like to try BSA-blocking of beads, could anyone help me to clarify the right sequence of blocking protocol steps? Incubate the beads with primary antibody, then add BSA, wash it and finally add the lysate? Or BSA first and then antibody? Or, if using lysate+antibody step first, may pre-blocking have any effect?
Thanks for sharing your experiences!
Barbora

First you block the beads with the BSA and the you add the lysate. Normally I dont do that preclearing, what I do is to incubate the cell lysate with beads for 1 hour, then discard the beads and start the Ip with the antibody (2h) and then new beads (1h). Maybe you can mix box: First you incubate your lysate with beads (and at the same time you incubate some beads with BSA), then you discards the beads from the lysate and you add your antibody for 2h, and after that you add your "blocked beads" for another hour.