Problems with Immunoprecipitation and Cross-linking

[SciMike]

Crosslinking is always tricky... often the antibodies' binding site is crosslinked when trying to crosslink the antibody to the bead. This can completely alter the specificity of the antibody for its target... and you never know what's going to happen when you crosslink.

There is a pretty new kit from Invitrogen made with dynabeads too..
Dynabeads Antibody Coupling Kit (14311D)... you get covalent antibody coupling to the beads and it's easy to use.. The background is lower than with protein A & G too.

good luck.

[ayalagrande]

Hi, I have crosslinked my antibodies but when I do immunoprecipitations, after elution with 0.1 M glycine pH 2.5 the antigens do not come out.
Any suggestions?

[MIKAH]

what type of crosslinker you have used??? because some crosslinker needs DTT to reverse their crosslinking so that they can be released.

Also go for dot blot of BCA protien analysis to make sure that there is elution or no elution when you treat with glycine