Smearing after immunoprecipitation - Smearing after immunoprecipitation (Feb/17/2005 )

To anyone who can help... We have been using homogenized mouse tissues and have immunoprecipitated pRb (the retinoblastoma protein) with a monoclonal antibody (linked to agarose), then immunoblotted with a polyclonal to E2F1. Although we get a band at the molecular weight of E2F1, the bands are heavily 'smeared'... both above and below the center of the band.

Has anyone experienced this?... and, can anyone give advise to what this phenomenon might be?

Thank you very much in advance...

You only get one band and it is smeared? If so this is probably too much protein loaded?

If there are several bands I would think it is interference from the antibody you IPed with - the heavy and light chains? If that is the case (and your protein of interest runs either higher than 55 kD or lower than 25 kD, merely cut away the heavy and light chain interfering regions on your membrane after transferring and your bands will "pop" and be much darker and clearer than before.