I’m wondering why in IP lanes, some bands are sharp, while some are smearing, even they are in the same lane. Originally I thought that is caused by non-specific binding to beads, but lane of IgG is most clean.
Is it possible that overloading of heavy and light chains of antibody leads to such a consequence? I directly boiled beads in 1X Laemmli buffer at 95 degree for 10 minute, so supposedly antibody would be loaded along with samples. I did not find concentration of the antibody in manufacturer's specification, and therefore I consider it 1ug/ul and mix 4ul of antibody with 1mg cell lysate in order to get abundant proteins which we postulate to associate with IP target.
(Coz I’m investigating protein-protein interaction, mild lysis buffer (pH 8.0) is currently used. They are run in different gels because my target proteins' molecular sizes range from 10kDa - 315 kDa. )
Thank you~
Could the smearing proteins be post-transitionally modified? eg Glycosylated ? if yes treatment with glycosidase such as PNGase or Endo H might help.