IP band slightly different size than input - (Jan/07/2008 )
I'v erecently been doing some IPs and noticed that my IP band is slightly larger than my
input. The antibody is great (only one band) so it isn't this. I have noticed this on occasion in the
literature and am wondering if anyone else has noticed this phenomenon and what they have done
about it. Is it due to the protein being in different buffers?
One possible explanation is that your interaction only involves a modified form of the protein (ie: phosphorylated). This could actually be a great piece of information! So, in the input, the most common form in the lysate is the non-modified form but in your IP the modified form is most prevalent. You can test this idea by splitting your IP in half at the end and treating half with a phosphatase. If the band drops back to the same size as the input, your interaction involves a phosphorylated protein. Just be sure that you split your IgG control and treat half of it as well. I did this once and had bands suddenly show up in the IgG.
Thanks! Great advice.
yes rkay is true. i also found the same phenomenon with my IP. it fitted greatly to the concept i was looking for, then you should capitalize on it.
are the salt concentrations in your input and IP samples the same when you run it on SDS-PAGE?. If not, this might infuence the migration of bands.