Posted 06 August 2009 - 07:46 PM
Posted 07 August 2009 - 08:06 AM
You might also try changing incubation times as well. Typically, we bind the ab/protein for 30 min to an hour at 4 degrees before adding the resin. We continue binding for 2 - 4 hours at 4 degrees before washing and loading on the gel. I have bound overnight and for only an hour depending on the antibody and/or protein. I think you will just have to play with it a bit to see if you can get signal with minimum amounts of HC "background".
When you say you have tried high and low stringency, what are you referring to, the binding buffer or the wash buffer? You should try different stringencies for both the binding buffer and the wash buffer. If they are one and the same then try changing one or the other.
I haven't used it, but a neighboring lab use to use True Blot for the ECL Westerns. It is supposed to eliminate the HC and LC bands that sometimes interfere. I thought Amershan used to sell it, but I found it at the posted link. True Blot
Happy developing! http://www.protocol-...icons/icon1.gif
Posted 07 August 2009 - 01:28 PM
Is the protein expressed in the cell type you are using?
Have you used a cell line that was previously used successfully by other groups?
Posted 09 September 2009 - 05:34 AM
Are you using Dynabeads for the IP? Have you tried both the direct and indirect method for IP?
When using Dynabeads Protein A or Protein G, one typically does not need to incubate for more than 10 min at room temperature, both for Ig binding and for beads-Ig-target coupling.
Do you have a positive control on your western blot? Perhaps the IP is working but the detection isn't?
Posted 09 September 2009 - 05:30 PM
Posted 22 September 2009 - 11:05 AM
If the Ip antibody is rabbit you should use a different species for the Western?
Do you have proper controls? Have you done the IP using a cell line that was previously published?