I was trying to Immunoprecipitation Drosha protein from mice C57 BJ/6 liver. I lysis 80mg in 1.2ml lysis buffer(from thermo +proteinase inhibitor cocktail). However, after Immunoprecipitation by using Drosha antibody(abcam 12286) and Magnetic beads protein G( invitrogen 10004), I can not see any bands of Drosha in 130~170kd region.
I want to know any body encounter with this situation?
You will have to do some troubleshooting.
What I would recommend is that you should analyze your supernatant by Western blot (WB) i.e. what is left in your tube after incubation with your beads, in order to know whether your IP has worked or not.
If Drosha is not detectable or significantly less present in the supernatant after incubation with beads and antibody than prior to the IP, then you could conclude that your IP has worked which, in turn, would suggest that the problem could come from the elution or any downstream operation.
In contrast, if Drosha's WB signal would we as strong in the samples prior and after IP, it would be indicative of an IP problem (dead antibody, problem with the beads etc...). We always have to include controls in order to be able to analyze and understand our experiments.
Best of luck.
It could better, if you add some sentences more to elaborate the the question. You can increase the time of interaction at room temperature rather than cold temperature. Are, getting WB of Drosha protein from crude protein before immuno- precipitation or not ?
You will have to do some troubleshooting.
What I would recommend is that you should analyze your supernatant by Western blot (WB) i.e. what is left in your tube after incubation with your beads, in order to know whether your IP has worked or not.
If Drosha is not detectable or significantly less present in the supernatant after incubation with beads and antibody than prior to the IP, then you could conclude that your IP has worked which, in turn, would suggest that the problem could come from the elution or any downstream operation.
In contrast, if Drosha's WB signal would we as strong in the samples prior and after IP, it would be indicative of an IP problem (dead antibody, problem with the beads etc...). We always have to include controls in order to be able to analyze and understand our experiments.
Best of luck.