Hello,
I am trying to conduct CO-IP experiment. I have IP: IgG , IP: Protein-A, and input. I am performing western for Protein-B. My results seem to get a band at the expected size in input , IP: Protein-A lane. However I also get this specific band in IgG, which ideally should not occur. The bands are quite strong, so I know these are not due to over exposing or just some background.
General protocol I used:
RIPA lysis for 30min
DNAase treatment 15min
Spinning and collecting the supernatant
Overnight incubation with Primary antibodies with the lysate
(I tried pre-clearing but it did not help)
1hr capture with Dynabeads G (40ul)
Washing*3 times
Western blot for specific antibody
Incubation of the PVDF membrane in 5% milk tbs-t
I would appreciate suggestions from the community!
Thank you!
Following elution, the primary antibody is also eluted (unless it is crosslinked to Protein A/G), which may arise two strong bands around 25 kDa and 50 kDa of the light chain and heavy chain, respectively.
Thank you for your reply!
I am observing a band specifically at 153kd where my protein of interest is suppose to appear, along with these bands for light and heavy chain.
So any idea why this might be?
If the elution is not performed in complete denatured condition, the native IgG band should appear approximately at 150 kDa (2 heavy chains + 2 light chains combined).
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