Does anyone have experience to avoid the IgG heavy/Light chains in immunoprepication assay to detect the endogenous targets? Because my target protein MW is close to the heavy chain (about 60 kd). and the strong band of H chain always looks bad especially when the endogenous protein expression is low. Thanks and all the suggestions will welcome.
Approaches to this include:
1. Use an antibody to your target for the blot that was generated in a different species as the antibody used for the precipitation. For instance, precipitate with rabbit anti-X and blot with mouse anti-X (or vice versa).
2. If you know the isotype, you can use a secondary antibody specific to light chain.
3. Biotinylate your antibody and use this for the blot only. Then use HRP-avidin as the secondary reagent. You can also directly link HRP to your antibody.
4. Cross link your antibody to beads for the precipitation.
5. If your antibody was generated to a peptide, you can use the same peptide to elute your protein off the beads while keeping the antibody attached. Then spin down the beads.
#1 is by far the best strategy, but the others could work if you are willing to optimize the conditions.
In addition to what Dr. Shapiro has suggested, you can make use of a secondary antibody that only recognizes primary antibodies in their native state. Use of such secondary antibody eliminates the detection of the denatured primary heavy and/or light chains during Western blotting. Many companies such as Abcam, Thermo, and Rockland sell such antibodies.
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