I did Flag M2 magnetic beads IP from some lysate. Negative control is the mouse magnetic beads. After Myc antibody blot, I always see the band a bit lower than 60KD. Is that background band heavy chain or something else? If it is, why does it appear and why is it so strong?
Hi, what are the species of your antibodies (antibody used for immunoprecipitation and detection) ? Most probably the band you see is because of the heavy chain of the antibody used for immunoprecipitation. Try this- after blotting and subsequent blocking, incubate the membrane directly with the secondary antibody (omitting primary antibody). Develop the blot and if you see the signal, then it is because of the antibody used for IP.
Yes, Bin Liu. The heavy chain of IP antibody is getting separated at 50 kDa. Your secondary antibody is anti mouse. It detects the heavy chain of IP antibody. This can take place even without primary antibody incubation.
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