In my Immuno-precipitation titration with the increase in the antibody, the intensity of main band in decreasing while of another band (has higher molecular weight than the main band) is increasing, not sure if it is the band of same protein. I started with the equal amount of protein (cell lysate), the equal volume of Agarose bead was added to it. Other steps were as per standard IP protocol. Could anybody can explain the reason for this?
OK, when we do immunoprecipitation (IP), the antibody should be present in excess relative to the antigen in the protein extract (and that's why you do an antibody titration because the antibody concentration is unknown). But you also want the Agarose beads to be in excess of the antibody, otherwise you loose sample. So if you increase antibody concentration you may reach a point when there are more antibodies than beads, and this will cause the signal to go down. The excess antibody simply saturates the beads and competes out antibody-antigen complexes. You should never exceed the binding capacity of the beads when you increase your antibody concentration.
But you didn't tell me how you detect the protein after IP. If you have in vivo labelling with 35S methionine and you detect the precipitated proteins by autoradiography, I cannot explain why one band should increase in intensity whilst another goes down. However, if you detect proteins after IP with western blots, then the increasing band could simply be your primary antibody that you have added in increasing amounts. Do a negative control with just beads and antibody (no protein extract) and if you see the same band increasing, that's your primary antibody then.
Thanks, Mr. Deneck for your answer. I get your point, and will probably help to resolve the problem.
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