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Non-specific band present after immunoprecipitation

Non-specific band present after immunoprecipitation - Protocols and Methods Forum

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Old 11-17-2009, 02:56 PM
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Unhappy Non-specific band present after immunoprecipitation



Hi, I want to raise 2 questions and hope someone can help me to solve it.

(1) Everytime I do IP, no matter using protein a agarose (Calbiochem) or protein g sepharose fast flow (GE), I also found out that there are consistently a band about 55 kDa present on the blot.

I have excluded the possibility of IgG heavy and light chain contamination since I always use different species of antibody for IP and Western blot.

I encounter the same problem in different cell lines, and using different lysis buffers also can't solve the problem.

this problem only appears in the IP sample instead of the total sample.

(2) Also, I want to know after boiling the IP samples in sample buffer, can I just freeze the beads together with the samples in -20c without separating them?

Many thanks,

Tom
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