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Co-IP
Posted by: EANON (IP Hidden, Junior member, 19)
Date: July 22, 2005 05:40PM
After lysing cells, I usually spin at high speeds to pellet cellular debris and then add antibody and protein G sepharose to the supernatant (for a co-IP). However, today I forgot to do this step and proceeded onwards.
Does anyone know of a method to 'save' my experiment? When running the samples on an SDS-PAGE, does the cellular debris interfere with other proteins? Thanks for responding to this poor poor mortal.
Re: Co-IP
Posted by: mito lab (IP Hidden, Unregistered user, )
Date: July 22, 2005 11:23PM
The insoluble proteins will certainly co-precipitate with your final pull-down complexes. There maybe a way to minimize the contamination: do not spin down your beads after incubation, in stead put the tube on ice for couple minutes to let beads settle down by gravity while insoluble debris is still in the solution. Aspirate and repeat wash two times and then follow normal wash steps with centrifugation. You may lose some beads but it should be ok if you are not doing quantification study. Good luck.
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