Immunoprecipitation

Zhonglin Chai Zhonglin.Chai at baker.edu.au
Tue Jul 24 18:45:14 EST 2001


One possible reason is the solubility of the protein as you mentioned in your message. But it is not clear how possibly you would be able to know it is a "soluble" nuclear factor by immunocytochemistry labelling. To determine whether your protein is present in the soluble fraction of the cell lysate, you may just do a Western using anti-myc antibody to detect your protein in the supernatant you will use for IP. 

Secondly you did not tell us how you detected the precipitated proteins. Did you label the proteins with 35S, or what? Not all the proteins are labelled equally well. To determine whether your protein is precipitated you may just load your precipitated proteins to gel and do another Western. You'd better have a negative control using isotype control antibody in your IP that should give you a negative result by Western.

To reduce the IP background, you may need to pre-clean your lysate using control antibody and beads, or use less antibody, or dilute your lysate, or incubate for a shorter time, etc.

Hope this of help.

Zhonglin Chai

Zhonglin Chai, PhD

Molecular Hypertension Laboratory
Baker Medical Research Institute
P.O. Box 6492
Melbourne 8008
Victoria, Australia
Telephone: +61 3, 9522 4357
mobile : 0413 58 1940, or international +61, 413 58 1940
Fax: +61 3, 9521 1362 
email: zhonglin.chai at baker.edu.au


>>> Natalie Sampson <nsampson at hgmp.mrc.ac.uk> 07/24/01 09:01PM >>>
I am trying (without success) to pull down a transiently expressed c-myc epitope tagged protein.  From immunocytochemistry labelling, my protein .  From the precipitation experiments I have done so far, I am getting several background (unwanted proteins) and not mine.  It is definitely present in the transfected cells (western blot analysis on total cell lysates).  Does anyone have any suggestions.  I have tried numerous washing buffers (RIPA, high salt etc) and am fast running o
ut of ideas.  Can anyone  help?

Thank you 

Natalie


nsampson at hgmp.mrc.ac.uk 

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