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Protocol for detecting soluble, secreted proteins on a Western?

il-1b western blot caspase-1

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#1 JYaron

JYaron

    PhD Candidate, Biological Design

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Posted 13 May 2013 - 05:55 PM

Does anybody have a good protocol for detecting soluble, secreted proteins on a Western after resolving by SDS-PAGE?

I want to detect interleukin 1-beta produced and processed by macrophages upon stimulation of the NLRP3 inflammasome.

Do I have to run the supernatant through a molecular weight cutoff filter in order to concentrate it? Or can I just add loading dye to the supernatant and run the gel?

Thanks!
Jordan Yaron, PhD Candidate
The Biodesign Institute at Arizona State University

www.jordanyaron.com

Ask me about cell biology, microscopy and cell death or find my writing at TheBioBlog.com!

#2 mdfenko

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Posted 14 May 2013 - 06:23 AM

it depends on the concentration of the target protein in the medium and how similar in molecular weight the target protein is to any of the major proteins in the medium (if cell culture medium then you'll have the proteins from serum) that may interfere with visualizing the target protein.

if necessary then you can concentrate by various means (ultrafiltration, precipitation with ammonium sulfate or acetone or ethanol or polyethylene glycol or tca or ...). the problem with many of these methods in the presence of cell culture medium is that you will concentrate more than just your protein of interest. you can fractionate with some of these methods. or you can selectively remove albumin (and igg) prior to concentration.

the western protocol is simple: separate on page (sds-page in your case), transfer to an appropriate membrane, block the membrane, incubate with antibody to your protein of interest, wash, incubate with antibody to the primary antibody (conjugated with something to allow you to visualize the result), wash, and visualize.

you can download a handbook on western blotting (and other useful handbooks) from this webpage at ge healthcare.

and also this handbook on protein blotting from millipore:

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