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immunoprecipation - (Jan/24/2010 )

Hi all!
I am 1st year phD student and new at this forum.
I will have to do some Immuprecipitation to see PFGFRA phosphorylation.
I ordered some Protein A immobilized on SepharoseŽ CL-4B (Product Code P 3391) from Sigma, it's lyophilized powder.
Now I don't know how I should use that powder, they say we should use buffer A, but they say the amounts???
When they talk about 50%slurry...what does it mean?



since the protein A sepharose is lyophilized you have to swell the gel. this is normally done in water but can be done in buffer.

after swelling, you would "define" (ie- remove small, broken particles) the gel by stirring, settling and decanting with water or buffer.

a 50% slurry is half of the total volume is swollen gel (after settling). then you stir it (with a rod, not a magnetic stir bar) until of uniform consistancy (which won't last long).

you can get more information from the (still?) free manuals provided by ge healthcare (they may be available by download from some of the threads here in bioforum). i recommend "gel filtration" and "affinity chromatography" for your purposes.