I'm doing an immunoprecipitation for a epitope-tagged protein expressed in mammalian cells (DH82- a dog cell line). I re-suspend my frozen cell pellet in lysis buffer (Tris-based with 100mM NaCl, 1mM EDTA, 1% Tx-100, 0.05% NP-40) and add a protease inhibitor cocktail and DNase. After lysis there is a lot of white clumpy precipitation floating around which I know is all the genomic DNA, but after I centrifuge my sample and the DNA/cell debris pellet, there is a cloudy white precipitate that floats at the top of my tube.
I couldn't get a good picture of it, but was wondering if anyone knows what causes this and if it may interfere with the rest of my IP. I've experienced it before but never had this much (the cell pellet I used this time was larger than what I've used in the past). It's impossible to avoid pipetting and doesn't pellet when I "clarify" my lysate by centrifugation at max speed (~20000G on tabletop centrifuge) for an hour.
Thanks.
I used to see this with CHO cell extracts. I think it was lipid, since it floated. I used to perform sucrose gradient ultracentrifugation (100,000 g for 18 hours) of the cell extract, then carefully remove the top white layer by suction using a very narrow transfer pipet with the tip placed just at the top surface of the liquid.
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