How to thoroughly solubilize proteins from tissue sample, for quantitation - (Jul/18/2012 )
I'm seeking advice for how to treat tissue samples to ensure that most of the proteins are solubilized, to improve the consistency of my protein quantitation results.
I am using the Bradford assay to quantitate my protein samples, which I use to normalize lipid data. I am seeing large variability in results, and by comparing the mass of the tissue with the protein concentrations, I think my problem is that I am not solubilizing all of the protein in my sample. I suspect the problems could be inconsistent homogenization, a non-optimal centrifugation step, or using an insufficient buffer.
I am working with planarian (flatworm) tissue. Based on C. elegans papers, I add 2 large worms (about 50 mg of tissue) and 1 mL PBS into a centrifuge tube, and use a pestle to dissociate the tissue. Then I pellet the tissue at 16000g for 5 minutes - this produces a sizable pellet (it looks like this contains the body pigments, the tough pharyngeal tissue, and cell debris). I reserve the supernatant and use it as-is for the Bradford assay.
I'm afraid that a lot of the protein may be going into the pellet! Can anyone advise how I can optimize this protocol to ensure thorough protein solubilization? E.g. add sonication, or spin at lower g's, or use lysis buffer? I have seen these referenced in various papers but would appreciate input whether you think it will help with processing planarian tissue (which is overall tougher than C elegans tissue, but relatively soft).
Thanks for your time!
Three major things - 1) add some detergents (SDS, tween 20, triton X-100, deoxycholate) to the PBS - most proteins won't easily dissolve without detergents, and the cell membranes possibly won't rupture enough to release the intracellular proteins. 2) Add protease and potentially phosphatase inhibitors to the lysis step to prevent protein degradation. 3) Bradford assy has a very limited sensitivity and small linear range where it is accurate for concentrations. You will be better off using the BCA assay if you can.