I'm trying to optimize Western blots for RBCs, to assess cytosolic proteins. I'm finding surprisingly little information on creating protein lysates, and any special tricks or tips. Any advice will be greatly appreciated.
If you want to get a concentrated lysate, I recommend to add one volume of distilled water, a quarter volume of CCl4, shake the mixture and centrifuge it at 10,000xg. The tetraclorocarbon goes down, the supernatant does no longer contain any ghost, but all cytosolic proteins and can easily be handled for whatever purposes. Of course, you may dilute it according to your demands.
Stephen, interesting to know that hemoglobin (Hb) and carbonic anhydrase (CA), comprise ~98% of total RBC constituents. Do you have a reference to share? Thanks
Dear Leopold: if the supernatant (aqueous phase) will not contain any ghost, does it mean all hemoglobin (Hb)-free RBCs will go down to the organic phase (CCL4)? Will the supernatant aqueous phase also contain Hb and membrane fraction with other cytosolic proteins? We are interested in cytosolic fraction without Hb, and also in membrane fraction separately. Your further clarification and response will be very helpful and highly appreciated Thanks
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