tissus proteins extraction - (Mar/05/2002 )
Hi,I would like to extract proteins from mouse tissus(kidney, brain, skeletic muscle.....) for western blot.
What is the best protocol for this extraction?
Does anyone help me?
I study bone tissue but the basics will be similar.
The fresh tissue should be rinsed thoroughly with cold PBS and cut up into small pieces (or alternatively frozen in liquid nitrogen and ground up).
The tissue fragments are extracted in a solution that contains either 4M Guanidine HCl or 6M Urea, plus inhibitors (PMSF, NEM and others). The soluble material is then dialysed against a buffer + inhibitors to remove the Gnd.HCl or Urea and can then be concentrated, lyophilized and run on SDS-PAGE gels.
Some references include:
Proteins LABFAX (1996) NC Price (ed) published by Academic Press. - has chapters on preparation of cell extracts, buffers, salting out etc.
There is also a Protein Protocols CD ROM that is excellent but I don't remember if it has a chapter on protein extraction from tissues.
Linde et al. (1980) Noncollagenous proteins of Dentin J.Biol.Chem 255:5931.
Termine et al. (1981) Mineral and collagen binding proteins of fetal calf bone. J. Biol. Chem. 256:10403.
Please e-mail me at firstname.lastname@example.org if you have any other specific questions.
just an addition to Sean's qualified reply:
Grinding of frozen sample is particularly useful, if you are looking for rather unstable proteins or in cases where you have a lot of proteases within the sample.
If available, a ball mill will do a good job for you.
i've used trizol to extract proteins from bone, but the result of western blotting was poor (molecular weights did not correspond to the stated values/no signal). any suggestions for me? should trizol be used for protein extraction? thanks~