Several questions about Western blotting of tissues - (Jan/31/2006 )
Dear colleagues,
I am going to analyze the expression of a protein in tumor tissue using Western blotting. My tissues will be fresh from mice. I have never worked on protein extraction from tissue, here are some questions I would like to ask. Please share your expertise.
1) What is the best ways of preserving tissue: liquid nitrogen, -70C, or processed immediately?
2) What is the best method of homogenizing the tissue? What cautions should be taken?
3) How is the result from tissue compared to that from cell lines?
Thank you in advance!
Kawaka
I am going to analyze the expression of a protein in tumor tissue using Western blotting. My tissues will be fresh from mice. I have never worked on protein extraction from tissue, here are some questions I would like to ask. Please share your expertise.
1) What is the best ways of preserving tissue: liquid nitrogen, -70C, or processed immediately?
2) What is the best method of homogenizing the tissue? What cautions should be taken?
3) How is the result from tissue compared to that from cell lines?
Thank you in advance!
Kawaka
1. any method should work different labs use different things I have done all 3 and unless you want to get enzymatic activity you can frezz the tissue.
2. either sonication (better for cells but still works for tissue) or homogenazing (high speed but not too high you will need to figure it out) with or with out detergent (is you protein on the membrane or not)
3. I don't know sometimes the same and sometimes not
good luck
Hi Kawaka,
I only have tried to extract protein from fish tissues, hope it also help.
1. In my lab, the tissues were snapped into liquid N2 and stored in -80 prior to protein extraction. And the tissues should be processed as easier as possible.
2. We use homogenizer and put the sample on ice during homogenization.
3. In my experience, protein from cell line gave lower background than that from tissues.
Hope it can help.
Pat