lysis buffer for generating lysate from ovarian and brain tumors? - (Oct/28/2013 )
Hi, I am new to basic science research. In my lab, there are a couple of people who are like me (little to no previous benchwork background). They have tried to produce lysates from frozen tissue with a gentleMACS dissociater (Miltenyi biotec) but have been unable to get a successful Western blot for any proteins, even for something like beta actin. We are all able to get great Western blots from cell cultures of established ovarian and brain tumor cell lines, however. They currently are not in the lab, and my mentor wants me to take over some of their work.
I was wondering what you thought the problem might be. My mentor suggested that we might not be using the right lysis buffer. We are using the Cell Signaling 10x cell lysis buffer, which seems to have Triton as the detergent. Can anyone help us out?
There are a lot of different detergents out there. I think you should search highwire.stanford.edu with you key words, "your cell line, cell lysis buffer, homogenization" or something along those lines. Find a good publication that has beautiful westerns and mimic their methods.