Extraction of membrane proteins and western on them - (Oct/28/2004 )
I'm working on a membrane protein (a receptor actually), I've transfected it into mammalian cells and I'm going to do some western Blots to assess its overexpression.
I'm looking for a protocol to extract specifically my membrane proteins. I've already done some litterature searches and I found many different protocols, but I wouldn't be able to perform many of them because I don't have all the apparatus in the lab.
Would you have an easy protocol to advice me to try ?
I also saw there were some kits available, what do you think about them ?
Thanks very much.
I worked with a membrane receptor and used a good protocol for extract protein. It didn´t need of sophisticated apparatus or expensive kits.
Prepare the next lysis buffer:
Tris-HCl 10mM pH 7.4, NaCl 50mM, EDTA 5mM, Triton X-100 1%, SDS 0.05%, NaF 50mM, Na3VO4 100microM, B-glicerophosphate 10mM, sodium pyrophosphate 10mM, phospho-serine 1mM, phospho-threonin 1mM, phospho-tyrosine 1mM, leupeptin 20microg/ml, bacitracine 500microg/ml, trypsin soybean inhibitor 50microg/ml and PMFS 100microg/ml.
All reagents from Sigma and must be prepared fresh before use it.
Check the buffer have a lot of proteases and phosphatases inhibitors. You can do modifications of course.
I cultured cells in 6-well dishes and lysed with 1 ml of lysis buffer but it was for a single experiment.
Lysis cells in ice-cold buffer. The P1000 pipette works great or you can use an insulin syringe.
Let lysed rest on ice 10 min.
Centrifuge 15 min at 4ºC and 13000 x g. Discard suppernatant.
This protocol works for IP and WB.
Sorry, I made a mistake up there: DO NOT DISCARD SUPPERNATANT!!!! he he, there is your receptor!! : )
good luck again.
good luck again.
Do you know the steps to extract tyrosinase, a transmembrane protein that present in an organelle (melanosome) in melanoma cells?