I'm trying to check the MHC 1 expression but I'm not sure if the general protocol for western blot can be applicable for transmembrane glycoproteins. For the sample preparation would RIPA buffer be ideal followed by centrifugation and boiling or does anyone have any other recommendations??
Perhaps you should look here:
Kimitaka Tanaka et al.
Down-regulation of Human Leukocyte Antigen class I heavy chain in tumors is associated with a poor prognosis in advanced esophageal cancer patients.
November 2011, International Journal of Oncology 40(4):965-74.
DOI: 10.3892/ijo.2011.1274
In principle, detergent lysis of cells should allow for solublization of transmembrane proteins and subsequent separation by SDS PAGE. MHC proteins are well solubilized with this technique. Not sure how many good antibodies there are for detecting MHC class I and II in westernblot. There are very good antibodies for IP of MHC class I and II (at least for human and mouse), and if this is an option, metabolic lanbeling of your cells, followed by IP. SDS-PAGE and radiograpgy might be an option. Alternatively, if you want to measure cell surface expressio, you could use flow cytometry (which is much more rapid and quantitative).
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- DNA