I am looking for some marker suggestions for using it in confocal as well as western blotting enriching the membrane. I work with rat glioma cell line as well as HEK293T cell line.
actually a negative marker for PM is actin - if you have in WB something in actin in the PM fraction, there is xcontamination.
Hi, Kalpita, here is a couple of membrane markers: http://www.abcam.com/index.html?pageconfig=resource&rid=10013&pid=14
I think that it is a problem to get pure membrane fraction, so, I would suggest to prove membrane markers only in immuno. Good luck!
actually a negative marker for PM is actin - if you have in WB something in actin in the PM fraction, there is xcontamination.
Good suggestions already. Appropriate markers can vary depending on the cell type of interest. Na+K+ ATPase is probably a pretty good general marker as suggested by Dr. Spittau, but you may find a different protein works better in your cells depending on abundance. Na+K+ATPase antibodies can work for both western blotting and IF. Another general marker often used in IF to tag the plasma membrane is Wheat Germ Agglutinin (WGA). Its a lectin available conjugated to a variety of fluorescent tags. We get ours from Molecular Probes. Its compatible with combining with antibody labeling. We apply it as if it were a secondary antibody in combination with the other secondary andibodies in double or triple label experiMents. Being a lecting labeling is improved by using buffers that contain some Ca++.
you may use Na/K ATPase as membrane marker. however, This is the marker not only for plasma membrane but also for total membrane including endosomes.
Depends on the cell type. I have used transferrion receptor and epidermal growth factor receptor successfully. For transferrin receptor, I cannot use trypsin to harvest the cells because the extracellular domain gets digested and that is what the antibody recognizes on western.
Above said cadherins and transferrins are subjected to endocytosis and hence will not reflect true plasma membrane fractions but also reflect endosomes (early, recycling), TGN and often lyso/autophagosomes. An immunofluorescence before Western blot will tell you these proteins are also located in endomembranes or not. After that proceed for Western blots if you see cadherins or transferrins are purely located at cell surface but not in endomembranes, in your cell system.
Hi, Kalpita, There is no 100% pure plasma membrane markers exist, If one consider highly dynamical protein exchange on plasma membrane (internalization, vesicle traffic etc). At least in WB after fractionation you can not be 100% sure. Perhaps, only IF give you a quite clear answer.
I have not done this myself, but how about using something like biotinylated annexin V? It binds to phosphatidyl serine in the plasma membrane - you should get strong positivity in your membrane extracts and no positivity in your cytosolic fractions. If you combine this with a cytosolic protein (e.g. actin, as suggested above), you should get minimal staining in your membrane fraction, but strong staining in your cytosolic fractions.
Hi Damir and Taras,
My western blotting technique was actually biotinylation experiment, labeling and pulling down specific fractions. I do see a 20% signal of actin in the blot if I take 100% of actin as other fractions say cytosolic. Is it completely absent from PM fraction or little bit is acceptable?
And also in IF of fixed as well as Live cells, the expression of my protein is predominant in perinuclear region and less on memebrane (80-20 ratio). Upon tweaking the signalling, I expect the PM fraction to increase and hence quatify with specific labels acting as markers.
Any suggestion on how to achieve that?
Thanks for the reply in advance.
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