Detecting intracellular membrane proteins.

Sat Apr 9 20:42:46 EST 1994

In responseto query from Haiyan Long, who states that he is in the process of
characterizing a putative membrane protein that is not expressed on the cell
surface.  If I understand your problem, it may be that the protein you seek
is in the ER, Golgi,secretory vesicles, or the vacuole.  If you have a good ant
ibody to the protein you can very gently lyse the cells (in such a way as to
keep organelles intact) and the treat with proteinase K.  If your protein is
in an organelle, it will be protected from proteolysis by the intact organelle
membrane.  If your protein is cytoplasmic, homogenize the hell out of the cells
 and do a high speed spinto drop out the membranes and you should see it with
the antibody in the supernatant fraction.

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