Hi all,
I am currently expressing a membrane protein on eukaryotic cell line, the morphology observed have similarities with other eukaryotic cell line expressing same protein, but is there any tracker for membrane protein to confirm my expression is indeed surrounding plasma membrane?
Thank you for answering my question.
If I understood well, you want to convince others that your protein is indeed on the plasma membrane, right? There many ways to state that a protein is a plasma membrane protein: in living cells you can stain lipids of PM with lipophilic dyes and simultaneously, stain your protein with an antibody to be detected with another fluorophore, then co-localization will proceed. Another is a pretty old technique called PM-Lawns, which take living cells cultured in glass coverslips to be exposed to hypo-osmotic solution and then, quickly frozen on a glacial acetic acid bath, so only the membrane get adhered to the glass (like a warm tongue in a cold metal bar). Then you fixed that covers with formaldehyde and proceed with a normal immunohistochemistry.
You can purify the plasma membrane from the cells using sonication followed by sucrose step-gradient ultracentrifugation. The plasma membrane is the lightest membrane fraction. You can use Western blotting to show that your protein is in this fraction.
Hi, you can prepare a membrane fraction from your over expressed cells, by sonication or cell lysis followed by centrifugation at 18K to remove cell debris, then centrifuge the supernatant at 43K , you get pure membrane fraction pellet. Solubilise the membranes with SDS or Triton, load and do western you can find out your protein.
If you have an antibody for your protein (or another way to identify it in a SDS-gel or Western blot) the easiest solution is to use a kit for membrane protein isolation. In our lab we routinely use Pierce Cell Surface Protein Isolation Kit to separate the membrane protein fraction (biotinylated) from the intracellular one (non biotinilated) to identify membrane localization.
Also, as a control we detect an intracellular protein in the membrane and intracellular fractions (for example actin).
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