Dear all,
I am using this kit for isolation of cell surface protein.
https://www.thermofisher.com/order/catalog/product/89881
In brief, this kit biotinylate the surface protein only, as the biotin reagent is impermeable to cell membrane, after cell lysis, we can collect the surface protein by Avidin resin.
However, I detected cytosolic protein in the surface protein fraction. The intracellular marker I used was beta-actin, but beta-actin is a part of the cytoskeleton which extend all the way from the cell membrane to nucleus, I am wondering whether it is possible that the beta-actin is sticked to some biotinylated surface protein, and be collected together with the surface protein. So I prefer to use a marker that is not a structural protein, for confirmation.
I have tried beta-catenin because the antibody is available in our lab and it is known as a nuclear protein. I detected beta-catenin in surface protein fraction also, but then I found that beta-catenin actually localize extracellularly also (http://www.uniprot.org/uniprot/P35222)
so it may not be a good choice for marker.
Did anyone do similar isolation? What intracellular marker did you used?
Hello KC,
you may try any mithocondrial marker such as COX4 (cytochrome C oxidase).
Alternatively you may use nuclear protein Ki-67 as marker.
To prevent endocytosis or any actin dependent membrane trafficking you may use cytochalasin D.
Hope it helps.
Cytosolic enzymes should be good markers for your purpose as they are synthesized by free ribosomes and do not enter the endoplasmic reticulum compartment for further distribution to other organelles or to the cell membrane. Why not choosing GAPDH as internal 'housekeeping' protein?
Peter
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