Hi, everyone!
I'm trying to select HEK293FS cells expressing SBP-LNGFR protein. The protein presence on the cell membrane is confirmed with immunofluorescence. But the MACS procedure with Streptavidin MicroBeads doesn't work. I don't get a pure fraction of cells, and also get a lot of cells out from the Mock transfection (cells not expressing SBP-LNGFR). Does anyone have an idea why this is happening?
Hello,
Could possibly be an issue with the Strep-beads if the mock cells are also attaching to the column. Do the mock transfection cells show expression with immunofluorescence? Protein expression could also be altered in the detached cells, hence the different expression patterns you see in immuno.
Other possibilities:
-Wash the cells prior to addition to column AND was the column more. This will remove background beads/cells.
-Run the eluted "positive" cells through another column (with additional washes) and see if the second elution of "positive" cells is purer.
Just some thoughts, but if you include more details about the procedure, it may be clarifying.
All the best,
Elliot
One possibility that you might have many dead/apoptotic cells that nonspecifcally bind to literally everything, including magnetic microbeads. If this is the case you can purify first your cell populations with magnetic dead cell removal kits or with Ficoll, sucrose gradient centrifugation, additional washings. Another possibility is high endogenous biotin levels in some cell types, but it's usually intracellular biotin. I would not rely on immunofluorescence alone in gene expression assays, try RT PCR, Western Blot as positive signal in IF can be just nonspecific binding of your antibodies to your cell line. Additional blocking steps with high concentrations of protein, Fc block can help, depending on cells type you work with. Also confirm that you do not overload the column with cells and have the column type suitable for the specific magnet. Too strong magnet will pick up cells with low nonspecific magnetic beads binding. Also during washing steps make sure that column is never dry, at the same time don't add washing buffer on the column before previous washing buffer completely leaves the column.
Thank you Elliot Glotfelty . The mock transfected cells don't show expression with IF. The cells are washed twice before being resuspended in labelling buffer. I have washed the column for twice as many times as recomended in the protocol to try and elute unbound cells but to no effect.
Thank you Natallia Mikhalkevich . As I mentioned above, the immuno signal is not nonspecific binding, since I don't see the signal in the mock transfected cells and the cells are blocked and not permeabilized before IF. High endogenous biotin levels are definitely a possibility and the problem is that I don't have enough info on the cell line itself since it's not available.
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