Detecting Biotinylated Proteins with Streptavidin

Biochemistry, Molecular Biology, and Cell Biology Protocols >> Detecting Biotinylated Proteins with Streptavidin

Detecting Biotinylated Proteins In Cells

1 Plate the cells onto glass cover slips, and let them attach for at least one night. Include a negative control that does not contain the biotinylated protein(s) of interest. Perform the biotinylation of the cells either after plating or prior to plating onto the cover slips.
2. Wash cells or tissue in buffer (such as phosphate-buffered saline).
3. Fix cells with 4% paraformaldehyde in 1x PBS (NaCl 137m M, 27 mM KCl, 80 mM Na₂HPO₄, 20 mM KH₂PO₄). (Make paraformaldehyde solution in the fume hood, using 9 parts water first, and then adding 1 part 10x PBS to make it in a solution with 1x PBS final concentration. It is also necessary to stir and heat the solution slightly, and add a few drops of 0.2 M NaOH to help dissolve. Do not remove from fume hood until the solution cools.)
4. Wash several times with PBS.
5. Block with 5% BSA in PBS.
6. Label with streptavidin-conjugated to a dye in the dark for 30 minutes (Try 1/1000 dilution first, if it gives a high background, try a lower dilution).
7. Wash 3-4 times 10-15 minutes each with PBS.
8. Add mowial and mount the cover slip upside down on a slide. Cover edges of the cover slip with nail polish, and allow the nail polish to dry. View on a fluorescence microscope after proper training from other people.

Detected Biotinylated Proteins Run on a Gel

Similar to above, include a negative control that does not contain the biotinylated protein(s) of interest.

1. Make a protein gel and transfer onto a blotting membrane as described.
2. Block the blot with 5% BSA in PBS (NaCl 137m M, 27 mM KCl, 80 mM Na₂HPO₄, 20 mM KH₂PO₄) for 1 hour.
3. Add streptavidin conjugated to horseradish peroxidase in PBS-Tween (PBS + 0.05% Tween 20) + 1% BSA at a streptavidin concentration between 1/5000-1/20,000.
4. Wash several times in PBS-Tween several minutes each and detect with ECL as described.