Molecular probes sells an anti-PentaHis antibody http://www.probes.com/servlets/product?region=Select+Region&item=21315 that crossreacts with hexahistidine-tagged proteins in most applications. It is a mouse IgG1 antibody, indicating that it will complex with our new Zenon One reagents http://www.probes.com/handbook/sections/0702.html to form labeled antibodies (one can make the complex either before or after if one uses a single color, except as below). We have had generally good success using Zenon labeling complexes for intracellular staining of antigens by flow cytometry (and imaging) although access may depend on your permeabilization techniques. However, if you also have cell surface expression of hexahistidine-tagged proteins (or other proteins that crossreact with the same primary antibody), it will be necessary to block these first while the cell is alive. We have never tried it but presumably one could block the cell-surface-expressed hexahistidine-tagged proteins with the same unlabeled mouse antibody to pentahistidine then, after fixation and permeabilization, use the pre-made Zenon One reagent-labeled complex of the same primary anti-pentahistidine antibody to label only the intracellular proteins. One could not do this with a secondary anti-mouse antibody because it would crossreact with the cell-surface-bound primary antibody but it should be possible with the Zenon One complex, which is essentially equivalent to the covalent dye conjugate of the mouse primary. The Zenon One label will not transfer to the anti-pentahistidine antibody used as the blocker, at least over the period of the experiment. The limitation would be whether the unlabeled primary has complete access to the cell-surface-expressed hexahistidine fusion proteins to block their binding activity step of the Zenon One reagent-labeled primary (if that is what is giving you background). Could be interesting to try this and I cannot think of another way to do the same experiment. Shelia R Alexander wrote: > Hello > I am searching for a 6His tag antibody that works by flow cytometry as a > marker for transfection efficiency. I have stably transfected K562 and > U937 cells and a mouse fibroblast line and put them under puromycin > antibiotic selection and have a population that has grown out but need to > prove that they express my protein of interest and aren't antibiotic > resistant. What I have seen so far is that the 6-His monoclonal > antibodies stain extracellular and permeabilized cells from my parent and > transfected cell line equally well. My 6-His tag is expressed > intracellular in this case but future studies would place the His tag on the > surface of the cells. Are there ways to block "native" HIS expression? > > Any suggestions would be appreciated. Thank you > > Shelia Alexander > Research Fellow > University of Minnesota > College of Veterinary Medicine > St Paul, MN 55113
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