I want to clone a tag onto protein X, a protein which we are expressing and purifying in the lab. We will than incubate protein X with cells to see which cells bind it. I want to stain the sample with an anti-tag antibody to stain all cells that are bound to protein X, which I can then analyze or sort by FACS.
Does anyone have experience with the best tag for this?
Say, Flag, or HA?
Hello Gil.
Could you rephrase what you are going to do to what to know? It's not clear from what you wrote.
Regards.
Hi Gil, I am not clear about the location of the protein you are going to clone and express in cell (whether it is surface, trans-membrane or internal).
I want to incubate a protein (that I am expressing in bacteria and purifying) with immune cells, and identify the cells that bind the protein by flow cytometry (FACS).
For this, I must do some cloning to add a tag to the protein.
I am wondering what is the best tag to add to a protein in order to detect it by flow cytometry.
I think I should use an HA-tag but not sure.
I have used both flag-tag and myc-tag successfully to tag proteins for flow cytometry. But I am sure other tags would work fine, too.
I think there are two issue you should be more concerned about when choosing the tag:
Good luck with your work
Hello Gil.
Any tags would be OK.
Since you would like to identify some of immune cells challenged and bound to the recombinant proteins on the cell surfaces, you have a plan to use another marker. Thus, you have two markers to be used for your cells. To evaluate safely, I would suggest fixing cells for label the cell surfaces to prevent the bound two biomarkers from being internalized by endocytotic membrane trafficking. Even the incubation of cells on ice cannot avoid dynamic endocytotic membrane trafficking due to the loads of heavy binding to the repeated tag domain, and void intervals of no control of low temperature during the whole procedure, leading to membrane endocytosis.
Best wishes.
Thank you for the detailed answer.
I chose to clone and HA tag in the mean time.
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