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MHC Tetramer labeling

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HugoTharinger

Participant

Posts: 6

Joined: Fri Nov 22, 2013 10:21 am

Post Thu Feb 27, 2014 9:44 pm

MHC Tetramer labeling

Dear CyTOFicionados,

I am looking for any information on how to label MHC-Tetramer.
Are you using DVS MaxPar labeling reagents and protocol ? what are the differences and precautions you need to take compare to IgG labeling? What is the protein yield recovery and number of metal atoms you are able to bind to it ? What is the ratio / concentration of polymer / protein you are using for the conjugation ...?
If you have a protocol that you are willing to share or any advices it would be greatly appreciated.

Hugo
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Feb 28, 2014 12:30 am

Re: MHC Tetramer labeling

Hi Hugo,

Unfortunately, I don't think that the main tetramer people (Evan Newell, Natalia Sigal, and Helen McGuire; all current or former members of the Mark Davis lab at Stanford) read the CyTOForum that much.

I do not use tetramers. However, as I understand it from the Davis Lab, yes, MAXPAR polymers are used. See the experimental section of this paper, for instance.

"Combinatorial tetramer staining and mass cytometry analysis facilitate T-cell epitope mapping and characterization."
Newell EW, Sigal N, Nair N, Kidd BA, Greenberg HB, Davis MM.
Nat Biotechnol. 2013, 31, 623-629
http://dx.doi.org/10.1038/nbt.2593

However, there's an issue: MAXPAR polymers are maleimide-terminated, which react with cysteines. Streptavidin has no native Cys. The Davis Lab made Cys point mutants, so that each streptavidin monomer can receive MAXPAR polymers. The advantage of this is that it creates a defined site for the polymer to attach, and is at a position that does not interfere with biotin binding. Alternative chemistries involving amine-linkage (NHS ester, etc) have the possibility of hitting a Lys in the biotin-active-site, which would keep you from having a full tetramer.

So, while there are ways to engineer an amine-reactive MAXPAR polymer to create a labeled streptavidin, that would be best used as an anti-biotinylated antibody secondary stain, rather than as the basis for a tetramer.


Mike

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