My peptide is labelled with TAMRA. I want to deliver that TAMRA coupled peptide to the cell lines and then i want to check the fluorescence by FACS. How much micro moles of peptide-tamra would i need to treat in order to get a clear signal?
Thanks in advance.
Hi Zaheer,
TAMRA itself will not bind to cells at working concentrations. It’s the affinity of your peptide that matters. If you have some previous data on peptide’s binding/ toxicity with cells, try TAMRA-peptide nearly 5-10 fold lesser concentration than that used with non-TAMRA peptide.
Getting fluorescence from cells with TAMRA-peptide is completely peptide dependent event, and you will have to check by yourself for your peptide.
For references, you can look at these articles; PMID: 21434868, 20857151, 19845398
Best
Saurabh
Hi Zaheer,
as pointed out by Saurabh, the ability of peptides to bind membranes depends on several factors related to the peptide sequence and architecture. Among the others, provided that the final peptide concentration is the same, the total available membrane area (i.e. the number of cells you use) will also have an impact on the final outcome. In terms of concentration, the threshold above which you start getting signal due to the adsorption to the membrane may vary greatly, though you might want to start with micro molar concentration, as a rough estimate. This is discussed in PMID:18069724, together with toxicity issues.
Depending on the endpoint you are looking at, there might be plenty of other issues to be addressed. In case, for instance, you want the peptide to penetrate the cell, you might want to have a look at PMID:19047062, 21878907, 25238106.
thank you so much for these nice and helpful suggestion. i will try that. thanks again/
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