I am planning to conjugate my peptides to BSA (using a cysteine added to the N-term) to plate on on a well so that I can do enzymatic reactions on my peptides.
I am finding a wide variety in the range of concentrations of peptide needed to ensure a good enzymatic reaction. The mass difference of the peptide and BSA is pretty large, but my peptides are about 2.5-3.3 kDa.
I have found some values for free peptides, but not that are fixed to a plate.
Any experience would be great! Thank you!
Hi Hanaa: Are you using your peptide as a substrate or as an antigen to coat on plate? Concentrations to each situation are different.
Actually, as both. First as a substrate, then as an antigen to detect modifications on the peptide substrate.
You probably can only do ELISA-based assay with coating (antigen type), as the binding of proteins/peptides on plastic surface is limited to sub micrograms level. On the other hand, enzymatic reaction requires miro- to mili- molar ranges. If I were you, I'd find a commercial substrate for enzyme assay and use your peptide as an inhibitor to determine its Ki, which indirectly suggest its binding to the enzyme. Hope it helps.
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