I have 3 synthetic peptides that I am using for ELISA: analysis indicate that all of the have good water solubility. They have net charges of -1, -3 and -1.9 at pH 7 with corresponding PIs of 4.73, 3.92 and 4.3 respectively. These are dissolved in carbonate buffer of pH 9.6 and stored in aliquots. Is this pH a good pH for coating my peptides?
And additionally, can UV activation of Corning plates for 30 seconds prior to coating improve on the results?
Thank you ALL in advance.
pH should be fine
make sure your peptides are big enough for direct coating, or weather you need to make use of eg a spacer to allow more efficient binding of antibodies in subsequent steps.
pH 9.6 (I assume you use 100 mM sodium carbonate/sodium bicarbonate buffer?) is common for coating. However you can also coat in PBS.If you think, the coating does not work properly do a test experiment where you can compare the performance of your ELISA in plate’s with different binding capacity (low, medium, high). You also may try different temperatures: for some proteins coating overnight at 4°C works better then 1-2h at the ambient temperature.
P.S. I would not recommend using coating buffer for a long-term storage of your proteins. Generally, more physiological conditions (pH 7.4, PBS) are better suitable for storage, especially when you will use your proteins for ELISA where the most antibodies are diluted in buffers with pH 7.4.
I would suggest that you couple all the peptides to BSA and use the conjugate for coating as usual. It also provides a better steric conformation for response.
If you add an additional C-terminal cysteine you can use this to provide polymeric peptides by conjugation to bovine serum albumin activated using N-succimidyl-3-[2-pyridyldithio]propionate
Rather a question than an answer: When coated peptides have charges at assay pH (means high or low pI), might that imply that they add some ion exchange properties to the plate and thus (depending on the analyte composition) might be blocked by unspecific binding (suggesting a specific signal that is too low for the actual titer) or increasing background (by eg binding secondaries unspecifically)? Is that concern real and what could one do about it (eg adding salt)?
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