1) I have to attach fluorescent dye to peptide. But the problem is sensitivity. Which would be the best dye to conjugate with peptide to detect it on 2-5 pmole scale?
2) Apart from fluorescence spectroscopy and fluorescence gel electrophoresis, what are the other most sensitive detection techniques?
Fluorescein has a very high quantum yield (0.95) at neutral pH. Amine-reactive fluorescein derivatives such as FITC are readily available and inexpensive. The drawbacks to fluorescein are that it is very sensitive to photobleaching and it loses fluorescence at acidic pH (such as in many reverse-phase HPLC mobile phases). If the labeled peptide will not be exposed to a long duration of exposure to bright light and will not be analyzed under acidic conditions, then fluorescein is a good choice.
Alexa Fluor 488-based amine-reactive dyes are way more expensive, but they also have high quantum yield (0.92). The advantages of Alexa Fluor 488 are photostability and retention of fluorescence at acidic pH.
https://www.thermofisher.com/us/en/home/references/molecular-probes-the-handbook/fluorophores-and-their-amine-reactive-derivatives/alexa-fluor-dyes-spanning-the-visible-and-infrared-spectrum.html
There are many other bright fluorophores that can be purchased with amine-reactive functionality. An important decision is what excitation and emission wavelengths you want to use. I suggested fluorescein and Alexa Fluor 488 because they can be excited with the light sources in instruments that are commonly available in laboratory instruments such as flow cytometers, microscopes, fluorescence detectors, and plate readers.
For ultrasensitive detection of the fluorescent peptide, such as in an LC system, I recommend using a laser-induced fluorescence detector. However, 2-5 pmoles is actually not such a small amount for detection: 5 µl of a 1 µM solution, which is easily detected by standard non-laser detectors, is 5 pmoles. You could easily detect 1/100 that amount with a decent fluorescence plate reader.
Dear Adam,
I am very grateful for your kind detailed response.
Thank you Sir.
I would suggest dyes from dyomics or atto tec at a corresponding wavelength. Addition of additives (Trolox, Etc.) might help to increase FL (doi 10.1002/anie.200801518).
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