I would like to label the N-termini of my peptides before electrophoresis. I found GE Healthcare's CyDye DIGE fluors, which can be used in this manner and imaged using the Typhoon scanner, but cannot be seen by eye in the gel. I am wondering if there is a dye available with a similar N-terminal covalent linking strategy, that can be seen by eye, so that I can cut out gel bands for mass spectrometry by viewing at a light box. The reason I am not using Coomassie or other protein stains is that I am looking at small peptides in a tricine gel, and I expect that Coomassie will not be sufficient to stain them to the point of being visible.
If you need to see somethinng, it has to absorb light in the visible range and it does not matter much whether that energy is emitted by fluorecence or dissipates some other way.
The reason why they suggest typhoon scanning is because it is much more sensitive so it is possible to detect really small amount of proteins. If you can use higher amount of protein/peptide, it will be visible in the gel as well. When I could see a band in my gel, I knew that the signal would be too strong for typhoon.
My suggestions:
a) Try to conjugate with FAM SE (it is cheap) and perhaps you will be able to see it. (Extinction coexxicient 80000 (M.cm)-1
or
b) Conjugate with UV absorbing fluorescent dye (like coumarine) and use UV lamp. The band should be visible as the fluorophore will emit at different wavelength.
we can offer FAM SE and other labeling, at high quality and good price! progress your research , email: [email protected]
Peptide may be in acidic condition. Please check pH before labeling. Adjust pH to 7 or above. Also clean up of peptide using C18 material, which may increase efficiency of labeling.
why do you need to do electrophoresis of Peptides. you can simply put them on HPLC and collect the fractions based on A280. You will be done in 1 hour. lyophilize the peptides and do MS, alternatively newer MS have a HPLC attached to them all you have to do is just add the peptide to the injector and let the machine do its job.
Hi,
If the objective is to identify the peptide, then Sharad is bang on. You can go for off line prefractionation or use nano LC as front end online coupled to QExactive.
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