I have two synthetic peptides (20-25 AA length) with terminal cysteines that I have been trying to conjugate to maleimide dyes. The maleimide-conjugation protocol has seemed easy enough, but I'm struggling to prove that my peptides have successfully tagged.
I had tried gel electrophoresis with Coomassie Blue, but later found out there was not sufficient peptide in my sample for the bound-tag to show. I then moved to HPLC but my lack of expertise and a lack of appropriate UV detector on the machine I used meant that was unsuccessful. Most recently I had submitted my tagged sample to an analytical service at my university for LCMS analysis, but the results have been inconclusive because there appears to be excessive fragmentation (and no obvious cysteine-tag fragments, or any fragments that differ by an m/z equal to the tag/tag+cysteine etc.).
I feel like this really shouldn't be that difficult, but it's absorbed months of my PhD project and I'm still no closer to proving the success of the maleimide tagging. Any advice?
Thank you.
Is your dye simply a chromophore or is it fluorescent? The latter case is usually much easier to detect. For my grad studies I had a similar cys labeled peptide that I conjugated to the fluorophore acrylodan. Using TCEP to assure I had no dimers was essential to efficient conjugation. My core facility then set me up to purify the labeled peptide quite easily on a prep C18 HPLC, but I was a novice and the expertise of the core lead was essential (thanks Deb!). In about a week I laid in enough labeled peptide for all my studies and gifted the rest to a collaborator : ) Good luck!
Edward Michelini It's a fluorophore, but I can't seem to find any HPLC machines available to me with an appropriate detector, or anybody with the right expertise in my school. I may have to have another think about doing preparative HPLC, I was getting quite good at the set up!!
Thank you :)
If you are conjugating the protein to a fluorophore, the simplest way to tell if the procedure worked is to run the sample on SDS-PAGE, do not stain with Coomassie Blue or fix the gel, and use a fluorescence gel imager (if there is one near you) to detect whether there is a fluorescent band on the gel at the correct molecular weight, compared to standards.
For peptides of such a small size, you will have to use a Tris-tricine gel.
Dialyze the sample or use PD MidiTrap G-10 desalting cartridge with a 700 Mr exclusion limit to get rid of excess and not reacted dye.
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