Hello all,
I recently started to use peptides for cellular studies. Unfortunately, when I was designing the peptides I did not have a FITC tag to them (the peptide was synthesized by an external company). The peptides still have an -NH2 terminal that I can react with FITC-NHS pretty easily. But due to the small size I would not be able to isolate the peptide from unreacted/hydrolyzed FITC-NHS by MW cutoff as I would for proteins.
The peptides are not very cheap so I would seek alternatives than purchasing more tagged peptides. While I know I could purify it by HPLC followed by concentration / lyophilization, it's adding a lot of procedures to the pipeline and I'm considering if there could be alternatives. And so, I'm wondering if peptides could be precipitated by TCA/acetone precipitation similar to proteins?
While I believe the precipitation is dependent on the peptide structure, I used one of our peptides and tried it out - the precipitation worked (I saw visual white fluffs) and the peptide absorbance curve showed peaks at 214/280. The curve looked identical to the peptide solution (unprecipitated) but with a lower absorbance unit (22 AU compared to 5 AU post-precipitation).
My question is, is this way of isolating peptides legit? And if I used FITC-NHS, would FITC also be precipitated by any chance? Will the peptide lose stability / increase cytotoxicity during TCA exposure? I am not using the peptide for functional studies, just trying to visualize cellular internalization.
- If this way makes no sense I will refer to other methods. Thanks for any suggestions.
I have had success purifying labeled peptide-like compounds away from the excess label using chromatography under gravity with Sephadex LH-20 in water. Besides the resin, all that is needed is a glass column with a stopcock such as an Econo-column (Bio-Rad).
Example here: https://journals.sagepub.com/doi/10.1177/1087057112436885?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%20%200pubmed
Leran:
FITC-labeled peptide and non -labeled peptide should be very different on HPLC, should be easily separeted.
Success depends a little on the hydrophobicity of your peptide.
I'd try dry Et2O first, esp. if the volume of reaction is small, so all water gets can dissolve in the ether. If it should not work, remove the ether and try isopropanol or ethanol: Begin with small amounts, cool and centrifuge, then increase the alcohol concentration until your labeled peptide precipitates. Fluorescein will precipitate sooner or later, too (pH dependent).
If you try Adam's suggestion, keep all fractions until you know that the operation was successful. For concentrating the peptide, use precipitation as above. There is a high chance that the fluorescein gets trapped on the column and is moving *very* slowly (which is in your favor).
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