Dear Margherita,

When you write :

xxxxxx

I should label peptides with cyanine dyes, which are very unstable molecules. Do you have any advice to handle and purify the product?

xxxxxx

please write : "I should label peptides with Cy 5 -cyanine dyes...." because cyanine dyes have not reactive groups for the conjugation,

When you read the following paper:

www.ncbi.nlm.nih.gov/pmc/articles/PMC3624894/

you can see the formula of Cy5 and related infos.

On other part, read the link of Lumiprobe: (labeling and purification)

https://de.lumiprobe.com/k/antibody-labeling-kit-cy5

This is a kit for the labeling of antibodies and proteins of similar size with sulfo-Cyanine5 fluorophore. The dye is attached by NHS ester chemistry, and then the excess of the fluorophore is removed by means of gel filtration using enclosed spin columns. The protocol requires only around 40 minutes of time, and provides desalted antibody in PBS buffer containing optimal number of fluorophores per Ab. The kit contains components for 10 reactions, up to 100 ug of antibody per reaction.

Please insert in Google Scholar or Google this part of your question :

Peptides labeling with Cy 5 cyanine dyes

and you ´ll obtain a big amount of hits.

Good luck

JRG

Margherita,

Cy5 is stable, unless in very basic condition(pH>8 or higher). You should use Cy5-NHS, not Cy5 acid, for label peptide with NH2 group. Cy5 NHS is not stable in basic condition, it is stable at dry/low temperature.

Normally, use Cy5 NHS in dry DMF/DMSO solution, drop in(add dropwise or portionwise under stiring/shaking) peptide in buffer(pH 7.5-8.0, NaHCO3 solution is the best). The reactivity of NH2 is much fast than OH- if pH

In addition to what Guobing Xiang has written, store the active ester in aliquots desiccated at -20°C or colder, prepare fresh solutions for each labeling experiment and use the solutions immediately. Don't use buffers with amino groups, as they'll happily compete with your protein for quenching the reactive dye.

If you should need to do a clean-up after the labeling, use an as small as possible amount of gel filtration material (e.g. a Sephadex G25 based column) and use as much protein as possible (ideally at least 5mg), as you might lose a lot of material when you start off with a few 100 micrograms.

Edit: When you need to label small molecules, have the label integrated in the peptide, synthesis if possible. Otherwise, purification by precipitation with an organic solvent (usually with ethanol or isopropanol) can do the trick.

Wolfgang Schechinger Guobing Xiang Jean Rene Grezes I thank you all for the answers. I need to label a small peptide with the Cy5 in the COOH form. It isn't preactivated with NHS. My question is if with HPLC is possible to purify it or if I'll lose everything due to the instability of the fluorophore. In some publication I saw purification with HPLC but I would like to understand the amount of the product or at least a range, that I will obtain because I didn't catch it by reading the published articles.

I thank you again.

Margherita Restori Margherita, you may activate the Cy5 yourself, of course, e.g. with EDC-HCl and NHS. Use slightly acidic conditions (e.g. 10mM HEPES or MOPS, pH 5.5) and after 10-15 min at RT, add it to your peptide, dissolved in e.g. 100mM NaHCO3. Clean-up by HPLC (I had not thought of it, previously) actually should be straight forward, just avoid harsh pH conditions.

The tricky part is to get rid of excess EDC in case your peptide has free carboxy groups, so you might need to purify the activated ester, which should be doable at acidic pH. NHS ester are more stable than you might assume, when kept at the right [i.e. acidic] conditions. The fluorochrome of Cy5 is quite stable. Just keep it out of direct sunlight. It's the last thing I'd be worried about.

Depending on how large or small your scale is and how precious your peptide is, it might be saving you a lot of time, hassle and frustration just to obtain some Cy5 NHS ester, though. If you really want or need to do the whole process yourself, I'd like to suggest to investigate and establish optimal conditions for coupling and clean/up with some cheap materials first, like another dye with a carboxyl group and a cheap peptide small molecule of similar properties or with a protein like insulin or BSA.

A short comment to the molecule Cy5: There are different types of Cy5 on the market. Some have sulfonic acid groups and some don't. Sulfonic acids are quite hydrophilic and hence the chromatographic properties may be challenging.