Janelia Fluor Shelf Life

Hello all,
I recently obtained some JF549-Halo ligand dye that I’d like to use in my experiments. The Promega protocol recommends diluting in DMSO, and cautions that this dilution is single-use. Given the high cost of the reagent, this seems slightly…insane.

Does anyone have any experience in aliquoting or storing these dyes long term? With the amount I currently have, if I could store it correctly I would never need to buy it again :slight_smile:

Thanks in advance!

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I got some JF-549 about a year ago (maybe more?) and diluted it down to 10 nM stocks and it still works just fine for me. Try to make lots of tubes so you can avoid freeze thawing the same tube over and over. You final stock concentration may vary depending on your application.

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I haven’t systematically checked to see whether the dyes have gotten at all dimmer (they certainly appear to behave exactly the same), but I’ve used JF549 that’s been frozen in DMSO at 1mM for probably over 18 months without any problems. I’ve also freeze-thawed vials of JF549 in DMSO at least 10 times without much ill effect. Perhaps it’s better to be on the safe side, but this does seems slightly overcautious.

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Depending on how much/how often you plan on using the JF dyes, we dilute ours down to 1 uM single (or short term) use stocks. We don’t ever freeze thaw the small aliquots.

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You can dissolve your dye in DMSO, make small one time use aliquots and dry them in a speed vac. You can then store the powder in -20 and redesolve it in DMSO as you need. The powder will last a long time.

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Our (Lavis lab) advice is to prepare a stock solution in DMSO at whatever concentration is convenient for you, then from that stock, make ~single use aliquots. We do recommend avoiding repeated freeze-thaw cycles on the same sample (hence the “single use” aliquots), but preparing a stock solution is, in and of itself, fine (and doesn’t require immediate, one-time use).

We’ve done a bit of work examining long-term storage and use of JF probes, and they seem to exhibit more degradation (not a lot, but still some… a few %) when the samples are repeatedly freeze-thawed. Oxidation of the dye and a minor amount of azetidine ring opening seem to be contributing decomposition pathways. This is more of an issue for some dyes than others… JF549 and bluer JFs seems quite resistant to such degradation, but JF646 and other red analogs are more prone to these issues.

If you are going to temporarily dissolve the JF dye in a solvent to aliquot and spin down back to solid, I actually recommend using MeOH (for all SNAP-tags, for all JF503 -> JF549 ligands, but NOT for NHS esters) and MeCN (for NHS esters and for JF585+ HaloTags), as it is much easier to remove these solvents. Even after extended time on a speed-vac, there will almost always be a little DMSO in the “dried” material, and DMSO is not the most innocuous solvent.

EDIT: I should also mention that we recommend using fresh, high-quality DMSO whenever possible. We prefer the Hybri-Max DMSO from Sigma, which can be ordered in boxes of 10 x 5 mL ampoules: https://www.sigmaaldrich.com/catalog/product/sigma/d2650?lang=en&region=US

IG: @dyerfulchymist
Twitter: @jonathangrimm

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Hello @JonGrimm and thanks for the advice! At what temperature do you store the single use aliquots?