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 
Thanks in advance!
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.
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.
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.
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.
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®ion=US
IG: @dyerfulchymist
Twitter: @jonathangrimm
Hello @JonGrimm and thanks for the advice! At what temperature do you store the single use aliquots?
Hello Jonathan Grimm,
I currently resuspend the Janelia JFX646 tubes in Acetonitrile and separate into tubes that I Speedvac to prepare dry aliquots. This seems to work for me.
Is it preferred to resuspend the 100 nmol tubes in DMSO and to separate into tubes of single-use aliquots, i.e., wet aliquots, no Speedvac? And if so, at what temperature and for how long (weeks, months?) is it ok to store the single-use aliquots for?
Also, if I add media to the tube that contains DMSO, to get a lower concentration of dye, will the dye, over a few hours, significantly decrease in concentration because of the dye sticking to the wall of the tube?
Best,
Yossi
Dear community -
I am adding a protocol for JFX650, that addresses the issues of stability, aliquot variability and resuspension. Its based on partial communication with Promega, the Lavis lab, and my own testing. But its in progress.
I would love to hear advice about step 4 in the usage of aliquots section. What tube or vial would be good for maintaining a constant effective concentration of the resuspended dye over several hours, i.e., where the effective concentration doesn’t decrease significantly due to sticking to the wall of the tube or vial?
I am copying the protocol below. Besides for step 4, the rest of the protocol is “grayed out”.
Thank you,
Yossi
Hi, Yossi, does it matter which active group the JFX650 has, for use of your posted protocol to make single-use aliquots?
Hello -
I think this is for both halotag or snaptag - although my personal experience is with halotag.
Please see the Lavis lab protocol paper below.
from -
Grimm JB, Brown TA, English BP, Lionnet T, Lavis LD. Synthesis of Janelia Fluor HaloTag and SNAP-Tag Ligands and Their Use in Cellular Imaging Experiments. Methods Mol Biol. 2017;1663:179-188. doi: 10.1007/978-1-4939-7265-4_15. PMID: 28924668.
best, Yossi
p.s., I miswrote in my own posted protocol that I use a speedvac. I am using the concentrator plus from Eppendorf - which is also a vacuum concentrator.
