Alexa Fluor 488 / solubility problem

Posted by Norman Watts on
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I'm seeking advice on how to dissolve the Alexa Fluor 488 phenylmercury dye from Molecular Probes?

I am finding it impossible. I have tried three different solvents: DMSO, DMF, and 100 mM HEPES pH 7.5. I have tried adding from 100-1000 ul of solvent to the vials (which contain 1 mg dye according to the manufacturer). I have vortexed, warmed, and waited while trying with a total of six different vials from two lots. In all cases the majority of the dye remains as solids at the bottom of the vial. In addition, the supernatant does not appear to be dissolved dye. I am unable to remove it with either a spin column, a 3000 MWCO, or even a 10000 MWCO dialysis lasting many days. I have heard that the dye molecules associate by stacking, making for a seemingly high molecular weight. Molecular Probes Tech Support claims ignorance of any problems.

Ideally I would like to be able to add 100 ul of solvent to 1 mg of dye (to obtain an ~11 mM solution, which I can then add in 1/10 volume to my sample, to have dye in ~10-fold molar excess over my ~100 uM protein). I would like to keep the solvent (if its DMF or DMSO) below 10% final concentration to minimize denaturation. I have seen reports in the literature where other Alexa 488 series dyes have supposedly been prepared as 10 mg/ml stock solutions in DMF.

Norman

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Norman Watts, Ph. D.

National Institutes of Health

50 South Drive, Rm. 1509

Bethesda, MD 20892-8025

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