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I can confirm the prior statement to Claire (being that I'm in Molecular Probes Tech Support).
Alexa Fluor 633 is more hydrophobic, which makes is less soluble in aqueous solutions, as well as more "sticky" for conjugation, leading to more difficulties with purifying conjugates.
Also, in my experience (having helped develop antifades here in R&D), AF633 is less photostable than AF647 in most environments, though I can't seem to dig up specific data from back then. This is the main reason I tend to recommend AF647 over AF633. Also, AF633 is a bit lower in wavelength, and therefore a bit more problematic with bleedthrough into lower wavelengths (such as combinations with Cy3 or AF555).
We perform a QY determination on some of the conjugates of these. If you compare the most recent lots of AF633 goat anti-mouse (A21050) and AF647 goat anti-mouse (A21235), both of which are determined relative to DDAO, AF633 has a value of 0.5 and AF647 has a value of 0.7.
Jason
Jason A. Kilgore
Technical Application Scientist
Molecular Probes / EVOS Tech Support
Thermo Fisher Scientific
1-800-955-6288 then option 4, then option 3, then option 2.
Or dial direct at +1 541 335 0353
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From: Confocal Microscopy List [mailto:
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Sent: Friday, June 16, 2017 8:35 AM
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Subject: Re: Fluorescence Extinction Coefficient vs Photostability - Alexa 633 vs 647
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Sometime ago a Molecular Probes rep told me the AF633 was not very soluble so they had a very hard time measuring the QY.
That is why they don't quote it on their webpage or in their literature.
She suspected it was much lower than AF647. We always recommend AF647.
I had remembered AF647 was more photostable but I see ThermoFisher doesn't have it on the dashboard. Not sure why.
https://www.thermofisher.com/ca/en/home/life-science/cell-analysis/fluorophores/alexa-fluor-647.htmlSincerely,
Claire
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