Can You Detect Alexa Fluor on Guava Easy Cyte Plus
 | For many years our technical support and scientific staff have been asked questions around the properties, use, and specific applications for Alexa Fluor dyes. Here is a comprehensive set of frequently asked questions and their answers as put together by the Molecular Probes technical support staff for the benefit of the researcher. |
Applications—Labeling Molecules
Do you have a protocol for conjugation of amine-reactive Alexa Fluor dyes to proteins?
What should I use to specifically label my antibody or protein?
How can I label my antibody/protein/oligo with an Alexa Fluor dye?
Applications—Cell Analysis
At what concentration should an Alexa Fluor IgG conjugate be used as a secondary antibody?
Do you offer a specific staining procedure (fix/permeabilization/block) for using Alexa Fluor conjugates in immunocytochemistry (ICC) and immunohistochemistry (IHC)?
Although staining protocols may vary with application, the Alexa Fluor conjugates can be used in place of any other secondary IgG conjugates that you are already using. They do not require a modification of your staining procedure.
Are Alexa Fluor dyes cell-permeant?
No, they are too highly charged.
Are Alexa Fluor dyes compatible with common fixatives, buffers, permeabilizing agents like formaldehyde, formalin, paraffin, xylene, Triton X-100, etc.?
Alexa Fluor dyes are compatible with Triton, formaldehyde, and formalin. Paraffin processing of tissues likely will extract dyes of almost any type, including Alexa Fluor dyes, though they are fine to use after the processing, such as for immunohistochemistry (IHC) imaging.
Is Alexa Fluor 405 the best dye for imaging at 405 nm?
The "best" dye really depends on the filter's emission specifications. Both Alexa Fluor 405 dye and Alexa Fluor 430 dye will efficiently excite at that wavelength.
Chemical Properties
How hydrophobic/hydrophilic are Alexa Fluor dyes compared to other standard counterparts?
All of the Alexa Fluor dyes have been engineered to be more hydrophilic than dyes such as fluorescein and Texas Red dye. Alexa Fluor 594 is the most hydrophobic of all of the Alexa Fluor dyes.
What can quench Alexa Fluor dyes?
Just like all dyes, Alexa Fluor dyes are susceptible to typical quenchers such as chromogens (trypan blue), quenchers (QSY), and other compounds such as 1, 4-diazabicyclooctane (DABCO) and N-propyl gallate (NPG), which, ironically, are also used as antifades.
What are the fluorescence quantum yields (QY) and lifetimes of the Alexa Fluor dyes?
What is the pH stability of Alexa Fluor dyes?
Most of the Alexa Fluor dyes are stable between pH 4 and 10.
What is the usable pH range of Alexa Fluor dyes?
For most of the Alexa Fluor dyes, we recommend a pH range of 6.5 to 8.5.
Performance
How are the Alexa Fluor dyes better in performance than their standard counterparts such as fluorescein (FITC), Cy3, Cy5, Texas Red, and allophycocyanin (APC) dyes?
What antifade mountant do you recommend for cells stained with Alexa Fluor conjugates?
For the best publication-quality imaging data, we recommend ProLong Gold Antifade Reagent, which requires letting the sample cure at least overnight, which helps obtain crisper images. If there is a need to image samples without waiting overnight, we recommend SlowFade Gold Antifade Reagent. For your convenience, these reagents come mixed with or without the nuclear stain 4', 6-diamidino-2-phenylindol (DAPI).
What is the brightest, most photostable, and best Alexa Fluor dye?
All things being equal with the appropriate filters, Alexa Fluor 488 is brighter and more photostable than other Alexa Fluor dyes.
What are the two best Alexa Fluor dyes?
If you have a typical fluorescence microscope with a BGR filter and no other constraints, then Alexa Fluor 488 and Alexa Fluor 594 are good choices, as the blue dyes generally have lower intensity.
Do I need to buy new emission filters to spectrally match the Alexa Fluor dyes?
Product Formulation/Availability
Are Alexa Fluor reactive dye vials (as in the Protein Labeling Kits or Monoclonal Antibody Kits) also available as stand-alone products?
Can I buy an Alexa Fluor dye without a reactive group?
Are the Alexa Fluor azides from the Click-iT EdU kits available separately?
Source: https://www.thermofisher.com/us/en/home/brands/molecular-probes/key-molecular-probes-products/alexa-fluor/alexa-fluor-frequently-asked-questions.html
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