🚀 go-pugleaf

On Tue, 30 May 1995, MARSHALL H. MONTROSE wrote:

> Jennifer,
>         Thank you for the informed reply. It was very helpful. Could I ask
> you to give this group a few key facts about Bodipy FL versus fluorescein
> so that we can end the discussion and avoid redundant inquiries to
> Mol.Probes (think how the people on the phone lines will thank you....). I
> promise we won't think you are just advertising.

My apologies for taking 2 days to reply.  I read this newsgroup from my
personal email account, and not at work, so it took me some time to
gather the data you have requested.

>   1. What is the quantum yield of Bodipy FL?

On an antibody conjugate, the quantum yield of BODIPY FL is 0.71.  For
reference, for fluorescein conjugated to an antibody, the quantum yield
is 0.56.  These numbers are based on antibody conjugates manufactured
and for sale by Molecular Probes.  The degrees of substitution for these
conjugates were optimized so that each resulted in the brightest, most
stable labeled antibody.

Unfortunately, I was not able to quickly find the information for the
quantum yield of the free dye in solution as compared to fluorescein.  I
will happily provide this information tomorrow afternoon.

>   2. What are the peak excitation and emission wavelengths?

For fluorescein (free dye) in a phosphate buffer at pH 8.4, the peak
excitation occurs at 491 nm, and emission is at 515 nm.

For BODIPY FL (free dye) in the same buffer, the peak excitation is at
503 nm and emission is at 512 nm.

>   3. If the peak emission is given a (normalized) value of one, what is the
> relative amount of emission at 560nm? at 600nm? It would be great if you
> could directly compare Bodipy FL values to fluorescein (at say pH 7-7.5).

Comparing the same free dye solutions described above:

When fluorescein is excited at its absorption peak (491 nm), if the
intensity of the emission peak (515 nm) is normalized to 1.0, then the
intensity at 560 nm is 0.22 and that at 600 nm is 0.04.

When BODIPY FL is excited at its absorption peak (503 nm), if the
intensity of the emission peak (512 nm) is normalized to 1.0, then the
intensity at 560 nm is 0.10 and that at 600 nm is 0.02.

The BODIPY FL fluorescence at waavelengths significantly greater than 550
nm is half or less than half of the corresponding fluorescein intensity
at the same wavelength.

>   4. If the peak excitation is normalized to 1, what is the relative
> excitation efficiency at 488nm?

When the fluorescein and BODIPY FL excitation peaks are normalized to
1.0, the relative excitation efficiencies at 488 nm are:

Fluorescein:  488 nm/491 nm = 95.5%
BODIPY FL:  488 nm/503 nm = 58.79%

>   5. Do any antibody vendors plan on adding this fluorophore to their
> armament?
>
There are other antibody vendors interested in these dyes, but at present
we are the only source of these conjugates.

We sell antibodies conjugated to BODIPY FL and a number of other dyes, as
well as anti-BODIPY FL antibodies.  We also sell kits and the free dye so
that you can do the conjugation yourself in your lab.

Unfortunately, the BODIPY dyes in these kits are quenched on conjugation to
many proteins (it is not a problem on oligonucleotides or peptides such
as phalloidin, however).  Molecular Probes uses special reactive BODIPY
derivatives to prepare BODIPY FL protein conjugates.  The reactive
derivatives, however, are not sufficiently stable for commercial sale.
We are still working to develop stable reactive derivatives that will
attenuate protein-induced quenching.  Molecular Probes does provide a
custom conjugation service using the proprietary chemistry we have
developed to prepare BODIPY FL conjugates.

Our technical assistance department would be delighted to help you with
more questions about BODIPY FL dye and conjugates.  They can be contacted
by phone at 503-465-8300, by fax at 503-344-6504, and by email at
tech@probes.mhs.compuserve.com.

I hope you find this information useful.
Jennifer Kramer
Molecular Probes, Inc.


>         Thanks for any information which you can offer.
>
> Chip Montrose
> Johns Hopkins University
>