http://confocal-microscopy-list.275.s1.nabble.com/Well-liked-405-secondary-antibodies-tp7589467p7589484.html
Published ext. coef. & quantum yield values from BD Biosciences:
> *****
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>
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> *****
>
> Hi Jeff,
>
> Excellent post.
>
> BD business model limitation ... can be dealt with. And the fluorescence
> moicroscopy community would be well served by using as many "flow
> cytometry" compatible reagents as possible.
>
> BD Biosciences and BioLegend each sell BV421 streptavidin conjugates
>
>
>
http://www.bdbiosciences.com/us/reagents/research/antibodies-buffers/second-step-reagents/avidinstreptavidin/bv421-streptavidin/p/563259>
>
>
https://www.biolegend.com/en-us/products/brilliant-violet-421-streptavidin-7297>
> I note that streptavidin has four binding sites for biotin, so may lose
> some efficiency in conjugation = need to optimize amount of BV-SA :
> biotin-mAb ... ideally get 1:1 conjugation, but if a complex forms.
>
> Maybe best counter to BD's business model is to use Fab (or scFv)
> instead of full length IgG. Or VHH nanobodies.
>
> enjoy,
>
> George
>
>
> On 5/2/2019 7:02 PM, Jeff Carmichael wrote:
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> > Post images on
http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Craig,
> >
> > You've probably heard some of this from me, and I know that George
> McNamara
> > has also been impressed with and written of the Brilliant Horizon fluors
> > from BD (BD owns this from Sirigen purchase).
> >
> > Here's a reference using Brilliant Violet 421 & Brilliant Violet 480:
> >
> > "Multilocus Imaging of the E. coli Chromosome by Fluorescent In Situ
> > Hybridization."
> >
>
https://www.researchgate.net/publication/319289637_Multilocus_Imaging_of_the_E_coli_Chromosome_by_Fluorescent_In_Situ_Hybridization> >
> > BV421 is approx. 20-40-fold brighter than AF405. BV480 is also approx.
> > 25-fold brighter than CFP (depending on who is measuring...)
> >
> > The problem remains the availability of reagents, as BD has decided that
> > their business model is better served by reserving these reagents for
> their
> > custom-conjugated Ab's for flow.....But I believe that BD still
> entertains
> > custom requests for their reagents.
> >
> > IMHO, directly conjugated, primary Ab's are the best way to leverage
> these
> > fluors because they're so bright that they don't need amplification. This
> > removes the problem of species specificity....Why wouldn't one try
> this???
> >
> > Here's a link to a presentation that illustrates the efficacy of this
> > approach of filling the gap of violet & blue fluors. Given the headlong
> > stampede towards increased multiplexing, I don't understand how these
> > fluors could be overlooked:
> >
https://www.chroma.com/5-channel-fluorescence-imaging-simplified> >
> > I hesitate using the "COMMERCIAL RESPONSE" qualifier, because Chroma
> > Technology has no formal relationship with BD Biosciences and receives no
> > credit for purchases of related products. Chroma Technology profits only
> > from sales of filter sets which are used to image these fluors.
> >
> > Jeff
> >
> > *Jeff Carmichael*
> >
> > *Director of Marketing*
> >
> > *
[hidden email] <
[hidden email]> | 802-428-2528*
> >
> >
> >
> > On Thu, May 2, 2019 at 4:47 PM Craig Brideau <
[hidden email]>
> > wrote:
> >
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> >> Post images on
http://www.imgur.com and include the link in your
> posting.
> >> *****
> >>
> >> What ever happened to Brilliant Violet? I remember a bit of marketing
> about
> >> it a while ago, but never got around to trying it myself. Did anyone
> get a
> >> chance to give it a go?
> >>
> >> Craig
> >>
> >> On Thu, May 2, 2019 at 2:27 PM Joe Lebowitz <
[hidden email]>
> wrote:
> >>
> >>> *****
> >>> To join, leave or search the confocal microscopy listserv, go to:
> >>>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> >>> Post images on
http://www.imgur.com and include the link in your
> >> posting.
> >>> *****
> >>>
> >>> Hello all,
> >>>
> >>> We have had some reproducibly disappointing results with Alexa Fluor
> 405
> >>> tagged secondary antibodies.
> >>>
> >>> Does anyone have a go-to secondary in this color other than the AF? We
> >> are
> >>> trying to multiplex as many channels as possible and this is the only
> one
> >>> we
> >>> struggle to get good brightness/SNR with.
> >>>
> >>> Thanks!
> >>>
> >>> joe
> >>>
> >>> --------------------------------
> >>>
> >>> Joe Lebowitz
> >>> Ph.D. Candidate
> >>> NINDS Pre-doctoral Fellow
> >>> Khoshbouei Lab
> >>> UF Department of Neuroscience
> >>> (561) 504-3810
> >>>
> >>>
> >>>
>