http://confocal-microscopy-list.275.s1.nabble.com/Well-liked-405-secondary-antibodies-tp7589467p7589488.html
Thanks for the interesting discussion. Interesting to note the potential
polymer. How does this compare to the coating chemistry of quantum dots?
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
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> *****
>
> Martin,
>
> Yes, I've had the misfortune of ruining perfectly good primary antibodies
> in conjugation reactions that resulted in labeled antibodies with excellent
> fluorophore:protein ratios, but no remainig antigenicity.
>
> So yes, this is largely hypothetical, but I do know that in the case of the
> reference I provided, BD Biosciences did offer assistance with the
> conjugation. I'm not sure if the author (David Bates) required the
> assistance or not, and I believe he conjugated oligos and not antibodies,
> but I believe BD will offer conjugation assistance, as the chemistry with
> these polymers can be challenging.
>
> My own take on the extra time is pick your poison....lots of preferred ways
> of doing things take more time, and it seems like many folks have a
> protocol or two that they are obsessive about. In the end it seems worth
> the effort to me. If imaging is the approach a worker has chosen to focus
> on to obtain some highly multiplexed data, then it seems that using the
> best tools is worth the trouble considering all of the time/effort going
> into doing the work at the bench, preparing samples/slides, acquiring the
> images, processing the images and then spending the time/effort doing image
> analysis. Being able to exploit this violet and blue fluorescence gap in
> the spectrum seems like an obvious approach to improve the reach of
> imaging.
>
> If the data is simply supporting in nature, and not central to the work,
> then one probably wouldn't do the extra work. It also seems that in this
> age of reproducibility crisis, better tools like these may be part of the
> solution.
>
> Jeff
>
> On Fri, May 3, 2019 at 10:38 AM Martin Wessendorf <
[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.
> > *****
> >
> > Hey, Jeff--
> >
> > As I see it, the main reasons for NOT going with directly conjugated
> > primary antibodies are (1) the extra time and effort required to perform
> > the conjugation and clean up the product and (2) the fact that in
> > principle, conjugation can alter the specificity of an antibody, which
> > would require repeating its characterization. However, the latter is a
> > theoretical issue rather than one that (to my knowledge) has been
> > documented. Anybody have data on this point? I've found nothing.
> >
> > Martin Wessendorf
> >
> >
> >
> >
> > On 5/2/2019 6:02 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
> > >>>
> > >>>
> > >>>
> >
> > --
> > Martin Wessendorf, Ph.D. office: (612) 626-0145
> > Assoc Prof, Dept Neuroscience lab: (612) 624-2991
> > University of Minnesota Preferred FAX: (612) 624-8118
> > 6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009
> > Minneapolis, MN 55455 e-mail:
[hidden email]
> > My preferred pronouns are "he", "him", and "his"
> >
>
> --
> <
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> *an employee owned
> company*
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>