http://confocal-microscopy-list.275.s1.nabble.com/Well-liked-405-secondary-antibodies-tp7589467p7589489.html
problem when chemically conjugating anything to an antibody. My unfortunate
molecules. Here's a link to a paper in the Biophysical Journal (2018)
a conductive polymer vs. a ZnS QDot shell vs. small organic fluorophore vs.
> *****
> 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.
> *****
>
> Thanks for the interesting discussion. Interesting to note the potential
> change in binding efficacy when you attach the fluorophore in the case of a
> polymer. How does this compare to the coating chemistry of quantum dots?
>
> Craig
>
> On Fri, May 3, 2019 at 10:07 AM Jeff Carmichael <
[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.
> > *****
> >
> > 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"
> > >
> >
> > --
> > <
https://www.chroma.com/>CHROMA TECHNOLOGY CORP®
> > *an employee owned
> > company*
> > 10 Imtec Lane, Bellows Falls, Vermont 05101 USA
> > 800-824-7662 |
> > FAX: 802-428-2525
> > www.chroma.com <
https://www.chroma.com/> |
> >
[hidden email] <mailto:
[hidden email]>
> >
>