Re: Well-liked 405 secondary antibodies **COMMERCIAL RESPONSE**

Posted by Jeffrey Carmichael on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Well-liked-405-secondary-antibodies-tp7589467p7589487.html

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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:

> *****
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> 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
> >>> [phone]
> >>>
> >>>
> >>>
>
> --
> Martin Wessendorf, Ph.D.                   office: [phone]
> Assoc Prof, Dept Neuroscience                 lab: [phone]
> University of Minnesota             Preferred FAX: [phone]
> 6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
> Minneapolis, MN  55455                    e-mail: [hidden email]
> My preferred pronouns are "he", "him", and "his"
>

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