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

Posted by 0000001ed7f52e4a-dmarc-request on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Well-liked-405-secondary-antibodies-tp7589467p7589479.html

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Interesting topic.

BD Biosciences and BioLegend each sell BV421 streptavidin conjugates 


Does this work? From my days in chemistry I remember that streptavidin sticks to almost anything, not what you want to combine with your highly specific antibody. Isn't t this why they developed neutravidin?

Maybe best counter to BD's business model is to use Fab (or scFv)
 instead of full length IgG. Or VHH nanobodies.


We used single Fab a lot for single molecule tracking and STORM. But they come off very easily, while you have less problems with inaccessible binding sites, labelling can be quite inefficient.
Best wishes
Andreas


-----Original Message-----
From: George McNamara <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Fri, 3 May 2019 5:08
Subject: Re: Well-liked 405 secondary antibodies **COMMERCIAL RESPONSE**

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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:

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>
> 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:
>
>> *****
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>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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>> *****
>>
>> 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:
>>
>>> *****
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>> 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]
>>>
>>>
>>>