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Alexa Fluor 405 Antibody

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Scott Wong

Alexa Fluor 405 Antibody

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Hi everyone,
I'm looking at getting some new antibodies and the Alexa Fluor 405
secondary lines up well with the filters and laser lines I have available.
Does anyone have experience with it, good or bad?
Thanks for the help,
Scott Wong

Romin, Yevgeniy/Sloan Kettering Institute

Re: Alexa Fluor 405 Antibody

> Mar 27, 2014, в 6:54 PM, "Wong, Scott" <[hidden email]> написал(а):
>
> *****
> 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.
> *****
>
> Hi everyone,
> I'm looking at getting some new antibodies and the Alexa Fluor 405
> secondary lines up well with the filters and laser lines I have available.
> Does anyone have experience with it, good or bad?
> Thanks for the help,
> Scott Wong
>

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Armstrong, Brian

Re: Alexa Fluor 405 Antibody

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Hi Scott, I agree that it is "pretty dim". However, I still recommend it as a "blue" fluorophore because it is actually better than most other options within this spectra.
Cheers,

Brian D Armstrong PhD
Associate Research Professor
Director, Light Microscopy Core
Beckman Research Institute
City of Hope
Dept of Neuroscience
1450 E Duarte Rd
Duarte, CA 91010
626-256-4673 x62872

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Yevgeniy Romin
Sent: Thursday, March 27, 2014 4:10 PM
To: [hidden email]
Subject: Re: Alexa Fluor 405 Antibody

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*****

Judging by experience in our facility, it's pretty dim.

mg_info.txt (1K) Download Attachment

Kurt Thorn

Re: Alexa Fluor 405 Antibody

In reply to this post by Scott Wong

> *****
> 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.
> *****
>
> Hi everyone,
> I'm looking at getting some new antibodies and the Alexa Fluor 405
> secondary lines up well with the filters and laser lines I have available.
> Does anyone have experience with it, good or bad?

I've summarized some of what I've heard about it and other 405 nm
excited dyes here: http://nic.ucsf.edu/dokuwiki/doku.php?id=dyes:405nm
(this includes some previous listserv discussion).

Kurt

> Thanks for the help,
> Scott Wong
>
>

--
Kurt Thorn
Director, Nikon Imaging Center
http://nic.ucsf.edu/blog/

Avi Perna

Re: Alexa Fluor 405 Antibody

In reply to this post by Romin, Yevgeniy/Sloan Kettering Institute

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**commercial interest disclosure**

Hi Scott,

You might check out the Brilliant Violet 421 conjugates as well. The ex/em profile is similar to other 405nm excited organic fluors, where BV421 is generally brighter and more resistant to photo-bleaching. I believe the first characterization and comparisons for imaging applications were described in Cytometry Part A.

http://onlinelibrary.wiley.com/enhanced/doi/10.1002/cyto.a.22043/#Survey

Kind regards,

Avi

Avi Perna
Applications Specialist
BioLegend
[hidden email]

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Yevgeniy Romin
Sent: Thursday, March 27, 2014 4:10 PM
To: [hidden email]
Subject: Re: Alexa Fluor 405 Antibody

*****
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.
*****

Judging by experience in our facility, it's pretty dim.

George McNamara

Re: Alexa Fluor 405 Antibody

In reply to this post by Kurt Thorn

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Brightness of a fluorophore is determined by extinction coefficient and
quantum yields of fluorescence and non-radiative decay pathways. Just
having excitation and emission maximum is not sufficient. Kevin
Belfield's lab is of the few who have quantified non-radiative decay
pathways with real photochemistry rates.

Brilliant Violet BV421 was very bright on my core facility's widefield
microscope (Zeiss Axiovert 200, Hg lamp, standard Zeiss DAPI cube) and
would be a lot brighter with optimized filter set.

As Kurt cites on his web page, the link
https://www.biolegend.com/brilliantviolet has a lot of information. The
"Intro" section
https://www.biolegend.com/brilliantviolet#intro
has details. Specifically for BV421:
Excitation max: 405 nm
Emission max: 421 nm
Molar extinction coefficient (Ec) = 2,500,000 M-1cm-1
Quantum yield = 0.6 in DPBS

For brightness, I usually use the formula (which I believe I originally
saw in Tsien, Ernst, Waggoner 1995 chapter in Pawley - I don't have the
book anymore):

B = Ec * QY / 1000

so,

BV421, B = 1500

This is comparable to several of the commercial quantum dots, that have
not had a big impact in our field. This is not to say that QDots are a
complete bust: Keith Lidke gave a nice talk at ABRF doing fast 8plex
QDots single molecules tracking on EGFR's - see Cutler 2013 for their
published work, http://www.ncbi.nlm.nih.gov/pubmed/23717596

For comparison,

Fluorescein, B = 0.9 * 90,000 = 81 (and undergoes NRD to non-fluorescent
molecules).

Information on BV421, its tandem acceptors, BV510, and the new BUV395
are available in

https://www.bdbiosciences.com/documents/webinar_071713_multicolor-bv.pdf

http://www.bdbiosciences.com/documents/BD_Brilliant_Violet395_Datasheet.pdf
and
https://www.bdbiosciences.com/documents/Horizon-Brilliant-Ultraviolet-DS-Reagents.pdf
... this also has BUV737 tandem.

BD has not posted Ec and QY for the BUV's, but I was told by a
BD/Sirigen researcher that the BUV395 Ec and QY are comparable to BV421.

PubSpectra has Ec and QY values for some fluorophores -
http://works.bepress.com/gmcnamara/9/

//

At ABRF this week I met Tom DiMatteo, whose new company, Epi Technology,
http://www.epitechnology.com/ has a product that promises to show
everyone how bad our widefield microscope epi-illumination paths are. My
thanks to Alison North for encouraging Tom to exhibit at the meeting.

I encourage everyone doing live cell fluorescence imaging to check out
Yuan, Shermoen and O'Farrell 2014's TALE-Lights paper:

http://www.ncbi.nlm.nih.gov/pubmed/24556431
Open access (thanks Pat!):
http://download.cell.com/current-biology/pdf/PIIS0960982214000244.pdf?intermediate=true
I encourage making popcorn before watching the videos:
http://www.cell.com/current-biology/supplemental/S0960-9822(14)00024-4

More on this topic can be found at
http://works.bepress.com/gmcnamara/42/ ... newer
http://works.bepress.com/gmcnamara/26 ... older

Financial disclosures: I have no financial connection with BD or Sirigen
or the QDot manufacturer. My only connection with Epi Technology will be
as a customer lab - as soon as I get a formal quote to order a CaliCube
for our lab's Leica DMI6000B microscope. I have never made any money on
PubSpectra and will happily deposit any thank you checks. Tattletales
was put in the public domain in October 2013
http://home.earthlink.net/~pubspectra/McNamara_20121023Tue_Tattletales_GFP_Public_Domain.jpg

(announced first here), and I welcome thank you checks for it and T-Bow too.

Enjoy,

George

On 3/27/2014 6:59 PM, Kurt Thorn 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.
> *****
>
> On 3/27/2014 3:51 PM, Wong, Scott 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.
>> *****
>>
>> Hi everyone,
>> I'm looking at getting some new antibodies and the Alexa Fluor 405
>> secondary lines up well with the filters and laser lines I have
>> available.
>> Does anyone have experience with it, good or bad?
>
> I've summarized some of what I've heard about it and other 405 nm
> excited dyes here: http://nic.ucsf.edu/dokuwiki/doku.php?id=dyes:405nm
> (this includes some previous listserv discussion).
>
> Kurt
>
>> Thanks for the help,
>> Scott Wong
>>
>>
>
>

--

George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/26/

rjpalmer

Re: Alexa Fluor 405 Antibody

In reply to this post by Scott Wong

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*****

We use labeled primaries. I love AFs in general, and we've tried this flavor more than once. Poor coupling and/or weak photon flux (blue diode emission). Your mileage may vary.

Robert J. Palmer Jr., Ph.D.
Microbial Receptors Section
Laboratory of Cell and Developmental Biology
Natl Inst Dental Craniofacial Res - Natl Insts Health
Bldg 30, Room 207
30 Convent Drive
Bethesda MD 20892
ph 301-594-0025
fax 301-480-0112

On Mar 27, 2014, at 6:51 PM, Wong, Scott 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.
> *****
>
> Hi everyone,
> I'm looking at getting some new antibodies and the Alexa Fluor 405
> secondary lines up well with the filters and laser lines I have available.
> Does anyone have experience with it, good or bad?
> Thanks for the help,
> Scott Wong

lechristophe

Re: Alexa Fluor 405 Antibody

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*****

I usually recommend DyLight 405 over Alexa Fluor 405 for secondary
antibodies. I don't have a direct comparison, but DyLight 406 is usable.

Christophe

2014-03-28 13:43 GMT+01:00 Rob Palmer <[hidden email]>:

> *****
> 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.
> *****
>
> We use labeled primaries. I love AFs in general, and we've tried this
> flavor more than once. Poor coupling and/or weak photon flux (blue diode
> emission). Your mileage may vary.
>
> Robert J. Palmer Jr., Ph.D.
> Microbial Receptors Section
> Laboratory of Cell and Developmental Biology
> Natl Inst Dental Craniofacial Res - Natl Insts Health
> Bldg 30, Room 207
> 30 Convent Drive
> Bethesda MD 20892
> ph 301-594-0025
> fax 301-480-0112
>
> On Mar 27, 2014, at 6:51 PM, Wong, Scott 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.
> > *****
> >
> > Hi everyone,
> > I'm looking at getting some new antibodies and the Alexa Fluor 405
> > secondary lines up well with the filters and laser lines I have
> available.
> > Does anyone have experience with it, good or bad?
> > Thanks for the help,
> > Scott Wong
>

Eric Griffis

Re: Alexa Fluor 405 Antibody

In reply to this post by Scott Wong

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*****

We've had good luck with Atto 425. Comparing phalloidins, Atto 425 significantly outperformed AlexaFluor-405 and CF-405 for OMX. However, the big caveat is that the preparation of the phalloidin probably plays as big of a role as the qualities of the fluor.

Best regards,

Eric

On 27 Mar 2014, at 22:51, "Wong, Scott" <[hidden email]<mailto:[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.
*****

Hi everyone,
I'm looking at getting some new antibodies and the Alexa Fluor 405
secondary lines up well with the filters and laser lines I have available.
Does anyone have experience with it, good or bad?
Thanks for the help,
Scott Wong

The University of Dundee is a registered Scottish Charity, No: SC015096

Alison J. North

Re: Alexa Fluor 405 Antibody

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*****

Hi,

I guess we all have different experiences! As discussed on a different
listserver recently, I just tested out a load of blue dyes on our OMX
and I found Atto 425 to be hopeless with our 405 laser/filter
combination - at least the conjugated antibody that I was using. But I
know other people have found it to be good. CF405M is good in our hands
as long as you use a low illumination intensity - otherwise there is
significant photobleaching. And CF405S is bright but I found that in
several mountants, including Prolong Gold, the second you hit the sample
with the 405 laser it photoconverts the dye into a green-emitting form.
The problem was much less obvious in Vectashield but is still a concern
for multiple labelling experiments. I certainly prefer CF405M over AF405.

Hope you find one that works for you!
Alison

On 3/28/2014 9:01 AM, Eric Griffis 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.
> *****
>
> We've had good luck with Atto 425. Comparing phalloidins, Atto 425 significantly outperformed AlexaFluor-405 and CF-405 for OMX. However, the big caveat is that the preparation of the phalloidin probably plays as big of a role as the qualities of the fluor.
>
> Best regards,
>
> Eric
>
>
>
>
> On 27 Mar 2014, at 22:51, "Wong, Scott" <[hidden email]<mailto:[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.
> *****
>
> Hi everyone,
> I'm looking at getting some new antibodies and the Alexa Fluor 405
> secondary lines up well with the filters and laser lines I have available.
> Does anyone have experience with it, good or bad?
> Thanks for the help,
> Scott Wong
>
>
> The University of Dundee is a registered Scottish Charity, No: SC015096

--
Alison J. North, Ph.D.,
Senior Director of the Bio-Imaging Resource Center and
Research Associate Professor,
The Rockefeller University,
1230 York Avenue,
New York,
NY 10065.
Tel: office ++ 212 327 7488
Tel: lab ++ 212 327 7486
Fax: ++ 212 327 7489

Scott Wong

Re: Alexa Fluor 405 Antibody

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

>Thanks so much for the information and links everyone, there's a lot of
good background information in them. It turns out that there is some
Alexa Fluor 405 that materialized at my work so I'm going to try a few
test runs with it. If it doesn't work well I'll look into some of the
other suggestions that were mentioned.
Cheers,
Scott

*****

> 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.
> *****
>
> Hi,
>
> I guess we all have different experiences! As discussed on a different
> listserver recently, I just tested out a load of blue dyes on our OMX
> and I found Atto 425 to be hopeless with our 405 laser/filter
> combination - at least the conjugated antibody that I was using. But I
> know other people have found it to be good. CF405M is good in our hands
> as long as you use a low illumination intensity - otherwise there is
> significant photobleaching. And CF405S is bright but I found that in
> several mountants, including Prolong Gold, the second you hit the sample
> with the 405 laser it photoconverts the dye into a green-emitting form.
> The problem was much less obvious in Vectashield but is still a concern
> for multiple labelling experiments. I certainly prefer CF405M over
> AF405.
>
> Hope you find one that works for you!
> Alison
>
>
> On 3/28/2014 9:01 AM, Eric Griffis 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.
>> *****
>>
>> We've had good luck with Atto 425. Comparing phalloidins, Atto 425
>> significantly outperformed AlexaFluor-405 and CF-405 for OMX. However,
>> the big caveat is that the preparation of the phalloidin probably plays
>> as big of a role as the qualities of the fluor.
>>
>> Best regards,
>>
>> Eric
>>
>>
>>
>>
>> On 27 Mar 2014, at 22:51, "Wong, Scott"
>> <[hidden email]<mailto:[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.
>> *****
>>
>> Hi everyone,
>> I'm looking at getting some new antibodies and the Alexa Fluor 405
>> secondary lines up well with the filters and laser lines I have
>> available.
>> Does anyone have experience with it, good or bad?
>> Thanks for the help,
>> Scott Wong
>>
>>
>> The University of Dundee is a registered Scottish Charity, No: SC015096
>
> --
> Alison J. North, Ph.D.,
> Senior Director of the Bio-Imaging Resource Center and
> Research Associate Professor,
> The Rockefeller University,
> 1230 York Avenue,
> New York,
> NY 10065.
> Tel: office ++ 212 327 7488
> Tel: lab ++ 212 327 7486
> Fax: ++ 212 327 7489
>