TAMRA use in acceptor photobleaching FRET

[Ben Abrams]

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Hi All,

I have recently been helping someone with a FRET experiment to verify
(yes/no) that two purified proteins are forming a complex. The donor is
FITC, the acceptor is TAMRA and we've been doing the experiment on a
Leica SP5 using the FRET-acceptor-photobleaching module.  We've gotten
reproducible efficiencies ~ 0.4 with a racemic mixture of the proteins
(n>10) and 0 for the FITC-conjugate alone.  However, when we tried the
TAMRA-conjugate alone as a negative control we see bright green
fluorescence in the donor channel after bleaching and "efficiencies"
~0.98.  This has been a surprise and I am not finding much discussion of
TAMRA being photo-convertible, but I'm not sure what else could explain
this. Has anyone else out there seen this behavior with TAMRA?  We're
thinking of relabeling the acceptor with Rhodamine 6G or B, but are not
sure either is chemically distinct enough to behave differently.   Does
anyone have any suggestions or explanation?

Thanks,
Ben

--
Benjamin Abrams,  Ph.D.
Facility Manager
UCSC Life Sciences Microscopy Center

University of California, Santa Cruz
1156 High Street
150 Sinsheimer labs
Santa Cruz, CA 95064

Office/voicemail: [phone]

http://stemcell.soe.ucsc.edu/facilities/microscopy

Training Request Form:
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[George McNamara]

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Hi Ben,

Consider replacing TAMRA with JF549 (reactive dye)

A general method to improve fluorophores for live-cell and
single-molecule microscopy.
Grimm JB, English BP, Chen J, Slaughter JP, Zhang Z, Revyakin A, Patel
R, Macklin JJ, Normanno D, Singer RH, Lionnet T, Lavis LD.
Nat Methods. 2015 Mar;12(3):244-50, 3 p following 250. doi:
10.1038/nmeth.3256.
PMID: 25599551

Price may be nice (maybe the cost of one email to Luke Lavis),

https://www.janelia.org/open-science/janelia-fluor-dyes

or you may be redirected to

https://www.tocris.com/dispprod.php?ItemId=524906#.WL3mvTvyuUk

Fluorescent dye; supplied as an NHS ester for coupling to primary amine
groups. Compatible with self-labeling tag systems,/e.g./HaloTag^® and
SNAP-tag^® . Suitable for confocal fluorescent imaging and super
resolution microscopy (SRM) techniques, such as dSTORM (live and fixed
cells). Cell permeable. Excitation maximum = 549 nm; emission maximum =
571 nm; Quantum yield = 0.88; Extinction coefficient = 101,000 M^-1 cm^-1 .


You may also find replacing fluorescein (the ITC should be long gone
before you image!) with JF646,

https://www.tocris.com/dispprod.php?ItemId=524907#.WL3nDDvyuUk

Fluorescent dye; supplied as an NHS ester for coupling to primary amine
groups. Compatible with self-labeling tag systems (/e.g./HaloTag^® and
SNAPtag^®). Suitable for confocal fluorescent imaging, super resolution
microscopy (SRM) techniques such as dSTORM (live and fixed cells) and
PAINT imaging. Can be multiplexed for two colour imaging withJanelia
Fluor^TM 549 SE <https://www.tocris.com/dispprod.php?ItemId=524906>(Cat.
No. 6147). Cell permeable. Excitation maximum = 646 nm; emission maximum
= 664 nm. Quantum yield = 0.54, Extinction coefficient = 152,000 M^-1
cm^-1 .

//

JF549 is synthesized and purified to be single isomer. My thanks to Jin
Wang, Baylor College of Medicine, for hosting Luke for a seminar in
Houston recently, and to Luke for speaking.


best wishes,
George


On 3/6/2017 4:19 PM, Ben Abrams 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 All,
>
> I have recently been helping someone with a FRET experiment to verify
> (yes/no) that two purified proteins are forming a complex. The donor
> is FITC, the acceptor is TAMRA and we've been doing the experiment on
> a Leica SP5 using the FRET-acceptor-photobleaching module.  We've
> gotten reproducible efficiencies ~ 0.4 with a racemic mixture of the
> proteins (n>10) and 0 for the FITC-conjugate alone.  However, when we
> tried the TAMRA-conjugate alone as a negative control we see bright
> green fluorescence in the donor channel after bleaching and
> "efficiencies" ~0.98.  This has been a surprise and I am not finding
> much discussion of TAMRA being photo-convertible, but I'm not sure
> what else could explain this. Has anyone else out there seen this
> behavior with TAMRA? We're thinking of relabeling the acceptor with
> Rhodamine 6G or B, but are not sure either is chemically distinct
> enough to behave differently.   Does anyone have any suggestions or
> explanation?
>
> Thanks,
> Ben
>

--


George McNamara, PhD
Houston, TX 77054
[hidden email]
https://www.linkedin.com/in/georgemcnamara
https://works.bepress.com/gmcnamara/75/
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650

[Aryeh Weiss]

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On 07/03/2017 0:19, Ben Abrams 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 All,
>
> I have recently been helping someone with a FRET experiment to verify
> (yes/no) that two purified proteins are forming a complex. The donor
> is FITC, the acceptor is TAMRA and we've been doing the experiment on
> a Leica SP5 using the FRET-acceptor-photobleaching module.  We've
> gotten reproducible efficiencies ~ 0.4 with a racemic mixture of the
> proteins (n>10) and 0 for the FITC-conjugate alone.  However, when we
> tried the TAMRA-conjugate alone as a negative control we see bright
> green fluorescence in the donor channel after bleaching and
> "efficiencies" ~0.98.  This has been a surprise and I am not finding
> much discussion of TAMRA being photo-convertible, but I'm not sure
> what else could explain this. Has anyone else out there seen this
> behavior with TAMRA? We're thinking of relabeling the acceptor with
> Rhodamine 6G or B, but are not sure either is chemically distinct
> enough to behave differently.   Does anyone have any suggestions or
> explanation?

TAMRA photoconverts to green. We saw this in our lab, and also saw
strong photoconversion with at least one of the orange bodipy dyes. If
you do acceptor photobleaching, you always have to heck for
photconversion of the acceptor.

--aryeh

--
Aryeh Weiss
Faculty of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph:  972-3-5317638
FAX: [phone]

[Ben Abrams]

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Hi Aryeh,

Thanks for your reply, I will certainly always check for conversion in the
future.

Best,
Ben



On 3/6/17 8:50 PM, Aryeh Weiss wrote:

> On 07/03/2017 0:19, Ben Abrams 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 All,
>>
>> I have recently been helping someone with a FRET experiment to verify
>> (yes/no) that two purified proteins are forming a complex. The donor
>> is FITC, the acceptor is TAMRA and we've been doing the experiment on
>> a Leica SP5 using the FRET-acceptor-photobleaching module.  We've
>> gotten reproducible efficiencies ~ 0.4 with a racemic mixture of the
>> proteins (n>10) and 0 for the FITC-conjugate alone.  However, when we
>> tried the TAMRA-conjugate alone as a negative control we see bright
>> green fluorescence in the donor channel after bleaching and
>> "efficiencies" ~0.98.  This has been a surprise and I am not finding
>> much discussion of TAMRA being photo-convertible, but I'm not sure
>> what else could explain this. Has anyone else out there seen this
>> behavior with TAMRA? We're thinking of relabeling the acceptor with
>> Rhodamine 6G or B, but are not sure either is chemically distinct
>> enough to behave differently. Does anyone have any suggestions or
>> explanation?
> TAMRA photoconverts to green. We saw this in our lab, and also saw
> strong photoconversion with at least one of the orange bodipy dyes. If
> you do acceptor photobleaching, you always have to heck for
> photconversion of the acceptor.
>
> --aryeh
>

--
Benjamin Abrams,  Ph.D.
Facility Manager
UCSC Life Sciences Microscopy Center

University of California, Santa Cruz
1156 High Street
150 Sinsheimer labs
Santa Cruz, CA 95064

Office/voicemail: [phone]

http://stemcell.soe.ucsc.edu/facilities/microscopy

Training Request Form:
http://goo.gl/forms/M9jd1ZHyohTnj0xk1