Best FRET pair
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Dear all,
We are planning to do FRET experiments with a fluorescent protein (YFP, mCherry or mOrange)and a directly labelled primary antibody. We are restricted to YFP, mCherry or mOrange for one of the proteins (the most abundant). Does anyone know what's the best FRET pair for any of these FPs, in terms of J (overlap), brightness ? Thanks for your help, Maria Calvo -- ___________________________________ Dra. Maria Calvo Unitat de Microscòpia Confocal Serveis Cientificotècnics-C.Casanova Facultat de Medicina Universitat de Barcelona- IDIBAPS C/ Casanova 143 Barcelona 08036 Tel: 34 934037159/39930 Fax: 34 934039946 E-mail: [hidden email] ___________________________________ |
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YFP-mOrange should work for you
We are using GFP-mOrange as FRET pair. You should have a better quantum yield for the donor compared to acceptor. Ammasi -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Maria Calvo Sent: Friday, March 26, 2010 11:48 AM To: [hidden email] Subject: Best FRET pair Dear all, We are planning to do FRET experiments with a fluorescent protein (YFP, mCherry or mOrange)and a directly labelled primary antibody. We are restricted to YFP, mCherry or mOrange for one of the proteins (the most abundant). Does anyone know what's the best FRET pair for any of these FPs, in terms of J (overlap), brightness ? Thanks for your help, Maria Calvo -- Ammasi Periasamy, Ph.D. Director, Keck Center for Cellular Imaging (KCCI) Professor of Biology and Biomedical Engineering Biology, Gilmer Hall (064), 485 McCormick Rd University of Virginia Charlottesville, VA 22904 Voice: 434-243-7602 (Office); 982-4869 (lab) Fax:434-982-5210; Email:[hidden email] http://www.kcci.virginia.edu ************************ 10th Annual Workshop on FRET Microscopy, March 8-12, 2011 http://www.kcci.virginia.edu/workshop/workshop2011/index.php *************************________ |
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Dear Ammasi,
Is that so important? CeFP-YFP is a great pair, though the quantum yield of the CeFP could be better, and it works better than the YFP-mRFP1 pair for example. Though I did not have a chance to compare that to GFP-mOrange or GFP-mRFP1. YFP-mRFP1 pair works quite good, nearly ten-fold better than the YFP-mCherry one. Having an optimal filter set is very important!, e.g. to keep the donor/FRET bleedthrough much below 40%.
Vitaly
301-515-7833 From: "Periasamy, Ammasi (ap3t)" <[hidden email]> To: [hidden email] Sent: Fri, March 26, 2010 1:54:49 PM Subject: Re: Best FRET pair YFP-mOrange should work for you We are using GFP-mOrange as FRET pair. You should have a better quantum yield for the donor compared to acceptor. Ammasi -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Maria Calvo Sent: Friday, March 26, 2010 11:48 AM To: [hidden email] Subject: Best FRET pair Dear all, We are planning to do FRET experiments with a fluorescent protein (YFP, mCherry or mOrange)and a directly labelled primary antibody. We are restricted to YFP, mCherry or mOrange for one of the proteins (the most abundant). Does anyone know what's the best FRET pair for any of these FPs, in terms of J (overlap), brightness ? Thanks for your help, Maria Calvo -- Ammasi Periasamy, Ph.D. Director, Keck Center for Cellular Imaging (KCCI) Professor of Biology and Biomedical Engineering Biology, Gilmer Hall (064), 485 McCormick Rd University of Virginia Charlottesville, VA 22904 Voice: 434-243-7602 (Office); 982-4869 (lab) Fax:434-982-5210; Email:[hidden email] http://www.kcci.virginia.edu ************************ 10th Annual Workshop on FRET Microscopy, March 8-12, 2011 http://www.kcci.virginia.edu/workshop/workshop2011/index.php *************************________ |
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In reply to this post by Maria Calvo-2
Dear all,
(and specially Ammasy and Vitaly, who answered my previous mail), Sorry if I didn't explain it well or I missed some details. We want to detect FRET by combining a fluorescent protein and a directly labelled primary antibody. The methods we are going to use are: sensitized emission and/or acceptor photobleaching (not lifetime imaging). 1) Fluorescent Proteins: We have only available the fusion protein constructs with YFP, mCherry or mOrange. 2) Fluorochromes: We will buy a kit to labell the primary antibody. We have to decide which fluorophore will be the best in combination with one of these fluorescent proteins. The combinations I thought were: -Donor - Acceptor -YFP -Alexa 594 or TR -YFP - A555 or Cy3 -mCherry - Cy5 or A647 -mOrange - Cy5 or A647 -FITC or A488 - mOrange -FITC or A488 - mCherry Which do you think is the best combination in terms of J (overlap), brightness? Thank you for your invaluable help! Maria Calvo Maria Calvo escribió: > Dear all, > > We are planning to do FRET experiments with a fluorescent protein > (YFP, mCherry or mOrange)and a directly labelled primary antibody. > We are restricted to YFP, mCherry or mOrange for one of the proteins > (the most abundant). > Does anyone know what's the best FRET pair for any of these FPs, in > terms of J (overlap), brightness ? > > Thanks for your help, > > Maria Calvo > -- ___________________________________ Dra. Maria Calvo Unitat de Microscòpia Confocal Serveis Cientificotècnics-C.Casanova Facultat de Medicina Universitat de Barcelona- IDIBAPS C/ Casanova 143 Barcelona 08036 Tel: 34 934037159/39930 Fax: 34 934039946 E-mail: [hidden email] ___________________________________ |
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Hi Maria,
This is sheng, working for Ammasi. From the pair list, I would recommend A488-mOrange, at least for sensitized FRET, assuming you have optimal Ex sources and Em filters. I did a quick calculation of the Ro for this pair - 5.889 nm, based on the following photophysical properties of the two fluorophores: Alexa 488 (Invitrogen T13342): QY = 0.92 (Be aware that the Alexa QY will likely drop down a lot when it is conjugated) mOrange: QY = 0.69, EC = 71,000, Brightness to EGFP = 146%. I can send you the spectra of the two fluorophores if you want. The photostability of mOrange is very poor and this might be a good thing for apFRET. However, it has been reported that mOrange may have a photoconversion issue (Nat.Methods, 2009, 6, 5, 355-358). Please do your controls carefully. If you use lasers in sensitized FRET, I do not suggest the DPSS 561 nm laser for the acceptor excitation, because it will limit the detection range of your FRET channel. To be honest, I have no experience on the A488-mOrange pair. Based on the paper, it should work. Another pair to try would be YFP-A555. sheng On Wed, Apr 7, 2010 at 12:14 PM, Maria Calvo <[hidden email]> wrote: > Dear all, > (and specially Ammasy and Vitaly, who answered my previous mail), > > Sorry if I didn't explain it well or I missed some details. > > We want to detect FRET by combining a fluorescent protein and a directly > labelled primary antibody. The methods we are going to use are: sensitized > emission and/or acceptor photobleaching (not lifetime imaging). > > 1) Fluorescent Proteins: We have only available the fusion protein > constructs with YFP, mCherry or mOrange. > 2) Fluorochromes: We will buy a kit to labell the primary antibody. > > We have to decide which fluorophore will be the best in combination with one > of these fluorescent proteins. > > The combinations I thought were: > > -Donor - Acceptor > > -YFP -Alexa 594 or TR > -YFP - A555 or Cy3 > -mCherry - Cy5 or A647 > -mOrange - Cy5 or A647 > -FITC or A488 - mOrange > -FITC or A488 - mCherry > > Which do you think is the best combination in terms of J (overlap), > brightness? > > Thank you for your invaluable help! > > Maria Calvo > > > > Maria Calvo escribió: >> >> Dear all, >> >> We are planning to do FRET experiments with a fluorescent protein (YFP, >> mCherry or mOrange)and a directly labelled primary antibody. >> We are restricted to YFP, mCherry or mOrange for one of the proteins (the >> most abundant). >> Does anyone know what's the best FRET pair for any of these FPs, in terms >> of J (overlap), brightness ? >> >> Thanks for your help, >> >> Maria Calvo >> > > > -- > > > ___________________________________ > > Dra. Maria Calvo > > Unitat de Microscòpia Confocal > Serveis Cientificotècnics-C.Casanova > Facultat de Medicina > Universitat de Barcelona- IDIBAPS > C/ Casanova 143 > Barcelona 08036 > > Tel: 34 934037159/39930 > Fax: 34 934039946 > E-mail: [hidden email] > ___________________________________ > |
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