combination of 3 fluorescence protein reporters
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Xinyu Zhao-2 |
combination of 3 fluorescence protein reporters
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear listers, One of our users wanted to image CFP and YFP labeled cells and would like to add a third fluorecence protein reporter later. I was wondering if anybody could make a recomendation on this third one. Anybody has ever used a CFP/YFP/mCherry combination ? Thank you very much. Xinyu Zhao Biomedical Imaging Core Lab School of Medicine University of Pennsylvania Tel: 215-898-6730 |
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Sam's Mail |
Do I hear 4? was: combination of 3 fluorescence protein reporters
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal We and many others have successfully used the combination of Cerulean(CFP)/Venus(YFP)/mCherry when we have needed three genetically encoded fluorescent proteins. Anyone care to comment on successes using four? -- Samuel A. Connell Director of Light Microscopy Cell & Tissue Imaging Center St. Jude Children's Research Hospital 332 North Lauderdale St., E7061 Memphis, TN 38105-2794 (901) 495-2536 [hidden email] -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Xinyu Zhao Sent: Thursday, November 08, 2007 9:07 AM To: [hidden email] Subject: combination of 3 fluorescence protein reporters. . Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear listers, One of our users wanted to image CFP and YFP labeled cells and would like to add a third fluorecence protein reporter later. I was wondering if anybody could make a recomendation on this third one. Anybody has ever used a CFP/YFP/mCherry combination ? Thank you very much. Xinyu Zhao Biomedical Imaging Core Lab School of Medicine University of Pennsylvania Tel: 215-898-6730 |
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Stephen Bunnell |
Re: combination of 3 fluorescence protein reporters
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In reply to this post by Xinyu Zhao-2
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Xinyu, We have had good luck with that combination, as well as mCerulean/mYFP/mCherry and mCerulean/mYFP/mStrawberry. Both of the latter sets work well on our fixed dichroic Ultraview spinning disk system, which has a custom built quad-pass dichroic that we acquired from Chroma. We have no detectable bleedthough between these three channels using 442/514/568 nm laser lines for excitation. By the book, Cherry looks like it should be brighter and more stable, but Strawberry 'fits' our existing filter sets (based on DsRed) much better. I had expected that mVenus should be much better that YFP, but in my hands (and filter sets) it did not offer any significant advantages. It actually bleached _faster_ than mYFP under constant 514nm illumination. Best regards, -Steve **************************************************************************** Stephen C. Bunnell, Ph.D. Assistant Professor Tufts University Medical School Department of Pathology Jaharis Bldg., Room 512 150 Harrison Ave. Boston, MA 02111 Phone: (617) 636-2174 Fax: (617) 636-2990 Email: [hidden email] On 11/8/07 10:06 AM, "Xinyu Zhao" <[hidden email]> wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Dear listers, > > One of our users wanted to image CFP and YFP labeled cells and would like to > add a third fluorecence protein reporter later. I was wondering if anybody > could make a recomendation on this third one. > > Anybody has ever used a CFP/YFP/mCherry combination ? > > Thank you very much. > > > Xinyu Zhao > Biomedical Imaging Core Lab > School of Medicine > University of Pennsylvania > Tel: 215-898-6730 |
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Catherine Degnin |
Re: combination of 3 fluorescence protein reporters
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Do you have suggestions on the best way to fix cells expressing mCherry-tagged proteins? I get gorgeous images using live cells, but I lose a lot of signal using a fixation protocol that works beautifully with eGFP and YFP... Thanks, Catherine On Nov 8, 2007, at 8:10 AM, Stephen Bunnell wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Dear Xinyu, > > We have had good luck with that combination, as well as > mCerulean/mYFP/mCherry and mCerulean/mYFP/mStrawberry. Both of the > latter > sets work well on our fixed dichroic Ultraview spinning disk > system, which > has a custom built quad-pass dichroic that we acquired from Chroma. > We have > no detectable bleedthough between these three channels using > 442/514/568 nm > laser lines for excitation. By the book, Cherry looks like it > should be > brighter and more stable, but Strawberry 'fits' our existing filter > sets > (based on DsRed) much better. > > I had expected that mVenus should be much better that YFP, but > in my > hands (and filter sets) it did not offer any significant > advantages. It > actually bleached _faster_ than mYFP under constant 514nm > illumination. > > Best regards, > > -Steve > > ********************************************************************** > ****** > Stephen C. Bunnell, Ph.D. > Assistant Professor > Tufts University Medical School > Department of Pathology > Jaharis Bldg., Room 512 > 150 Harrison Ave. > Boston, MA 02111 > > Phone: (617) 636-2174 > Fax: (617) 636-2990 > Email: [hidden email] > > > On 11/8/07 10:06 AM, "Xinyu Zhao" <[hidden email]> wrote: > >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> Dear listers, >> >> One of our users wanted to image CFP and YFP labeled cells and >> would like to >> add a third fluorecence protein reporter later. I was wondering if >> anybody >> could make a recomendation on this third one. >> >> Anybody has ever used a CFP/YFP/mCherry combination ? >> >> Thank you very much. >> >> >> Xinyu Zhao >> Biomedical Imaging Core Lab >> School of Medicine >> University of Pennsylvania >> Tel: 215-898-6730 |
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Michael Schell |
Re: combination of 3 fluorescence protein reporters
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Catherine,
We observe the same decrease in mCherry fluorescence following standard 4% PFA fixation. One solution is to stain with an antibody to RFPs. We've been using a polyclonal from Rockland with excellent results. Works with mCherry and tdTomato. No x-reaction with GFP variants. No commercial interest. Michael Michael J. Schell, Ph.D Assist. Professor Dept. of Pharmacology Uniformed Services University 4301 Jones Bridge Rd. Bethesda, MD 20814-3220 tel: (301) 295-3249 On Nov 8, 2007, at 2:24 PM, Catherine Degnin wrote:
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George McNamara |
Re: combination of 3 fluorescence protein reporters
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In reply to this post by Xinyu Zhao-2
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal If your microscope can image Cy5 well, try mKate from www.evrogen.com At 10:06 AM 11/8/2007, you wrote: >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >Dear listers, > >One of our users wanted to image CFP and YFP labeled cells and would like to >add a third fluorecence protein reporter later. I was wondering if anybody >could make a recomendation on this third one. > >Anybody has ever used a CFP/YFP/mCherry combination ? > >Thank you very much. > > >Xinyu Zhao >Biomedical Imaging Core Lab >School of Medicine >University of Pennsylvania >Tel: 215-898-6730 George McNamara, Ph.D. University of Miami, Miller School of Medicine Image Core Miami, FL 33010 [hidden email] [hidden email] 305-243-8436 office http://home.earthlink.net/~pubspectra/ http://home.earthlink.net/~geomcnamara/ http://www.sylvester.org/health_pro/shared_resources/index.asp (see Analytical Imaging Core Facility) |
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Adrian Smith-6 |
Re: combination of 3 fluorescence protein reporters
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Note - evrogen are selling mKate as "TagFP635" (took me a while to spot that). Also, isn't it somewhat shorter than Cy5? (mKate = excitation max 588, emission max 635) Regards, Adrian Smith Centenary Institute, Sydney, Australia On 12/11/2007, at 1:13 AM, George McNamara wrote: > > > If your microscope can image Cy5 well, try mKate from www.evrogen.com > > > > At 10:06 AM 11/8/2007, you wrote: >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> Dear listers, >> >> One of our users wanted to image CFP and YFP labeled cells and >> would like to >> add a third fluorecence protein reporter later. I was wondering if >> anybody >> could make a recomendation on this third one. >> >> Anybody has ever used a CFP/YFP/mCherry combination ? >> >> Thank you very much. >> >> >> Xinyu Zhao >> Biomedical Imaging Core Lab >> School of Medicine >> University of Pennsylvania >> Tel: 215-898-6730 > > > > > > > George McNamara, Ph.D. > University of Miami, Miller School of Medicine > Image Core > Miami, FL 33010 > [hidden email] > [hidden email] > 305-243-8436 office > http://home.earthlink.net/~pubspectra/ > http://home.earthlink.net/~geomcnamara/ > http://www.sylvester.org/health_pro/shared_resources/index.asp (see > Analytical Imaging Core Facility) |
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Adrian, I see we have a tender now in process - you were building an RFP, did the situation change on your end? We will proceed to submit the tender naturally but just wanted to touch base with you. Kind Regards, Brian Brian Jones Business Manager Micro-Imaging Systems Group Precision Instruments Division Australia & New Zealand Tel: +61 03 9265 5419 Mobil 0437 848 180
Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Note - evrogen are selling mKate as "TagFP635" (took me a while to spot that). Also, isn't it somewhat shorter than Cy5? (mKate = excitation max 588, emission max 635) Regards, Adrian Smith Centenary Institute, Sydney, Australia On 12/11/2007, at 1:13 AM, George McNamara wrote: > > > If your microscope can image Cy5 well, try mKate from www.evrogen.com > > > > At 10:06 AM 11/8/2007, you wrote: >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> Dear listers, >> >> One of our users wanted to image CFP and YFP labeled cells and >> would like to >> add a third fluorecence protein reporter later. I was wondering if >> anybody >> could make a recomendation on this third one. >> >> Anybody has ever used a CFP/YFP/mCherry combination ? >> >> Thank you very much. >> >> >> Xinyu Zhao >> Biomedical Imaging Core Lab >> School of Medicine >> University of Pennsylvania >> Tel: 215-898-6730 > > > > > > > George McNamara, Ph.D. > University of Miami, Miller School of Medicine > Image Core > Miami, FL 33010 > [hidden email] > [hidden email] > 305-243-8436 office > http://home.earthlink.net/~pubspectra/ > http://home.earthlink.net/~geomcnamara/ > http://www.sylvester.org/health_pro/shared_resources/index.asp (see > Analytical Imaging Core Facility) This message is intended only for the addressee. If you are not the intended recipient you are notified that disclosing, copying, distributing or taking any action in reliance on the contents of this information is strictly prohibited
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi Brian,
Yes - the procurement situation changed here due to several factors and as a result it was decided that we should use the University process for the confocal purchase. From a technical point of view our requirements are as we have been discussing (and as I hope are captured in the tender documentation). Regards, Adrian On 19/11/2007, at 11:51 AM, Brain Jones wrote:
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