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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Would anyone be willing to recommend a source for YFP and CFP plasmid vectors for FRET, preferably compatible with D5Hα. Any feedback would be most appreciated. Thanks!! Mike DISCLAIMER: The information in this electronic communication and attachments, if any, is confidential matter, and is intended only for the use of the recipient individual or entity named above. If the reader of this message is not the intended recipient you are hereby notified that distribution, dissemination, or copying or review by, unauthorized persons is strictly prohibited. All personal messages express views solely of the sender, which are not to be attributed to any organization. If you have received this transmission in error, immediately notify the sender and permanently delete this transmission including attachments. |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Contact Richard Day, Indiana Univ, ask for the constructs published in: Characterization of an improved donor fluorescent protein for Forster resonance energy transfer microscopy. </pubmed/18601527> Day RN, Booker CF, Periasamy A. J Biomed Opt. 2008 May-Jun;13(3):031203.PMID: 18601527 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2483694/?tool=pubmed On 1/28/2011 11:18 AM, Mike Tighe wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Would anyone be willing to recommend a source for YFP and CFP plasmid > vectors for FRET, preferably compatible with D5Hα. Any feedback would be > most appreciated. > > Thanks!! > Mike > > > > > DISCLAIMER: > > The information in this electronic communication and attachments, if > any, is confidential matter, and is intended only for the use of the > recipient individual or entity named above. If the reader of this > message is not the intended recipient you are hereby notified that > distribution, dissemination, or copying or review by, unauthorized > persons is strictly prohibited. All personal messages express views > solely of the sender, which are not to be attributed to any > organization. If you have received this transmission in error, > immediately notify the sender and permanently delete this transmission > including attachments. > > -- George McNamara, PhD Analytical Imaging Core Facility University of Miami |
mTFP might be a good donor, however based on my experience, v
***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Mike, mTFP might be a good donor, however based on my experience, venusYFP is not the best acceptor for the FRET studies. EYFP with the F64L substitution worked better for me. ...though in the very beginning I was in a "blind" love with the venusYFP too. Also, the paper did not show any advantage of mTFP over the CeFP. In addition, though the CeFP carries A206K substitution, I would recommend to add well known monomeric mutations in positions 221 and 223 to CeFP and all three to EYFP(F64L). As it is difficult to monitor fluorophore concentration on a voxel-by-voxel basis, highly accurate, quantitative, comparative FRET measurements study is still a challenging task (FLIM inclusive). George, do you have a good reference to? As to the vectors, just do it yourself - it is very simple and quick, PCR is a powerful tool and Dave Piston is the best source for the CeFP(A206K). Good luck, Vitaly 301-515-7833 ________________________________ From: George McNamara <[hidden email]> To: [hidden email] Sent: Fri, January 28, 2011 9:32:26 PM Subject: Re: CFP YFP plasmid vectors ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Contact Richard Day, Indiana Univ, ask for the constructs published in: Characterization of an improved donor fluorescent protein for Forster resonance energy transfer microscopy. </pubmed/18601527> Day RN, Booker CF, Periasamy A. J Biomed Opt. 2008 May-Jun;13(3):031203.PMID: 18601527 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2483694/?tool=pubmed On 1/28/2011 11:18 AM, Mike Tighe wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Would anyone be willing to recommend a source for YFP and CFP plasmid > vectors for FRET, preferably compatible with D5Hα. Any feedback would be > most appreciated. > > Thanks!! > Mike > > > > > DISCLAIMER: > > The information in this electronic communication and attachments, if > any, is confidential matter, and is intended only for the use of the > recipient individual or entity named above. If the reader of this > message is not the intended recipient you are hereby notified that > distribution, dissemination, or copying or review by, unauthorized > persons is strictly prohibited. All personal messages express views > solely of the sender, which are not to be attributed to any > organization. If you have received this transmission in error, > immediately notify the sender and permanently delete this transmission > including attachments. > > -- George McNamara, PhD Analytical Imaging Core Facility University of Miami |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Vitaly, With respect to monomers, you and other fans of ECFP should enjoy the following paper: Cyan Fluorescent Protein Carries a Constitutive Mutation That Prevents Its Dimerization. </pubmed/21175224> Espagne A, Erard M, Madiona K, Derrien V, Jonasson G, Lévy B, Pasquier H, Melki R, Mérola F. Biochemistry. 2011 Feb 1;50(4):437-439. Epub 2010 Dec 30.PMID: 21175224 (page 3 mentions the N146I mutation does not prevent YFP dimerization ... FLIM fans may find of interest PubMed 18975974, especially Fig 2A-C vs 2D and mTFP's lifetime). Quantitative FRET references: see other papers from from Richard Day's, papers from Steve Vogel's lab, and Periasamy's lab. Also: FRET imaging. </pubmed/14595367> Jares-Erijman EA, Jovin TM. Nat Biotechnol. 2003 Nov;21(11):1387-95. Review.PMID: 14595367 Enjoy, George On 1/29/2011 9:21 AM, Vitaly Boyko wrote: > mTFP might be a good donor, however based on my experience, v > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi Mike, > > mTFP might be a good donor, however based on my experience, venusYFP is not the > best acceptor for the FRET studies. EYFP with the F64L substitution worked > better for me. ...though in the very beginning I was in a "blind" love with the > venusYFP too. Also, the paper did not show any advantage of mTFP over the CeFP. > In addition, though the CeFP carries A206K substitution, I would recommend to > add well known monomeric mutations in positions 221 and 223 to CeFP and all > three to EYFP(F64L). > As it is difficult to monitor fluorophore concentration on a voxel-by-voxel > basis, highly accurate, quantitative, comparative FRET measurements study is > still a challenging task (FLIM inclusive). George, do you have a good reference > to? > > As to the vectors, just do it yourself - it is very simple and quick, PCR is a > powerful tool and Dave Piston is the best source for the CeFP(A206K). > > Good luck, > > Vitaly > 301-515-7833 > > > > > ________________________________ > From: George McNamara<[hidden email]> > To: [hidden email] > Sent: Fri, January 28, 2011 9:32:26 PM > Subject: Re: CFP YFP plasmid vectors > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > > Contact Richard Day, Indiana Univ, ask for the constructs published in: > > Characterization of an improved donor fluorescent protein for Forster > resonance energy transfer microscopy.</pubmed/18601527> > > Day RN, Booker CF, Periasamy A. > > J Biomed Opt. 2008 May-Jun;13(3):031203.PMID: 18601527 > > http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2483694/?tool=pubmed > > > > On 1/28/2011 11:18 AM, Mike Tighe wrote: > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> Would anyone be willing to recommend a source for YFP and CFP plasmid >> vectors for FRET, preferably compatible with D5Hα. Any feedback would be >> most appreciated. >> >> Thanks!! >> Mike >> >> >> >> >> DISCLAIMER: >> >> The information in this electronic communication and attachments, if >> any, is confidential matter, and is intended only for the use of the >> recipient individual or entity named above. If the reader of this >> message is not the intended recipient you are hereby notified that >> distribution, dissemination, or copying or review by, unauthorized >> persons is strictly prohibited. All personal messages express views >> solely of the sender, which are not to be attributed to any >> organization. If you have received this transmission in error, >> immediately notify the sender and permanently delete this transmission >> including attachments. >> >> >> > > -- George McNamara, PhD Analytical Imaging Core Facility University of Miami |
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