CFP and GFP proteins
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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, One of my collaborators has a mouse endogenously expressing 2 proteins (both are expressed in the same cells), one coupled to CFP and the other to GFP. I am curious about the spectrum- how does one best evaluate both proteins in the same sample without concerns about cross-reactivity. Thanks, Ramana SIdhaye |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Separating the two proteins should not be too difficult if you have the right filter sets, or if you have a spectral microscope system. If you check the spectra (e.g. invitrogen spectra viewer which you can Google), you will see that GFP emission starts at ~ 500 nm, so a narrow CFP filter should give litttle or no GFP signal. Conversely, if you excite GFP at around 488 nm, there should be little or no CFP excitation. Your institution probably has a Microscopy Core facility, so the best is to talk to them... I'm sure they can point you in the right direction. -- Julio Vazquez Fred Hutchinson Cancer Research Center Seattle, WA 98109-1024 http://www.fhcrc.org On Aug 1, 2012, at 12:55 PM, Ramana Sidhaye wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi, > One of my collaborators has a mouse endogenously expressing 2 proteins (both are expressed in the same cells), one coupled to CFP and the other to GFP. I am curious about the spectrum- how does one best evaluate both proteins in the same sample without concerns about cross-reactivity. > > Thanks, > Ramana SIdhaye |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Ramana, We addressed this issue on a stereo dissecting microscope by detecting the GFP using a YFP filter set. If you're using filters for your imaging, you can try this route. Good luck, ~Gregg *Gregg Sobocinski* Microscope Imaging Specialist University of Michigan, MCDB Dept. Ann Arbor, Michigan, USA On Wed, Aug 1, 2012 at 4:44 PM, Julio Vazquez <[hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Separating the two proteins should not be too difficult if you have the > right filter sets, or if you have a spectral microscope system. If you > check the spectra (e.g. invitrogen spectra viewer which you can Google), > you will see that GFP emission starts at ~ 500 nm, so a narrow CFP filter > should give litttle or no GFP signal. Conversely, if you excite GFP at > around 488 nm, there should be little or no CFP excitation. Your > institution probably has a Microscopy Core facility, so the best is to talk > to them... I'm sure they can point you in the right direction. > > -- > Julio Vazquez > Fred Hutchinson Cancer Research Center > Seattle, WA 98109-1024 > > > http://www.fhcrc.org > > > On Aug 1, 2012, at 12:55 PM, Ramana Sidhaye wrote: > > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > ***** > > > > Hi, > > One of my collaborators has a mouse endogenously expressing 2 proteins > (both are expressed in the same cells), one coupled to CFP and the other to > GFP. I am curious about the spectrum- how does one best evaluate both > proteins in the same sample without concerns about cross-reactivity. > > > > Thanks, > > Ramana SIdhaye > - |
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In reply to this post by Ramana Sidhaye
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello Ramana, Your question poses a rather tricky problem. If you excite both proteins then no filter can possibly separate them, as CFP has a broad emission peak that entirely encompasses the emission of GFP. You thus need to excite them sequentially. A 488 nm laser will excite GFP with decent speficity, and you can detect that with a normal GFP filter. However, CFP is often excited with the 457 line of a gas laser, and this is no good. It will excite GFP almost as well as CFP, and it leaves you very little room at the blue end of the spectrum for a filter to detect the parts of CFP emission that do not overlap with GFP. 442 nm lasers are much better for specifically exciting CFP but you may have a hard time finding a lab that uses one. Further, it is _very_ hard to do a confocal experiment in which you alternate excitations at 442 and 488 nm and you can not do 457/488, full stop. The emission-side dichroic mirror is just too tricky to engineer. If you have to do it, you could try to find a modern spectral scope that has a non-specific emission-side dichroic such as the Nikon BS20/80. _If_ you can find such a scope with a 442 nm laser then this dichroic will let you alternate excitations at 442 and 488 nm. However, the cost in terms of light loss is pretty steep, and mouse transgenic proteins are often relatively dim relative to something nuked off the CMV promoter in a HeLa cell. On the other hand, perhaps you could use a two-photon scope to alternate 442/488 excitations without the scan head dichroic being such an obstacle. This falls outside my experience so the 2p folks will have to tell you whether or not you can do that. cheers, TF Timothy Feinstein, PhD Postdoctoral Fellow Laboratory for GPCR Biology Dept. of Pharmacology & Chemical Biology University of Pittsburgh, School of Medicine BST W1301, 200 Lothrop St. Pittsburgh, PA 15261 On Aug 1, 2012, at 3:55 PM, Ramana Sidhaye wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi, > One of my collaborators has a mouse endogenously expressing 2 proteins (both are expressed in the same cells), one coupled to CFP and the other to GFP. I am curious about the spectrum- how does one best evaluate both proteins in the same sample without concerns about cross-reactivity. > > Thanks, > Ramana SIdhaye |
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In reply to this post by Gregg Sobocinski
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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 Gregg, Most epifluorescence YFP cubes pass light from 500-530 nm, and only the bluer part of that excites GFP. A 514 nm laser will have a very hard time with GFP. However, you raise an excellent point. If this experiment can be done using non-confocal means then it would be a great idea to do so. I'd even risk expulsion from this board and suggest that is a good rule in general (e.g., if intensity quantitation is more of a concern than fine-fine detail). cheers, TF Timothy Feinstein, PhD Postdoctoral Fellow Laboratory for GPCR Biology Dept. of Pharmacology & Chemical Biology University of Pittsburgh, School of Medicine BST W1301, 200 Lothrop St. Pittsburgh, PA 15261 On Aug 1, 2012, at 5:05 PM, Gregg Sobocinski wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Ramana, > We addressed this issue on a stereo dissecting microscope by detecting the > GFP using a YFP filter set. If you're using filters for your imaging, you > can try this route. > > Good luck, > ~Gregg > *Gregg Sobocinski* > Microscope Imaging Specialist > University of Michigan, MCDB Dept. > Ann Arbor, Michigan, USA > > On Wed, Aug 1, 2012 at 4:44 PM, Julio Vazquez <[hidden email]> wrote: > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> Separating the two proteins should not be too difficult if you have the >> right filter sets, or if you have a spectral microscope system. If you >> check the spectra (e.g. invitrogen spectra viewer which you can Google), >> you will see that GFP emission starts at ~ 500 nm, so a narrow CFP filter >> should give litttle or no GFP signal. Conversely, if you excite GFP at >> around 488 nm, there should be little or no CFP excitation. Your >> institution probably has a Microscopy Core facility, so the best is to talk >> to them... I'm sure they can point you in the right direction. >> >> -- >> Julio Vazquez >> Fred Hutchinson Cancer Research Center >> Seattle, WA 98109-1024 >> >> >> http://www.fhcrc.org >> >> >> On Aug 1, 2012, at 12:55 PM, Ramana Sidhaye wrote: >> >>> ***** >>> To join, leave or search the confocal microscopy listserv, go to: >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>> ***** >>> >>> Hi, >>> One of my collaborators has a mouse endogenously expressing 2 proteins >> (both are expressed in the same cells), one coupled to CFP and the other to >> GFP. I am curious about the spectrum- how does one best evaluate both >> proteins in the same sample without concerns about cross-reactivity. >>> >>> Thanks, >>> Ramana SIdhaye >> > > > > - |
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