Photobleaching mechanism question
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John Runions |
Photobleaching mechanism question
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Hi Everyone, this question follows on from a helpful discussion that we had about photobleaching back in November. I have recently tried to explain to a group of colleagues about the mechanism of photobleaching. The answer is based on the transition of molecules from the excited singlet state (S1) to the triplet state (T1) which is long-lived and therefore more susceptible to bleaching by free radicals (my entire discussion of this is below). My question that arises from my attempted answer is: why are excited molecules more susceptible to oxidative attack than ground state molecules. I hope I'm not completely mucking up the mechanism here. Would the physicists out there please help. Thanks, John. The original answer: When excited, fluorophores generally transition from singlet ground state (S0) to singlet excited state (S1). Relaxation from S1 to S0 results in emission of heat and light (fluorescence). Lifetime in S1 is in the nano to pico second range and allows very little time for the excited molecule to interact with free radicals. Periodically, however, an excited molecule will do a transition from S1 to the triplet excited state (T1 - the physics of this is a bit difficult to understand). T1 is a very long-lived state - molecules can remain in T1 for up to the microsecond range - i.e. a thousand to a million times longer than for normal S1 state. It is during this long T1 state that molecules are attacked by free radicals and destroyed. --
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Oxygen is naturally a triplet molecule.
Triplet-triplet reactions are particularly
likely to occur, and so a triplet excited state is more
likely to get oxidised.
That's the explanation I have always been given, and it
does seem to make
sense.
Guy
Optical Imaging Techniques in Cell Biology From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Runions Sent: Monday, 16 February 2009 10:29 PM To: [hidden email] Subject: Photobleaching mechanism question Hi Everyone, this question follows on from a helpful discussion that we had about photobleaching back in November. I have recently tried to explain to a group of colleagues about the mechanism of photobleaching. The answer is based on the transition of molecules from the excited singlet state (S1) to the triplet state (T1) which is long-lived and therefore more susceptible to bleaching by free radicals (my entire discussion of this is below). My question that arises from my attempted answer is: why are excited molecules more susceptible to oxidative attack than ground state molecules. I hope I'm not completely mucking up the mechanism here. Would the physicists out there please help. Thanks, John. The original answer: When excited, fluorophores generally transition from singlet ground state (S0) to singlet excited state (S1). Relaxation from S1 to S0 results in emission of heat and light (fluorescence). Lifetime in S1 is in the nano to pico second range and allows very little time for the excited molecule to interact with free radicals. Periodically, however, an excited molecule will do a transition from S1 to the triplet excited state (T1 - the physics of this is a bit difficult to understand). T1 is a very long-lived state - molecules can remain in T1 for up to the microsecond range - i.e. a thousand to a million times longer than for normal S1 state. It is during this long T1 state that molecules are attacked by free radicals and destroyed. -- (Sent from my cra%#y
non-Blackberry electronic device that still has wires) *********************************
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Tobias Baskin |
Re: Photobleaching mechanism question
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In reply to this post by John Runions
John,
From the non physicist's point of view the answer could go something like this. If you have a power surge it can fry your computer. But if your computer is not plugged into the mains then it would take a very big power surge indeed to do the damage. A molecule in the ground state can of course be damaged by free radical attack but no more or less than other molecules. But once a molecule has absorbed a photon then it is not in the ground state any more. To continue my hoaky analogy, a chromophore in light is like your computer plugged in to the mains. Hope this helps. The physicists (and musicians) can go for the triplets. Tobias >Hi Everyone, this question follows on from a helpful discussion >that we had about photobleaching back in November. I have recently >tried to explain to a group of colleagues about the mechanism of >photobleaching. The answer is based on the transition of molecules >from the excited singlet state (S1) to the triplet state (T1) which >is long-lived and therefore more susceptible to bleaching by free >radicals (my entire discussion of this is below). > >My question that arises from my attempted answer is: why are excited >molecules more susceptible to oxidative attack than ground state >molecules. I hope I'm not completely mucking up the mechanism here. >Would the physicists out there please help. > >Thanks, John. > >The original answer: When excited, fluorophores generally transition >from singlet ground state (S0) to singlet excited state (S1). >Relaxation from S1 to S0 results in emission of heat and light >(fluorescence). Lifetime in S1 is in the nano to pico second range >and allows very little time for the excited molecule to interact >with free radicals. Periodically, however, an excited molecule will >do a transition from S1 to the triplet excited state (T1 - the >physics of this is a bit difficult to understand). T1 is a very >long-lived state - molecules can remain in T1 for up to the >microsecond range - i.e. a thousand to a million times longer than >for normal S1 state. It is during this long T1 state that molecules >are attacked by free radicals and destroyed. > >-- >Runions signature > >(Sent from my cra%#y non-Blackberry electronic device that still has wires) > > > >********************************* >John Runions, Ph.D. >School of Life Sciences >Oxford Brookes University >Oxford, UK >OX3 0BP > >email: <mailto:[hidden email]>[hidden email] >phone: +44 (0) 1865 483 964 > ><http://www.brookes.ac.uk/lifesci/runions/HTMLpages/index.html%21>Runions' >lab web site > > > >Visit <http://www.illuminatedcell.com/ER.html>The Illuminated Plant >Cell dot com >Oxford Brookes Master's in ><http://www.brookes.ac.uk/studying/courses/postgraduate/2007/bmt>Bioimaging >with Molecular Technology |
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Mark Cannell |
Re: Photobleaching mechanism question
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While I am all for analogies to convey the basis of complicated
processes to the ignorant, the analogy must be accurate. But in this case, if a _scientist_ asks about bleaching from the triplet state why does the explanation have to be dumbed down to such an (inaccurate) level? Surely the basis of chemical reactions in terms of electrons and electron pairing should not be so unfamiliar after undergraduate physics and chemistry? A good explanation is given in Encyclopeda Britannica and should be within the grasp of most I think. http://www.britannica.com/EBchecked/topic/457736/photochemical-reaction/277509/Consequences-of-photoexcitation#ref=ref499215 Even if you can't remember the simple reason for more rapid oxidation from the excited state (due to singlet oxygen production, I think, but probably other possibilities also exist in complex molecular systems), you point your undergraduates there rather than use horribly inaccurate analogies. If nothing else, a reason to learn this explanation is that it is the basis of life on earth as it explains how light can be harnessed to chemical reactions! Cheers Mark P.S. Tobias, if you wonder why I object to your analogy it is because a power surge does not involve switching off the computer!!!! > John, > From the non physicist's point of view the answer could go > something like this. If you have a power surge it can fry your > computer. But if your computer is not plugged into the mains then it > would take a very big power surge indeed to do the damage. A molecule > in the ground state can of course be damaged by free radical attack > but no more or less than other molecules. But once a molecule has > absorbed a photon then it is not in the ground state any more. To > continue my hoaky analogy, a chromophore in light is like your > computer plugged in to the mains. > > Hope this helps. The physicists (and musicians) can go for the > triplets. > > Tobias > > >> Hi Everyone, this question follows on from a helpful discussion that >> we had about photobleaching back in November. I have recently tried >> to explain to a group of colleagues about the mechanism of >> photobleaching. The answer is based on the transition of molecules >> from the excited singlet state (S1) to the triplet state (T1) which >> is long-lived and therefore more susceptible to bleaching by free >> radicals (my entire discussion of this is below). >> >> My question that arises from my attempted answer is: why are excited >> molecules more susceptible to oxidative attack than ground state >> molecules. I hope I'm not completely mucking up the mechanism here. >> Would the physicists out there please help. >> >> Thanks, John. >> >> The original answer: When excited, fluorophores generally transition >> from singlet ground state (S0) to singlet excited state (S1). >> Relaxation from S1 to S0 results in emission of heat and light >> (fluorescence). Lifetime in S1 is in the nano to pico second range >> and allows very little time for the excited molecule to interact with >> free radicals. Periodically, however, an excited molecule will do a >> transition from S1 to the triplet excited state (T1 - the physics of >> this is a bit difficult to understand). T1 is a very long-lived state >> - molecules can remain in T1 for up to the microsecond range - i.e. a >> thousand to a million times longer than for normal S1 state. It is >> during this long T1 state that molecules are attacked by free >> radicals and destroyed. >> >> -- >> Runions signature >> >> (Sent from my cra%#y non-Blackberry electronic device that still has >> wires) >> >> >> >> ********************************* >> John Runions, Ph.D. >> School of Life Sciences >> Oxford Brookes University >> Oxford, UK >> OX3 0BP >> >> email: <mailto:[hidden email]>[hidden email] >> phone: +44 (0) 1865 483 964 >> >> <http://www.brookes.ac.uk/lifesci/runions/HTMLpages/index.html%21>Runions' >> lab web site >> >> >> >> Visit <http://www.illuminatedcell.com/ER.html>The Illuminated Plant >> Cell dot com >> Oxford Brookes Master's in >> <http://www.brookes.ac.uk/studying/courses/postgraduate/2007/bmt>Bioimaging >> with Molecular Technology |
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Ignatius, Mike |
Re: Photobleaching mechanism question
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Boy, now I am really glad I didn't share my Luke Skywalker, avoid the
Dark Side/State analogy, that I use with students. When Luke/Fluors are activated, riled with hate, they are most vulnerable to going to the dark side/state. Yoda: "But beware of the dark side. Anger, fear, aggression, PHOTOTOXICITY, SIGNAL LOSS, the dark side of the Force are they." Luke: "Is the dark side stronger?" Yoda: "No, no, no. Quicker, easier, more seductive, HARDER TO PREVENT IN LIVE CELLS." Anakin -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Mark Cannell Sent: Monday, February 16, 2009 1:03 PM To: [hidden email] Subject: Re: Photobleaching mechanism question While I am all for analogies to convey the basis of complicated processes to the ignorant, the analogy must be accurate. But in this case, if a _scientist_ asks about bleaching from the triplet state why does the explanation have to be dumbed down to such an (inaccurate) level? Surely the basis of chemical reactions in terms of electrons and electron pairing should not be so unfamiliar after undergraduate physics and chemistry? A good explanation is given in Encyclopeda Britannica and should be within the grasp of most I think. http://www.britannica.com/EBchecked/topic/457736/photochemical-reaction/ 277509/Consequences-of-photoexcitation#ref=ref499215 Even if you can't remember the simple reason for more rapid oxidation from the excited state (due to singlet oxygen production, I think, but probably other possibilities also exist in complex molecular systems), you point your undergraduates there rather than use horribly inaccurate analogies. If nothing else, a reason to learn this explanation is that it is the basis of life on earth as it explains how light can be harnessed to chemical reactions! Cheers Mark P.S. Tobias, if you wonder why I object to your analogy it is because a power surge does not involve switching off the computer!!!! > John, > From the non physicist's point of view the answer could go > something like this. If you have a power surge it can fry your > computer. But if your computer is not plugged into the mains then it > would take a very big power surge indeed to do the damage. A molecule > in the ground state can of course be damaged by free radical attack > but no more or less than other molecules. But once a molecule has > absorbed a photon then it is not in the ground state any more. To > continue my hoaky analogy, a chromophore in light is like your > computer plugged in to the mains. > > Hope this helps. The physicists (and musicians) can go for the > triplets. > > Tobias > > >> Hi Everyone, this question follows on from a helpful discussion that >> we had about photobleaching back in November. I have recently tried >> to explain to a group of colleagues about the mechanism of >> photobleaching. The answer is based on the transition of molecules >> from the excited singlet state (S1) to the triplet state (T1) which >> is long-lived and therefore more susceptible to bleaching by free >> radicals (my entire discussion of this is below). >> >> My question that arises from my attempted answer is: why are excited >> molecules more susceptible to oxidative attack than ground state >> molecules. I hope I'm not completely mucking up the mechanism here. >> Would the physicists out there please help. >> >> Thanks, John. >> >> The original answer: When excited, fluorophores generally transition >> from singlet ground state (S0) to singlet excited state (S1). >> Relaxation from S1 to S0 results in emission of heat and light >> (fluorescence). Lifetime in S1 is in the nano to pico second range >> and allows very little time for the excited molecule to interact with >> free radicals. Periodically, however, an excited molecule will do a >> transition from S1 to the triplet excited state (T1 - the physics of >> this is a bit difficult to understand). T1 is a very long-lived state >> - molecules can remain in T1 for up to the microsecond range - i.e. a >> thousand to a million times longer than for normal S1 state. It is >> during this long T1 state that molecules are attacked by free >> radicals and destroyed. >> >> -- >> Runions signature >> >> (Sent from my cra%#y non-Blackberry electronic device that still has >> wires) >> >> >> >> ********************************* >> John Runions, Ph.D. >> School of Life Sciences >> Oxford Brookes University >> Oxford, UK >> OX3 0BP >> >> email: <mailto:[hidden email]>[hidden email] >> phone: +44 (0) 1865 483 964 >> >> s' >> lab web site >> >> >> >> Visit <http://www.illuminatedcell.com/ER.html>The Illuminated Plant >> Cell dot com >> Oxford Brookes Master's in >> <http://www.brookes.ac.uk/studying/courses/postgraduate/2007/bmt>Bioimag ing >> with Molecular Technology |
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Mark Cannell |
Re: Photobleaching mechanism question
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LOL Mike
But Luke skywalker only existed in a Galaxy far far away and long ago even if he did have lots of possibilities in his mitochlorians -or so we are told... Cheers Ignatius, Mike wrote: > Boy, now I am really glad I didn't share my Luke Skywalker, avoid the > Dark Side/State analogy, that I use with students. When Luke/Fluors are > activated, riled with hate, they are most vulnerable to going to the > dark side/state. > > Yoda: "But beware of the dark side. Anger, fear, aggression, > PHOTOTOXICITY, SIGNAL LOSS, the dark side of the Force are they." > Luke: "Is the dark side stronger?" > Yoda: "No, no, no. Quicker, easier, more seductive, HARDER TO PREVENT IN > LIVE CELLS." > > Anakin > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] > On Behalf Of Mark Cannell > Sent: Monday, February 16, 2009 1:03 PM > To: [hidden email] > Subject: Re: Photobleaching mechanism question > > While I am all for analogies to convey the basis of complicated > processes to the ignorant, the analogy must be accurate. But in this > case, if a _scientist_ asks about bleaching from the triplet state why > does the explanation have to be dumbed down to such an (inaccurate) > level? Surely the basis of chemical reactions in terms of electrons and > > electron pairing should not be so unfamiliar after undergraduate physics > > and chemistry? > > A good explanation is given in Encyclopeda Britannica and should be > within the grasp of most I think. > > http://www.britannica.com/EBchecked/topic/457736/photochemical-reaction/ > 277509/Consequences-of-photoexcitation#ref=ref499215 > > Even if you can't remember the simple reason for more rapid oxidation > from the excited state (due to singlet oxygen production, I think, but > probably other possibilities also exist in complex molecular systems), > you point your undergraduates there rather than use horribly inaccurate > analogies. If nothing else, a reason to learn this explanation is that > it is the basis of life on earth as it explains how light can be > harnessed to chemical reactions! > > Cheers Mark > P.S. Tobias, if you wonder why I object to your analogy it is because a > power surge does not involve switching off the computer!!!! > > > >> John, >> From the non physicist's point of view the answer could go >> something like this. If you have a power surge it can fry your >> computer. But if your computer is not plugged into the mains then it >> would take a very big power surge indeed to do the damage. A molecule >> in the ground state can of course be damaged by free radical attack >> but no more or less than other molecules. But once a molecule has >> absorbed a photon then it is not in the ground state any more. To >> continue my hoaky analogy, a chromophore in light is like your >> computer plugged in to the mains. >> >> Hope this helps. The physicists (and musicians) can go for the >> triplets. >> >> Tobias >> >> >> >>> Hi Everyone, this question follows on from a helpful discussion that >>> > > >>> we had about photobleaching back in November. I have recently tried >>> to explain to a group of colleagues about the mechanism of >>> photobleaching. The answer is based on the transition of molecules >>> from the excited singlet state (S1) to the triplet state (T1) which >>> is long-lived and therefore more susceptible to bleaching by free >>> radicals (my entire discussion of this is below). >>> >>> My question that arises from my attempted answer is: why are excited >>> molecules more susceptible to oxidative attack than ground state >>> molecules. I hope I'm not completely mucking up the mechanism here. >>> Would the physicists out there please help. >>> >>> Thanks, John. >>> >>> The original answer: When excited, fluorophores generally transition >>> from singlet ground state (S0) to singlet excited state (S1). >>> Relaxation from S1 to S0 results in emission of heat and light >>> (fluorescence). Lifetime in S1 is in the nano to pico second range >>> and allows very little time for the excited molecule to interact with >>> > > >>> free radicals. Periodically, however, an excited molecule will do a >>> transition from S1 to the triplet excited state (T1 - the physics of >>> this is a bit difficult to understand). T1 is a very long-lived state >>> > > >>> - molecules can remain in T1 for up to the microsecond range - i.e. a >>> > > >>> thousand to a million times longer than for normal S1 state. It is >>> during this long T1 state that molecules are attacked by free >>> radicals and destroyed. >>> >>> -- >>> Runions signature >>> >>> (Sent from my cra%#y non-Blackberry electronic device that still has >>> wires) >>> >>> >>> >>> ********************************* >>> John Runions, Ph.D. >>> School of Life Sciences >>> Oxford Brookes University >>> Oxford, UK >>> OX3 0BP >>> >>> email: <mailto:[hidden email]>[hidden email] >>> phone: +44 (0) 1865 483 964 >>> >>> >>> > <http://www.brookes.ac.uk/lifesci/runions/HTMLpages/index.html%21>Runion > s' > >>> lab web site >>> >>> >>> >>> Visit <http://www.illuminatedcell.com/ER.html>The Illuminated Plant >>> Cell dot com >>> Oxford Brookes Master's in >>> >>> > <http://www.brookes.ac.uk/studying/courses/postgraduate/2007/bmt>Bioimag > ing > >>> with Molecular Technology >>> |
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Mark Cannell |
Re: Photobleaching mechanism question
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Ok John, I'll bite:
It comes from the degeneracy = 2s+1 where s is the total spin angular momentum. Having all paired electrons (i.e. as many up electrons as down) gives s=0 so the degeneracy is 1 = singlet. If you have two unpaired up electrons the degeneracy is 2*(+1/2 + 1/2)+1 =3 Cheers Mark > >Luke Skywalker aside, what really confuses everyone is that none of these explanations states what is single in "singlet" and triple in "triplet". > >-- John J. Lemasters, MD, PhD > LOL Mike > > But Luke skywalker only existed in a Galaxy far far away and long ago > even if he did have lots of possibilities in his mitochlorians -or so > we are told... > > Cheers > > Ignatius, Mike wrote: >> Boy, now I am really glad I didn't share my Luke Skywalker, avoid the >> Dark Side/State analogy, that I use with students. When Luke/Fluors are >> activated, riled with hate, they are most vulnerable to going to the >> dark side/state. >> Yoda: "But beware of the dark side. Anger, fear, aggression, >> PHOTOTOXICITY, SIGNAL LOSS, the dark side of the Force are they." >> Luke: "Is the dark side stronger?" Yoda: "No, no, no. Quicker, >> easier, more seductive, HARDER TO PREVENT IN >> LIVE CELLS." >> Anakin >> >> -----Original Message----- >> From: Confocal Microscopy List [mailto:[hidden email]] >> On Behalf Of Mark Cannell >> Sent: Monday, February 16, 2009 1:03 PM >> To: [hidden email] >> Subject: Re: Photobleaching mechanism question >> >> While I am all for analogies to convey the basis of complicated >> processes to the ignorant, the analogy must be accurate. But in this >> case, if a _scientist_ asks about bleaching from the triplet state >> why does the explanation have to be dumbed down to such an >> (inaccurate) level? Surely the basis of chemical reactions in terms >> of electrons and >> >> electron pairing should not be so unfamiliar after undergraduate physics >> >> and chemistry? >> >> A good explanation is given in Encyclopeda Britannica and should be >> within the grasp of most I think. >> >> http://www.britannica.com/EBchecked/topic/457736/photochemical-reaction/ >> 277509/Consequences-of-photoexcitation#ref=ref499215 >> >> Even if you can't remember the simple reason for more rapid oxidation >> from the excited state (due to singlet oxygen production, I think, >> but probably other possibilities also exist in complex molecular >> systems), you point your undergraduates there rather than use >> horribly inaccurate analogies. If nothing else, a reason to learn >> this explanation is that it is the basis of life on earth as it >> explains how light can be harnessed to chemical reactions! >> >> Cheers Mark >> P.S. Tobias, if you wonder why I object to your analogy it is because >> a power surge does not involve switching off the computer!!!! >> >> >> >>> John, >>> From the non physicist's point of view the answer could go >>> something like this. If you have a power surge it can fry your >>> computer. But if your computer is not plugged into the mains then it >>> would take a very big power surge indeed to do the damage. A >>> molecule in the ground state can of course be damaged by free >>> radical attack but no more or less than other molecules. But once a >>> molecule has absorbed a photon then it is not in the ground state >>> any more. To continue my hoaky analogy, a chromophore in light is >>> like your computer plugged in to the mains. >>> >>> Hope this helps. The physicists (and musicians) can go for the >>> triplets. >>> >>> Tobias >>> >>> >>> >>>> Hi Everyone, this question follows on from a helpful discussion that >>>> >> >> >>>> we had about photobleaching back in November. I have recently >>>> tried to explain to a group of colleagues about the mechanism of >>>> photobleaching. The answer is based on the transition of molecules >>>> from the excited singlet state (S1) to the triplet state (T1) which >>>> is long-lived and therefore more susceptible to bleaching by free >>>> radicals (my entire discussion of this is below). >>>> >>>> My question that arises from my attempted answer is: why are >>>> excited molecules more susceptible to oxidative attack than ground >>>> state molecules. I hope I'm not completely mucking up the >>>> mechanism here. Would the physicists out there please help. >>>> >>>> Thanks, John. >>>> >>>> The original answer: When excited, fluorophores generally >>>> transition from singlet ground state (S0) to singlet excited state >>>> (S1). Relaxation from S1 to S0 results in emission of heat and >>>> light (fluorescence). Lifetime in S1 is in the nano to pico second >>>> range and allows very little time for the excited molecule to >>>> interact with >>>> >> >> >>>> free radicals. Periodically, however, an excited molecule will do a >>>> transition from S1 to the triplet excited state (T1 - the physics >>>> of this is a bit difficult to understand). T1 is a very long-lived >>>> state >>>> >> >> >>>> - molecules can remain in T1 for up to the microsecond range - i.e. a >>>> >> >> >>>> thousand to a million times longer than for normal S1 state. It is >>>> during this long T1 state that molecules are attacked by free >>>> radicals and destroyed. >>>> >>>> -- >>>> Runions signature >>>> >>>> (Sent from my cra%#y non-Blackberry electronic device that still >>>> has wires) >>>> >>>> >>>> >>>> ********************************* >>>> John Runions, Ph.D. >>>> School of Life Sciences >>>> Oxford Brookes University >>>> Oxford, UK >>>> OX3 0BP >>>> >>>> email: <mailto:[hidden email]>[hidden email] >>>> phone: +44 (0) 1865 483 964 >>>> >>>> >>>> >> <http://www.brookes.ac.uk/lifesci/runions/HTMLpages/index.html%21>Runion >> s' >>>> lab web site >>>> >>>> >>>> >>>> Visit <http://www.illuminatedcell.com/ER.html>The Illuminated Plant >>>> Cell dot com >>>> Oxford Brookes Master's in >>>> >> <http://www.brookes.ac.uk/studying/courses/postgraduate/2007/bmt>Bioimag >> ing >>>> with Molecular Technology >>>> > > |
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In reply to this post by Tobias Baskin
Mark Cannell sent the following addition to my explanation
but so far as I can see just to me, not the list. So I'll add it here: I said: Oxygen is naturally a triplet molecule. Triplet-triplet reactions are particularly likely to occur, and so a triplet excited state is more likely to get oxidised. Mark adds: Your answer is very close. The final part is that the triplet-triplet interaction produces two singlet molecules and singlet oxygen is very reactive. Since it is in close proximity to the fluorochrome it is likely to oxidize it. As to bizarre explanations, I can't see the point. How do they help? Singlet just means all electrons are paired with ones of opposite 'spin' - think of them as tiny magnets, each one that has its north pole upwards is matched with one with its south pole upwards, so there's no overall magnetic field. Triplet means that there are unpaired electrons. Mark has explained the reason for the names, but they're not so important. One possible decay route for an excited singlet molecule is to go into a triplet state. It's relatively unlikely for an electron to change its spin so usually not many will decay that way. But since we're exciting a lot of molecules some will chance to take that path. Again, getting from there back to a non-excited state means changing an electron's spin, and since this is an infrequent occurrence the molecule will remain in a triplet state for a while. If nothing happens in this time the molecule will go back to the ground state, unbleached, but it is very vulnerable to oxygen attack while waiting in the triplet state. None of this is difficult to follow. The explanation that 'It's easy to switch to the dark side' is simply wrong. It's quite hard to switch to the dark side, it's just that if you keep on exciting a molecule, over and over, eventually it'll end up there. The best analogy I can think of is a fly-paper. It's quite unlikely that a fly will bump into the fly-paper, but when it does, it can't get off. So eventually you'll get rid of all the flies in the room. Guy Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 ______________________________________________ Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Tobias Baskin Sent: Monday, 16 February 2009 11:47 PM To: [hidden email] Subject: Re: Photobleaching mechanism question John, From the non physicist's point of view the answer could go something like this. If you have a power surge it can fry your computer. But if your computer is not plugged into the mains then it would take a very big power surge indeed to do the damage. A molecule in the ground state can of course be damaged by free radical attack but no more or less than other molecules. But once a molecule has absorbed a photon then it is not in the ground state any more. To continue my hoaky analogy, a chromophore in light is like your computer plugged in to the mains. Hope this helps. The physicists (and musicians) can go for the triplets. Tobias >Hi Everyone, this question follows on from a helpful discussion that >we had about photobleaching back in November. I have recently tried to >explain to a group of colleagues about the mechanism of photobleaching. >The answer is based on the transition of molecules from the excited >singlet state (S1) to the triplet state (T1) which is long-lived and >therefore more susceptible to bleaching by free radicals (my entire >discussion of this is below). > >My question that arises from my attempted answer is: why are excited >molecules more susceptible to oxidative attack than ground state >molecules. I hope I'm not completely mucking up the mechanism here. >Would the physicists out there please help. > >Thanks, John. > >The original answer: When excited, fluorophores generally transition >from singlet ground state (S0) to singlet excited state (S1). >Relaxation from S1 to S0 results in emission of heat and light >(fluorescence). Lifetime in S1 is in the nano to pico second range and >allows very little time for the excited molecule to interact with free >radicals. Periodically, however, an excited molecule will do a >transition from S1 to the triplet excited state (T1 - the physics of >this is a bit difficult to understand). T1 is a very long-lived state - >molecules can remain in T1 for up to the microsecond range - i.e. a >thousand to a million times longer than for normal S1 state. It is >during this long T1 state that molecules are attacked by free radicals >and destroyed. > >-- >Runions signature > >(Sent from my cra%#y non-Blackberry electronic device that still has >wires) > > > >********************************* >John Runions, Ph.D. >School of Life Sciences >Oxford Brookes University >Oxford, UK >OX3 0BP > >email: <mailto:[hidden email]>[hidden email] >phone: +44 (0) 1865 483 964 > ><http://www.brookes.ac.uk/lifesci/runions/HTMLpages/index.html%21>Runions' >lab web site > > > >Visit <http://www.illuminatedcell.com/ER.html>The Illuminated Plant >Cell dot com Oxford Brookes Master's in ><http://www.brookes.ac.uk/studying/courses/postgraduate/2007/bmt>Bioima >ging >with Molecular Technology No virus found in this incoming message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.10.24/1954 - Release Date: 15/02/2009 6:09 PM No virus found in this outgoing message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.10.24/1954 - Release Date: 15/02/2009 6:09 PM |
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I realised on seeing the message on the list that it wasn't clear what was me and what was Mark.
Mark Cannell sent the following addition to my explanation but so far as I can see just to me, not the list. So I'll add it here: I said: Oxygen is naturally a triplet molecule. Triplet-triplet reactions are particularly likely to occur, and so a triplet excited state is more likely to get oxidised. Mark adds: Your answer is very close. The final part is that the triplet-triplet interaction produces two singlet molecules and singlet oxygen is very reactive. Since it is in close proximity to the fluorochrome it is likely to oxidize it. [from here on it's me, simple dumb biologist, again] As to bizarre explanations, I can't see the point. How do they help? Singlet just means all electrons are paired with ones of opposite 'spin' - think of them as tiny magnets, each one that has its north pole upwards is matched with one with its south pole upwards, so there's no overall magnetic field. Triplet means that there are unpaired electrons. Mark has explained the reason for the names, but they're not so important. One possible decay route for an excited singlet molecule is to go into a triplet state. It's relatively unlikely for an electron to change its spin so usually not many will decay that way. But since we're exciting a lot of molecules some will chance to take that path. Again, getting from there back to a non-excited state means changing an electron's spin, and since this is an infrequent occurrence the molecule will remain in a triplet state for a while. If nothing happens in this time the molecule will go back to the ground state, unbleached, but it is very vulnerable to oxygen attack while waiting in the triplet state. None of this is difficult to follow. The explanation that 'It's easy to switch to the dark side' is simply wrong. It's quite hard to switch to the dark side, it's just that if you keep on exciting a molecule, over and over, eventually it'll end up there. The best analogy I can think of is a fly-paper. It's quite unlikely that a fly will bump into the fly-paper, but when it does, it can't get off. So eventually you'll get rid of all the flies in the room. Guy Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 ______________________________________________ Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Tobias Baskin Sent: Monday, 16 February 2009 11:47 PM To: [hidden email] Subject: Re: Photobleaching mechanism question John, From the non physicist's point of view the answer could go something like this. If you have a power surge it can fry your computer. But if your computer is not plugged into the mains then it would take a very big power surge indeed to do the damage. A molecule in the ground state can of course be damaged by free radical attack but no more or less than other molecules. But once a molecule has absorbed a photon then it is not in the ground state any more. To continue my hoaky analogy, a chromophore in light is like your computer plugged in to the mains. Hope this helps. The physicists (and musicians) can go for the triplets. Tobias >Hi Everyone, this question follows on from a helpful discussion that >we had about photobleaching back in November. I have recently tried to >explain to a group of colleagues about the mechanism of photobleaching. >The answer is based on the transition of molecules from the excited >singlet state (S1) to the triplet state (T1) which is long-lived and >therefore more susceptible to bleaching by free radicals (my entire >discussion of this is below). > >My question that arises from my attempted answer is: why are excited >molecules more susceptible to oxidative attack than ground state >molecules. I hope I'm not completely mucking up the mechanism here. >Would the physicists out there please help. > >Thanks, John. > >The original answer: When excited, fluorophores generally transition >from singlet ground state (S0) to singlet excited state (S1). >Relaxation from S1 to S0 results in emission of heat and light >(fluorescence). Lifetime in S1 is in the nano to pico second range and >allows very little time for the excited molecule to interact with free >radicals. Periodically, however, an excited molecule will do a >transition from S1 to the triplet excited state (T1 - the physics of >this is a bit difficult to understand). T1 is a very long-lived state - >molecules can remain in T1 for up to the microsecond range - i.e. a >thousand to a million times longer than for normal S1 state. It is >during this long T1 state that molecules are attacked by free radicals >and destroyed. > >-- >Runions signature > >(Sent from my cra%#y non-Blackberry electronic device that still has >wires) > > > >********************************* >John Runions, Ph.D. >School of Life Sciences >Oxford Brookes University >Oxford, UK >OX3 0BP > >email: <mailto:[hidden email]>[hidden email] >phone: +44 (0) 1865 483 964 > ><http://www.brookes.ac.uk/lifesci/runions/HTMLpages/index.html%21>Runions' >lab web site > > > >Visit <http://www.illuminatedcell.com/ER.html>The Illuminated Plant >Cell dot com Oxford Brookes Master's in ><http://www.brookes.ac.uk/studying/courses/postgraduate/2007/bmt>Bioima >ging >with Molecular Technology No virus found in this incoming message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.10.24/1954 - Release Date: 15/02/2009 6:09 PM No virus found in this outgoing message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.10.24/1954 - Release Date: 15/02/2009 6:09 PM No virus found in this incoming message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.10.24/1954 - Release Date: 15/02/2009 6:09 PM No virus found in this outgoing message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.10.24/1954 - Release Date: 15/02/2009 6:09 PM |
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Emmanuel Gustin |
Re: Photobleaching mechanism question
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In reply to this post by Guy Cox
Hi,
To give a phycist's point of view: Photobleaching is a quite complex process and oxydative attack is just of the possible pathways for it, although one that is fairly common in biological samples. From the physical perspective, a molecule's stability is defined in terms of energy. To radically simplify it, think of all the possible chemical structures of the molecule and its close neighbours as a landscape: The elevation represents chemical energy, X and Y coordinates represent "chemical structure". Of course there are actually N coordinates involved, with N the rather large number we arrive at by taking three for every atom and substracting translation, rotation, and perhaps internal changes that we still choose to call the same molecule, such as rotations around bonds. The human mind is more comfortable with just two coordinates. Stable molecules are represented by valleys in the landscape, where the depth of the valley below "sea level" is the binding energy of the molecule. There may be a few "mountain passes" allowing transit to different chemical configurations by a simpler way than taking the entire molecule apart and rebuilding it from scratch. Now if we look at the electronic states, the ground state of a stable molecule is near the bottom of the valley of chemical energy -- not at the bottom, for quantum-mechanical reasons, but still quite low in it. It is like a trapped cloud bank. Perhaps a nearby valley, representing a reaction with a nearby oxygen molecule, is deeper, but there is no easy way of getting there. The change in chemical structure can only be achieved by crossing a high barrier, the height of which is the "activation energy" of that reaction. The probability of crossing the barrier decreases exponentially with its height, making it an unlikely event. Unless you can put in extra energy, for example in the form of light -- something chemists do all the time when they perform photochemistry. The singlet excited state has an extra amount of energy that is, in chemical terms, significant. A 488 nm photon has 2.54 eV of energy; the binding energy of for example a C-C single bond is 3.6 eV. So the corresponding "cloud bank" sits higher in the valley. For a normal fluorophore it is still trapped, surrounded by energy barriers, but from the new perspective these are a lot lower. Getting over that barrier is now much more likely than for the ground state, and the excited state may be metastable rather than stable. However, for the singlet excited state there is another event that is still much more likely, at that is fast return to the ground state. The two processes compete for the fate of the excited molecule, and that makes the chemical reaction a relatively rare event, which will only happen in significant amounts if you try often enough. The triplet excited state, to be of importance, actually has to be lower in energy than the singlet state. From the perspective of the triplet state, the energy barriers are a bit higher than for the singlet state, and therefore a chemical reaction may actually be in itself, per time unit, less likely. (Various quantum-mechanical selection rules and interactions make it more complicated than that.) But for the triplet state, the probability of the competing process of return to the ground state has shrunk into insignificance, becoming anything up to a few billion times less likely. Because the two processes still compete for the fate of the molecule, the odds that it will react are a lot better. However, virtually eliminating the return to ground state improves the odds for /any/ process that can interact with an excited state. Oxydation is not the only process that can happen, and not the only one that can cause photobleaching. For example, excited states can also absorb light, with a different absorption spectrum than the ground state. Light might be present at the same wavelength as the original excitation or, if you use multiple lasers or a multiband excitation filter, at a different one. The additional energy supplied by that light may be enough to permit a different chemical reaction, perhaps through ionisation. On the other hand, the photo-excitation of a triplet state to another triplet state may finally allow the molecule to return to a singlet state, followed by a rapid return to the ground state, thus actually reducing the probability of photobleaching. Feedback loops like that probably make the dependency of photobleaching on intensity, exposure time, and wavelength so complicated. Best Regards, Emmanuel |
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Knecht, David |
Re: Photobleaching mechanism question
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Very nice discussion, guys. I think I may finally be getting a handle on this stuff. If I can put this together, the previous discussion of this topic left us with continuous excitation pushing more molecules into the triplet state because you hit a singlet excited molecule with another photon. Thus there was an argument for pulsed excitation (or presumably turning your laser power down) to reduce bleaching so that molecules could decay productively (ie the recent Hell lab publication). Also we have the oxidative attack issue raised here. So is it safe to say that by turning down the intensity of your excitation, you are both reducing the number of molecules that you put into the triplet state, and reducing the amount of ROS generated which would attack that triplet state and oxidize it to a non-fluorescent form? Dave
On Feb 18, 2009, at 5:26 AM, Gustin, Emmanuel [TIBBE] wrote:
Dr. David Knecht Department of Molecular and Cell Biology Co-head Flow Cytometry and Confocal Microscopy Facility U-3125 91 N. Eagleville Rd. University of Connecticut Storrs, CT 06269 860-486-2200 860-486-4331 (fax) |
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Quick question:
Does someone knows if the antibody anti-GFP would cross-react also with the mCherry? I assume the answer is no but i cannot find any specification. I could only found no cross-reactivity of DsRed antibodies with GFP...... Thanks in advance Valeria |
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Along those same lines what about anti-DsRed cross reacting with RFP?
Thanks. ------Original Message------ From: Valeria Berno Sender: Confocal Microscopy List To: [hidden email] ReplyTo: Confocal Microscopy List Sent: Apr 7, 2009 8:19 AM Subject: GFP and mCherry Quick question: Does someone knows if the antibody anti-GFP would cross-react also with the mCherry? I assume the answer is no but i cannot find any specification. I could only found no cross-reactivity of DsRed antibodies with GFP...... Thanks in advance Valeria |
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In reply to this post by Valeria Berno
Cheers, Fred On 7-Apr-09, at 6:19 AM, Valeria Berno wrote:
Fred D. Mast Department of Cell Biology Medical Sciences Building Room 5-14 University of Alberta Edmonton, Alberta, T6G 2H7 Canada Tel: 1-780-492-7407 |
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Hi Valeria, mCherry carries additional 7 aa peptides of GFP origin, i.e.
identical to GFP, at both the N- and C-terminus of the protein. Thus, it could
be a cross-reactivity. mRFP1 is similar to mCherry, and does not carry GFP peptides
suggesting you a simple solution to the problem. Cheers, Vitaly NCI-Frederick, 301-846-6575 From: Confocal Microscopy
List [mailto:[hidden email]] On Behalf Of Fred Mast It depends on the antibody. GFP and mCherry share similar
residues in their N-terminus (the first 16 I believe). You can test this by
conducting a western with your sample. Cheers, Fred On 7-Apr-09, at 6:19 AM, Valeria Berno wrote:
Quick question: Fred D. Mast Department of Cell Biology Medical Sciences Building Room 5-14 University of Alberta Edmonton, Alberta, T6G 2H7 Canada Tel: 1-780-492-7407 |
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In reply to this post by Valeria Berno
We have been working a little with an a-RedFP from Rockland (600-401-379) to
probe for Cherry and the Living Colors JL-8 for GFP. On westerns they both look specific. Marc On 7/4/09 05:19, "Valeria Berno" <[hidden email]> wrote: > Quick question: > > Does someone knows if the antibody anti-GFP would cross-react also with > the mCherry? > > I assume the answer is no but i cannot find any specification. I could > only found no cross-reactivity of DsRed antibodies with GFP...... > > Thanks in advance > > Valeria ------------------ Marc D Green [hidden email] Pombe.net |
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In reply to this post by Valeria Berno
I don't believe you could answer that question without testing. One anti-GFP could cross-react when another wouldn't ... they are similar molecules so it just depends which epitopes you are targeting.
Guy Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 ______________________________________________ Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Valeria Berno Sent: Tuesday, 7 April 2009 10:19 PM To: [hidden email] Subject: GFP and mCherry Quick question: Does someone knows if the antibody anti-GFP would cross-react also with the mCherry? I assume the answer is no but i cannot find any specification. I could only found no cross-reactivity of DsRed antibodies with GFP...... Thanks in advance Valeria No virus found in this incoming message. Checked by AVG. Version: 7.5.557 / Virus Database: 270.11.44/2044 - Release Date: 6/04/2009 6:59 PM No virus found in this outgoing message. Checked by AVG. Version: 7.5.557 / Virus Database: 270.11.44/2044 - Release Date: 6/04/2009 6:59 PM |
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Stephen Bunnell |
Re: GFP and mCherry: Hands on Data
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In reply to this post by Valeria Berno
As noted, DsRed, mRFP1, mCherry, mStrawberry, and tdTomato are derived from
a common source. Of these, the rabbit polyclonal anti-DsRed antibody from BD, #632397 recognizes all but tdTomato by western blotting. It will also IP these proteins. Similarly, GFP, EGFP, CFP, Cerulean, YFP, Citrine, and Venus are derived from a common source, as are the common monomeric variants of the same (the A206K mutation is most typical). We have confirmed that the JL-8 monoclonal from BD recognizes Venus, Cerulean, CFP, YFP, mCFP, mYFP, and EGFP by western blotting. We have not seen it crossreact against DsRed, mRP1, or mCherry. For IPs we use the Abcam antibody 'ab290', which works well for EGFP, YFP, mYFP, CFP, and mCFP, and does not capture DsRed, mRFP1, or mCherry. Good luck, -Steve On 4/7/09 8:19 AM, "Valeria Berno" <[hidden email]> wrote: > Quick question: > > Does someone knows if the antibody anti-GFP would cross-react also with > the mCherry? > > I assume the answer is no but i cannot find any specification. I could > only found no cross-reactivity of DsRed antibodies with GFP...... > > Thanks in advance > > Valeria **************************************************************************** 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] -- To argue with a person who has renounced the use of reason is like administering medicine to the dead. Thomas Paine |
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Boswell, Carl A - (cboswell) |
Re: GFP and mCherry: Hands on Data
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Wow. Ask and ye shall receive.
Thanks Steve. Carl Carl A. Boswell, Ph.D. Molecular and Cellular Biology University of Arizona 520-954-7053 FAX 520-621-3709 ----- Original Message ----- From: "Stephen Bunnell" <[hidden email]> To: <[hidden email]> Sent: Tuesday, April 07, 2009 4:53 PM Subject: Re: GFP and mCherry: Hands on Data As noted, DsRed, mRFP1, mCherry, mStrawberry, and tdTomato are derived from a common source. Of these, the rabbit polyclonal anti-DsRed antibody from BD, #632397 recognizes all but tdTomato by western blotting. It will also IP these proteins. Similarly, GFP, EGFP, CFP, Cerulean, YFP, Citrine, and Venus are derived from a common source, as are the common monomeric variants of the same (the A206K mutation is most typical). We have confirmed that the JL-8 monoclonal from BD recognizes Venus, Cerulean, CFP, YFP, mCFP, mYFP, and EGFP by western blotting. We have not seen it crossreact against DsRed, mRP1, or mCherry. For IPs we use the Abcam antibody 'ab290', which works well for EGFP, YFP, mYFP, CFP, and mCFP, and does not capture DsRed, mRFP1, or mCherry. Good luck, -Steve On 4/7/09 8:19 AM, "Valeria Berno" <[hidden email]> wrote: > Quick question: > > Does someone knows if the antibody anti-GFP would cross-react also with > the mCherry? > > I assume the answer is no but i cannot find any specification. I could > only found no cross-reactivity of DsRed antibodies with GFP...... > > Thanks in advance > > Valeria **************************************************************************** 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] -- To argue with a person who has renounced the use of reason is like administering medicine to the dead. < Thomas Paine |
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Valeria Berno |
Re: GFP and mCherry: Hands on Data
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True
thanks all Valeria Carl Boswell wrote: > Wow. Ask and ye shall receive. > Thanks Steve. > > Carl > > Carl A. Boswell, Ph.D. > Molecular and Cellular Biology > University of Arizona > 520-954-7053 > FAX 520-621-3709 > ----- Original Message ----- From: "Stephen Bunnell" > <[hidden email]> > To: <[hidden email]> > Sent: Tuesday, April 07, 2009 4:53 PM > Subject: Re: GFP and mCherry: Hands on Data > > > As noted, DsRed, mRFP1, mCherry, mStrawberry, and tdTomato are derived > from > a common source. > > Of these, the rabbit polyclonal anti-DsRed antibody from BD, #632397 > recognizes all but tdTomato by western blotting. It will also IP these > proteins. > > Similarly, GFP, EGFP, CFP, Cerulean, YFP, Citrine, and Venus are derived > from a common source, as are the common monomeric variants of the same > (the > A206K mutation is most typical). > > We have confirmed that the JL-8 monoclonal from BD recognizes Venus, > Cerulean, CFP, YFP, mCFP, mYFP, and EGFP by western blotting. We have not > seen it crossreact against DsRed, mRP1, or mCherry. For IPs we use the > Abcam > antibody 'ab290', which works well for EGFP, YFP, mYFP, CFP, and mCFP, > and > does not capture DsRed, mRFP1, or mCherry. > > Good luck, > > -Steve > > > On 4/7/09 8:19 AM, "Valeria Berno" <[hidden email]> wrote: > >> Quick question: >> >> Does someone knows if the antibody anti-GFP would cross-react also with >> the mCherry? >> >> I assume the answer is no but i cannot find any specification. I could >> only found no cross-reactivity of DsRed antibodies with GFP...... >> >> Thanks in advance >> >> Valeria > > **************************************************************************** > > 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] > |
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