Freezing fluorophores
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Hi, What’s your opinion about freezing fluorescently conjugated antibodies? I know for example that the Qdots are destroyed by freezing, but how about others? /fredrik -------------------------------------- Fredrik Wermeling, PhD-student Karolinska Institutet Department of Medicine Unit of Clinical Allergy Research L2:04 Karolinska Hospital SE-171 76 Stockholm Sweden phone: +46-8-51776696 mail: [hidden email] fax: +46-8-335724 -------------------------------------- |
 
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Martin Wessendorf-2 |
Re: Freezing fluorophores
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Fredrik Wermeling wrote: > What’s your opinion about freezing fluorescently conjugated antibodies? > I know for example that the Qdots are destroyed by freezing, but how > about others? Dear Fredrik-- In my experience, freezing is almost always bad. The signal-to-noise ratio of the secondary antibodies that I use (C2, Cy3 and Cy5 conjugates) goes from being superb to being poor with one freeze-thaw cycle. The problem with freezing appears to be denaturation of the IgG to which they're conjugated. The explanation that I've heard is that as an antibody solution freezes, the pure water crystallizes first. What remains has a higher concentration of salts. As freezing progresses, the concentration of salt in the liquid fraction becomes higher and higher, ultimately resulting in denaturation of the protein present in that fraction. By that logic, if you need to freeze, very rapid freezing should be better than slow freezing. As an alternative to freezing, you can store your antibodies at -20 C without freezing by diluting them 1:1 in glycerol. (That trick comes from Bill Stegeman at Jackson.) In my experience that works well. Good luck! Martin Wessendorf -- Martin Wessendorf, Ph.D. office: (612) 626-0145 Assoc Prof, Dept Neuroscience lab: (612) 624-2991 University of Minnesota Preferred FAX: (612) 624-8118 6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009 Minneapolis, MN 55455 **MY E-MAIL ADDRESS HAS CHANGED. PLEASE USE [hidden email]** |
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In reply to this post by Fredrik Wermeling
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal We aliquot and freeze our dye-conjugated secondaries at -20 (not a frost-free freezer, but real -20C) all the time. They last for at least a year under these conditions, and in most cases, several years. Julian >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >Hi, > >What's your opinion about freezing fluorescently conjugated >antibodies? I know for example that the Qdots are destroyed by >freezing, but how about others? > >/fredrik > > > >-------------------------------------- >Fredrik Wermeling, PhD-student >Karolinska Institutet >Department of Medicine >Unit of Clinical Allergy Research >L2:04 Karolinska Hospital >SE-171 76 Stockholm >Sweden > >phone: +46-8-51776696 >mail: [hidden email] >fax: +46-8-335724 >-------------------------------------- -- Julian P.S. Smith III Dept. of Biology Winthrop University 520 Cherry Rd. Rock Hill, SC 29733 803-323-2111 x6427 (vox) 803-323-3448 (fax) 803-524-2347 (cell) |
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Mullen, Steven Francis (UMC-Student) |
FW: Freezing fluorophores
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In reply to this post by Fredrik Wermeling
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Martin is probably correct in his description of the potential damage of freezing on antibodies. When one assesses a phase diagram for NaCl, the salt concentration will go from ~ 0.15 Molar (in an isotonic solution) to about 4 Molar when freezing to -20. The concentration will increase further at lower temperatures. Adding the glycerol at 50% will prevent ice crystallization and not allow the freeze-concentration of the salts (and any other solutes), at least up to certain temperatures and cooling rates. I routinely freeze conjugated antibodies in a -80 freezer after adding 50% glycerol with no apparent loss in function (however, I do not use a wide variety of antibodies and conjugates, so this may not be universal). However, the colligative effect of glycerol should be universal. Withough "running the numbers" (i.e. do the calculations), the rate of freezing may not matter in 50% glycerol. I don't have a phase diagram for glycerol/NaCl/H2O handy, but the homogenous nucleation temperature of such a solution is likely to be very low, perhaps even below -80. Hence, one should expect no ice formation in such a solution. My antibody aliquots appear liquid when I pull them out of the -80, but I can't tell visually if any ice has formed. What appears to be the case is that, if any ice does form, it is very little. I doubt if any ice forms at -20. It would be an interesting exercise to use first principles and calculate how much glycerol is needed to prevent ice formation at -20 (should be less than 50%) and test that against no glycerol, or a dilution series of glycerol and determine at what salt concentration one begins to see failure of the immunochemistry reaction. Sounds like a nice science fair project. For those interested in learning more about the use of phase diagram information to predict solution composition during freezing of biological samples, and how this was done successfully to predict the loss of red blood cells as a function of glycerol concentration, refer to the original papers by Lames Lovelock: [1] J.E. Lovelock, The denaturation of lipid-protein complexes as a cause of damage by freezing. The Proceedings of the Royal Society of London B 147 (1957) 427-434. [2] J.E. Lovelock, The haemolysis of human red blood cells by freezing and thawing. Biochim Biophys Acta 10 (1953) 414-426. [3] J.E. Lovelock, The mechanism of the protective action of glycerol against haemolysis by freezing and thawing. Biochim Biophys Acta 11 (1953) 28-36. ...and the following paper is a nice review of the utility of phase diagrams in cryobiology: [1] F.H. Cocks, and W.E. Brower, Phase diagram relationships in cryobiology. Cryobiology 11 (1974) 340-58. Steven F. Mullen Ph.D. Postdoctoral Research Fellow The University of Missouri Department of Veterinary Pathobiology Cryobiology Laboratory E124 Vet Med 1600 E Rollins St Columbia MO 65201 Phone: 573-882-5506 Fax: 573-884-7521 [hidden email] Fredrik Wermeling wrote: > What's your opinion about freezing fluorescently conjugated antibodies? > I know for example that the Qdots are destroyed by freezing, but how > about others? Dear Fredrik-- In my experience, freezing is almost always bad. The signal-to-noise ratio of the secondary antibodies that I use (C2, Cy3 and Cy5 conjugates) goes from being superb to being poor with one freeze-thaw cycle. The problem with freezing appears to be denaturation of the IgG to which they're conjugated. The explanation that I've heard is that as an antibody solution freezes, the pure water crystallizes first. What remains has a higher concentration of salts. As freezing progresses, the concentration of salt in the liquid fraction becomes higher and higher, ultimately resulting in denaturation of the protein present in that fraction. By that logic, if you need to freeze, very rapid freezing should be better than slow freezing. As an alternative to freezing, you can store your antibodies at -20 C without freezing by diluting them 1:1 in glycerol. (That trick comes from Bill Stegeman at Jackson.) In my experience that works well. Good luck! Martin Wessendorf -- Martin Wessendorf, Ph.D. office: (612) 626-0145 Assoc Prof, Dept Neuroscience lab: (612) 624-2991 University of Minnesota Preferred FAX: (612) 624-8118 6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009 Minneapolis, MN 55455 **MY E-MAIL ADDRESS HAS CHANGED. PLEASE USE [hidden email]** -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Martin Wessendorf Sent: Thursday, January 17, 2008 10:01 AM To: [hidden email] Subject: Re: Freezing fluorophores Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Fredrik Wermeling wrote: > What's your opinion about freezing fluorescently conjugated antibodies? > I know for example that the Qdots are destroyed by freezing, but how > about others? Dear Fredrik-- In my experience, freezing is almost always bad. The signal-to-noise ratio of the secondary antibodies that I use (C2, Cy3 and Cy5 conjugates) goes from being superb to being poor with one freeze-thaw cycle. The problem with freezing appears to be denaturation of the IgG to which they're conjugated. The explanation that I've heard is that as an antibody solution freezes, the pure water crystallizes first. What remains has a higher concentration of salts. As freezing progresses, the concentration of salt in the liquid fraction becomes higher and higher, ultimately resulting in denaturation of the protein present in that fraction. By that logic, if you need to freeze, very rapid freezing should be better than slow freezing. As an alternative to freezing, you can store your antibodies at -20 C without freezing by diluting them 1:1 in glycerol. (That trick comes from Bill Stegeman at Jackson.) In my experience that works well. Good luck! Martin Wessendorf -- Martin Wessendorf, Ph.D. office: (612) 626-0145 Assoc Prof, Dept Neuroscience lab: (612) 624-2991 University of Minnesota Preferred FAX: (612) 624-8118 6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009 Minneapolis, MN 55455 **MY E-MAIL ADDRESS HAS CHANGED. PLEASE USE [hidden email]** |
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In reply to this post by Fredrik Wermeling
Search the CONFOCAL archive at
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Hi,
by keeping them at 4C and opening the tubes in the sterile hood and using
sterile tips, we manage to keep them functional for a year or
so.
Marc
------------------------------------------------ De : Fredrik Wermeling [mailto:[hidden email]] Envoyé : 17 janvier 2008 10:51 À : [hidden email] Objet : Freezing fluorophores Hi, Whats your opinion about freezing
fluorescently conjugated antibodies? I know for example that the Qdots are
destroyed by freezing, but how about others? /fredrik -------------------------------------- Fredrik Wermeling, PhD-student Karolinska Institutet Department of Medicine Unit of Clinical Allergy Research L2:04 Karolinska Hospital SE-171 76 Stockholm Sweden phone: +46-8-51776696 mail: [hidden email] fax: +46-8-335724 -------------------------------------- |
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In reply to this post by Julian Smith III
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal We have the same experience as Julian - fluorescently-tagged antibodies in 2-5 microlitre aliquots frozen at -80 have lasted fine since I bought them in 2000 with no perceptible increase in background though some decrease in staining intensity. cheers, Rosmeary Rosemary White [hidden email] CSIRO Plant Industry ph. 61 (0)2-6246 5475 GPO Box 1600 fax. 61 (0)2-6246 5334 Canberra, ACT 2601 Australia On 18/1/08 3:01 AM, "Julian Smith III" <[hidden email]> wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > We aliquot and freeze our dye-conjugated secondaries at -20 (not a > frost-free freezer, but real -20C) all the time. They last for at > least a year under these conditions, and in most cases, several years. > Julian > >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> Hi, >> >> What's your opinion about freezing fluorescently conjugated >> antibodies? I know for example that the Qdots are destroyed by >> freezing, but how about others? >> >> /fredrik >> >> >> >> -------------------------------------- >> Fredrik Wermeling, PhD-student >> Karolinska Institutet >> Department of Medicine >> Unit of Clinical Allergy Research >> L2:04 Karolinska Hospital >> SE-171 76 Stockholm >> Sweden >> >> phone: +46-8-51776696 >> mail: [hidden email] >> fax: +46-8-335724 >> -------------------------------------- > |
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