Re: [ccp4bb] co-crystallization
As said, your Km is different in mother liquor than in your reaction conditions, but even that is not the end of it: You ligand/substrate might be inducing the slightest of all conformations in your protein that interferes with crystallization, or might block crystal contacts. Then, you can either (1) get no crystals any longer, or (2) get no substrate in your crystals, depending on whichever wins energetically, substrate binding or crystal formation. Therefore, being orders of magnitude above physiological Km/Kd is still not a guarantee for getting crystals of a complex. So, dwelling obsessively on molar ratios and scratching your head becomes irrelevant after a while, one should just do the experiment with a good molar ratio as suggested in this thread, and accept the verdict of the gods of crystallization. Interestingly enough, your substrate-protein complex might crystallize more efficiently, or crystallize in different conditions, so not all is negative. Engin P.S. You are talking about Km and a substrate, but I hope you have a substrate analog, inactive enzyme or you are trying to capture the product complex, otherwise you might be in for a surprise when you solve the structure. - Original Message - From: Juergen Bosch [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, December 1, 2008 6:35:24 AM GMT -08:00 US/Canada Pacific Subject: Re: [ccp4bb] co-crystallization The Km changes with your reservoir, so predictions are limited. In general if you have a low Km this is favourable but not a given that your ligand will be found in the electron density map. As a starting point try a molar ratio of 3 of the ligand to your protein and you can go as high as you want (assuming it's soluble and does not cost you an arm and a leg). 50 fold excess has been used in some cases. And remember for your cryo-step to include your ligand in sufficient amounts ! Jürgen On 1 Dec 2008, at 05:31, yangliuqing wrote: Hello,everyone, I have a question for cocrystallization, is there some relationship between Km value and substrate concentration when making cocrystallization? How can I know the substrate is enough for binding? Thank you very much! liuqing 八卦娱乐包打听，MSN资讯速递帮你忙！ 了解详细！ - Jürgen Bosch University of Washington Dept. of Biochemistry, K-426 1705 NE Pacific Street Seattle, WA 98195 Box 357742 Phone: +1-206-616-4510 FAX: +1-206-685-7002 Web: http://faculty.washington.edu/jbosch
Re: [ccp4bb] co-crystallization
mesters wrote: Yes!, there is: the fraction of occupied protein with substance can be calculated: S / (S + Km) with S being the concentration of the compound. So, if S = Km, half of the sites are occupied (it follows from Michaelis-Menten theory). But- one warning (perhaps obvious but I think worth pointing out): S here is the _free_ concentration. The total concentration you have to add must include the bound ligand also. To achieve 50% saturation you need to add 50% of the protein concentration plus 1 x Km. In enzyme assays, where the enzyme may be picomolar, the amount bound is often insignificant. But in crystallization experiments the protein is often hundreds of micromolar, and Ki's 1 uM, the Ki becomes irrelevant- just add stoichiometric ligand and a little extra. Ed In order to saturate the enzyme for 90,90909 % with the compound: 1) S = 10 x Kd (concentration of S at least 10 times the Kd) and 2) S P (total concentration of S must be larger than total concentration of protein or binding sites) Depending on the solubility of the compound, this is not always possible. In such a case, you need to use DMSO and/or add solid compound to the protein solution and leave it for quite some time for the compound to finally bind to the protein. - J. - yangliuqing wrote: Hello,everyone, I have a question for cocrystallization, is there some relationship between Km value and substrate concentration when making cocrystallization? How can I know the substrate is enough for binding? Thank you very much! liuqing 八卦娱乐包打听，MSN资讯速递帮你忙！ 了解详细！ http://im.live.cn/newsexpress
Re: [ccp4bb] Program to fill unitcell randomly
Ethan A Merritt wrote: On Friday 28 November 2008, Mueller, Juergen-Joachim wrote: Dear all, does anybody know a program to fill an unit cell a,b,c randomly by an arbitrary number of spheres (atoms)? First you would need to define random. Uniform density throughout the lattice? Uniform distribution of neighbor-neighbor distances? Uniform fractional coodinates? Must the placement conform to space group symmetry? Although I am sure it was not intended, this might suggest to some that uniform is equivalent to random- actually they are the opposite: a random distribution would have large areas with nothing and other places where two or three spheres are almost on top of each other. A uniform distribution is, well, uniform. Most programming languages have a function to generate a random number evenly distributed between 0 and 1. Decide how many atoms you want, get three random numbers for each atom, and those are your fractional coordinates of your random spheres. Coordconv will convert to orthogonal angstroms given your cell parameters. Applesoft came with a BASIC program to do this in two dimensions- start with a white screen and paint random pixels black for as long as you let it run. Some black patches developed early on, and some pixels remained white for hours, not because the random number generator was faulty, but because that is the nature of randomness.
Re: [ccp4bb] Program to fill unitcell randomly
On Monday 01 December 2008 10:28:34 Edward A. Berry wrote: Ethan A Merritt wrote: On Friday 28 November 2008, Mueller, Juergen-Joachim wrote: Dear all, does anybody know a program to fill an unit cell a,b,c randomly by an arbitrary number of spheres (atoms)? First you would need to define random. Uniform density throughout the lattice? Uniform distribution of neighbor-neighbor distances? Uniform fractional coodinates? Must the placement conform to space group symmetry? Although I am sure it was not intended, this might suggest to some that uniform is equivalent to random- actually they are the opposite: a random distribution would have large areas with nothing and other places where two or three spheres are almost on top of each other. A uniform distribution is, well, uniform. I fear you are muddying the waters rather than clarifying. What you refer to as random distribution is better described as random sampling from a uniform distribution. Most programming languages have a function to generate a random number evenly distributed between 0 and 1. My point was that simple random sampling is not correct in the context of crystallographic symmetry. If you use this procedure to fill the unit cell, as originally requested, you will violate the crystal symmetry. If you use it to fill the asymmetric unit, then the distribution that describes placement within the full unit cell is no longer the same distribution as you sampled from, since it is now perturbed by the additional placements generated by crystallographic symmetric rather than by random sampling. That may be acceptable, or it may not, depending on the intended application. Decide how many atoms you want, get three random numbers for each atom, and those are your fractional coordinates of your random spheres. Coordconv will convert to orthogonal angstroms given your cell parameters. That was the uniform fractional coordinates case that I listed. It is unlikely to be the correct choice (although as always it depends on the question). This problem is that since it is based on fractional coordinates rather than the true cartesian coordinates, the resulting density of atomic centers will be strongly anisotropic. The density along each axis will be inversely proportional to the cell edge. You would do better to define a cartesian coordinate grid that fills the region of interest, and then assign an atom to each grid point with probability 1/N. This produces artifacts of its own, of course, since the distribution of interatomic distances is now discrete rather than continuous. The question what is random? is very deep, and the answer depends strongly on the intended application. -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742
[ccp4bb] Diffraction problems
Hi All, I am working on a 80 KDa single amino acid mutant of a protein expressed in E.Coli. The protein is very pure and I get very nice looking crystals in different conditions. I used different cryo protectants and tried room temp testing of diffraction for these crystals. I don't get any diffraction at all in most of them. A few were salt, so I was sure that there is no other instrumental problem. If anyone has faced the same problem or someone can give some insight about it other than the papers that suggest post crystallization treatments , something that has worked for them, I would be grateful Thanks Garima
[ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?
Dear fellow crystallographers, This is a question which is not CCP4-related. Is anybody aware of a protein which is known to be a dimer in solution (say by SEC), and yet crystallizes as a monomer? Wouldn't the high concentration in the crystallization drop further favor dimerization? In other words, if a protein crystallizes as a monomer, can I conclude that it does not form biologically relevant dimers in solution? Thank you in advance for your replies. Thierry * This message and any attachments are solely for the intended recipient. If you are not the intended recipient, disclosure, copying, use or distribution of the information included in this message is prohibited -- Please immediately and permanently delete.
Re: [ccp4bb] co-crystallization
Just to be a pedantic pain - Km is not necessarily Kd. I think that assumption only holds if the chemical step following substrate binding is rate-limiting. Phoebe Original message Date: Mon, 1 Dec 2008 15:34:59 +0100 From: mesters [EMAIL PROTECTED] Subject: Re: [ccp4bb] co-crystallization To: CCP4BB@JISCMAIL.AC.UK Yes!, there is: the fraction of occupied protein with substance can be calculated: S / (S + Km) with S being the concentration of the compound. So, if S = Km, half of the sites are occupied (it follows from Michaelis-Menten theory). In order to saturate the enzyme for 90,90909 % with the compound: 1) S = 10 x Kd (concentration of S at least 10 times the Kd) and 2) S P (total concentration of S must be larger than total concentration of protein or binding sites) Depending on the solubility of the compound, this is not always possible. In such a case, you need to use DMSO and/or add solid compound to the protein solution and leave it for quite some time for the compound to finally bind to the protein. - J. - yangliuqing wrote: Hello,everyone, I have a question for cocrystallization, is there some relationship between Km value and substrate concentration when making cocrystallization? How can I know the substrate is enough for binding? Thank you very much! liuqing 八卦娱乐包打听，MSN资讯速递帮你忙！ 了解详细！ http://im.live.cn/newsexpress -- Dr. Jeroen R. Mesters Gruppenleiter Strukturelle Neurobiologie und Kristallogenese Institut für Biochemie, Universität zu Lübeck Zentrum für Medizinische Struktur- und Zellbiologie Ratzeburger Allee 160, D-23538 Lübeck Tel: +49-451-5004065, Fax: +49-451-5004068 Http://www.biochem.uni-luebeck.de Http://www.iobcr.org Http://www.selfish-brain.org Http://www.opticryst.org -- If you can look into the seeds of time and say which grain will grow and which will not - speak then to me (Macbeth) -- Phoebe A. Rice Assoc. Prof., Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 RNA is really nifty DNA is over fifty We have put them both in one book Please do take a really good look http://www.rsc.org/shop/books/2008/9780854042722.asp
Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?
Thank you Patrick for the reply, as well to another person who has replied directly to me. Please provide with examples if you know of any (say a reference or a PDB id ), as it would allow for comparison between published results and my own crystallization system. To answer your points: The crystallization conditions in the case at hand are as follow : the precipitant is PEG, the amount of salt is relatively low (0.1 M buffer + some NaCl etc.), and the pH is 8.0. The SEC experimental conditions are not too far away from the crystallization conditions except, of course, for the presence of PEG. Thierry From: Patrick Loll [mailto:[EMAIL PROTECTED] Sent: Monday, December 01, 2008 05:59 PM To: Fischmann, Thierry Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer? depends on the crystallization conditions (and the SEC conditions). E.g., if the crystals grow in 4 M salt at pH 3, maybe that disrupts a complex that forms under more physiological conditions On 1 Dec 2008, at 5:47 PM, Fischmann, Thierry wrote: Dear fellow crystallographers, This is a question which is not CCP4-related. Is anybody aware of a protein which is known to be a dimer in solution (say by SEC), and yet crystallizes as a monomer? Wouldn't the high concentration in the crystallization drop further favor dimerization? In other words, if a protein crystallizes as a monomer, can I conclude that it does not form biologically relevant dimers in solution? Thank you in advance for your replies. Thierry * This message and any attachments are solely for the intended recipient. If you are not the intended recipient, disclosure, copying, use or distribution of the information included in this message is prohibited -- Please immediately and permanently delete. --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 [EMAIL PROTECTED] * This message and any attachments are solely for the intended recipient. If you are not the intended recipient, disclosure, copying, use or distribution of the information included in this message is prohibited -- Please immediately and permanently delete.
Re: [ccp4bb] Quikchange cloning: Insert length
Have you seen these papers: M Geiser, R Cebe, D Drewello, and R Schmitz. Integration of pcr fragments at any specific site within cloning vectors without the use of restriction enzymes and dna ligase. Biotechniques, 31(1):88–90, 2001. W Wang and B A Malcolm. Two-stage pcr protocol allowing introduction of multiple mutations, deletions and insertions using quikchange site-directed mutagenesis. Biotechniques, 26(4):680–682, 1999. If i recall correctly, Geiser el al inserted a 1kb fragment with a modified Quickchange method. On Mon, Dec 1, 2008 at 12:54 PM, Raji Edayathumangalam [EMAIL PROTECTED] wrote: Hi Folks, Sorry for the non-xtallo posting. I am curious to hear what is the longest insert anyone has cloned using a modification of the Quikchange cloning strategy. Basically, ligation-independent cloning by strapping on homologous regions of the vector onto the primers which also generate the initial PCR product. I plan to proceed with my insert which is ~ 2kb and am curious to get some feedback if you have successfully cloned inserts 1.5kb using the above strategy. Many thanks. Raji Michael Giffin The Scripps Research Institute Department of Molecular and Experimental Medicine 10550 North Torrey Pines Road, MEM-131 La Jolla, CA 92037 email: [EMAIL PROTECTED] lab: 858-784-7758
Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?
Yo Thierry: The periplasmic domain of the aspartate receptor, in the absence of ligand, 1lih, is a dimer, but crystallizes as a monomer in the sense that there is one monomer per asymmetric unit. There is a disulphide bond between two Cys36 that maintains it as a dimer (and indeed reduction of this bond inhibits crystallization). Each of two ligand binding sites spans both monomers. So based on that, the biologically relevant form is definitely a dimer, so you can't conclude otherwise based on the fact that it crystallizes as one monomer per asymmetric unit. Now if it were to crystallize in a space group lacking a crystallographic 2-fold coincident with the natural dimer axis, that might be a different story. When you add aspartate, it crystallizes as a dimer with only one of two potential binding sites occupied by the ligand. Bill William G. Scott Contact info: http://chemistry.ucsc.edu/~wgscott/ On Dec 1, 2008, at 2:47 PM, Fischmann, Thierry wrote: Dear fellow crystallographers, This is a question which is not CCP4-related. Is anybody aware of a protein which is known to be a dimer in solution (say by SEC), and yet crystallizes as a monomer? Wouldn't the high concentration in the crystallization drop further favor dimerization? In other words, if a protein crystallizes as a monomer, can I conclude that it does not form biologically relevant dimers in solution? Thank you in advance for your replies. Thierry * This message and any attachments are solely for the intended recipient. If you are not the intended recipient, disclosure, copying, use or distribution of the information included in this message is prohibited -- Please immediately and permanently delete.
Re: [ccp4bb] Quikchange cloning: Insert length
I am curious to hear what is the longest insert anyone has cloned using a modification of the Quikchange cloning strategy. Basically, ligation-independent cloning by strapping on homologous regions of the vector onto the primers which also generate the initial PCR product. I plan to proceed with my insert which is ~ 2kb and am curious to get some feedback if you have successfully cloned inserts 1.5kb using the above strategy. Here, the maximum we've tried was 3 kbp and it worked just as well as with the the smaller fragments. Most of the Quickchange cloning we did involved 1500-300 bp and no obvious differences in success rate vs size come to mind. Dima
Re: [ccp4bb] Quikchange cloning: Insert length
It helps to remember that PCR does have an upper limit of total double-stranded DNA content (regardless of its molarity!) after which it does not work any more (due to the competition of the polymerase for non-specific dsDNA versus primer-substrate pairs). Therefore the theoretical limits on this form of cloning are imposed by the molarity of two DNA fragments, the fidelity/processivity of your PCR enzyme, and the quality of the reaction mix. In practice it helps to also consider the frequency of PCR errors as well as certain other factors. I've done this sort of cloning with ~4 KB inserts, however the use of such long inserts required optimization of conditions and was not 100% successful (unlike the relatively simple insertion of 0.5-1.5 KB). With long inserts it is *critical* to have highly purified and highly homogenous starter DNA as well as (individually selectable) a correct template/insert ratio. Artem -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Raji Edayathumangalam Sent: Monday, December 01, 2008 3:54 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Quikchange cloning: Insert length Hi Folks, Sorry for the non-xtallo posting. I am curious to hear what is the longest insert anyone has cloned using a modification of the Quikchange cloning strategy. Basically, ligation-independent cloning by strapping on homologous regions of the vector onto the primers which also generate the initial PCR product. I plan to proceed with my insert which is ~ 2kb and am curious to get some feedback if you have successfully cloned inserts 1.5kb using the above strategy. Many thanks. Raji
Re: [ccp4bb] Program to fill unitcell randomly
On Monday 01 December 2008 15:07:56 Edward A. Berry wrote: Thanks, Ethan, For your third point- I realized (after sending) that the distribution would be stretched along the long axis- but actually I'm having a hard time coming to grips with that conceptually- if there are n atoms in the cell, they will necessarily be distributed more sparsely in projection along the long cell axis than the short axes, and you can't add more atoms along the long axis to increase it's density without increasing density along the other two. Heh. A new Monty Hall problem to demonstrate how probability distributions mess with our minds. You are of course correct that you cannot increase the number of atoms in a fixed cell without increasing the density in all directions through the crystal. And I phrased my original description badly, if I made it sound like somehow this was possible. I should not have used the word density; perhaps mean distance between particles along the path is a better wording. The main point I was trying to make is that if you ask for a random distribution in 3-space without mentioning crystals, you are probably expecting certain properties. In particular, unless otherwise stated, the criteria for random would normally include isotropy. Or at least that's what I would assume. A distribution that had significantly different properties in different directions would not be considered random in this context. But a crystal lattice is the antithesis of random in this sense, because it imposes by definition a requirement for an exact direction-dependent repeat spacing determined by the lattice. You cannot simultaneously satisfy a requirement for uniform isotropic distribution in 3-space and a requirement for crystalline symmetry, except in the degenerate case of density = 0. As for the rest, I think it is semantics or a question how precisely we want to say something. Yes, what I was describing was a randomly chosen sample from a uniform probability distribution, but it is this sample that the OP is requesting- so I would rephrase your question: does he want _a random sampling from_ a uniform probability distribution throughout the lattice, or ... Ed Ethan Merritt wrote: On Monday 01 December 2008 10:28:34 Edward A. Berry wrote: Ethan A Merritt wrote: On Friday 28 November 2008, Mueller, Juergen-Joachim wrote: Dear all, does anybody know a program to fill an unit cell a,b,c randomly by an arbitrary number of spheres (atoms)? First you would need to define random. Uniform density throughout the lattice? Uniform distribution of neighbor-neighbor distances? Uniform fractional coodinates? Must the placement conform to space group symmetry? Although I am sure it was not intended, this might suggest to some that uniform is equivalent to random- actually they are the opposite: a random distribution would have large areas with nothing and other places where two or three spheres are almost on top of each other. A uniform distribution is, well, uniform. I fear you are muddying the waters rather than clarifying. What you refer to as random distribution is better described as random sampling from a uniform distribution. Most programming languages have a function to generate a random number evenly distributed between 0 and 1. My point was that simple random sampling is not correct in the context of crystallographic symmetry. If you use this procedure to fill the unit cell, as originally requested, you will violate the crystal symmetry. If you use it to fill the asymmetric unit, then the distribution that describes placement within the full unit cell is no longer the same distribution as you sampled from, since it is now perturbed by the additional placements generated by crystallographic symmetric rather than by random sampling. That may be acceptable, or it may not, depending on the intended application. Decide how many atoms you want, get three random numbers for each atom, and those are your fractional coordinates of your random spheres. Coordconv will convert to orthogonal angstroms given your cell parameters. That was the uniform fractional coordinates case that I listed. It is unlikely to be the correct choice (although as always it depends on the question). This problem is that since it is based on fractional coordinates rather than the true cartesian coordinates, the resulting density of atomic centers will be strongly anisotropic. The density along each axis will be inversely proportional to the cell edge. You would do better to define a cartesian coordinate grid that fills the region of interest, and then assign an atom to each grid point with probability 1/N. This produces artifacts of its own, of course, since the distribution of interatomic distances is now discrete rather than continuous. The question what is random? is very deep, and the answer
Re: [ccp4bb] Program to fill unitcell randomly
(I don't remember the motivation for the original question.) Shake-and-Bake used to generate random atoms in an asymmetric unit, and the program kept the atoms spaced by at least a bond length. Since PDB entry 2erl, I am not up to date on Shake-and-Bake's current set of tricks. The crystal for 2erl was so densely packed that random atoms spaced by 1.5A produced very good starting phase sets. (but I still don't know what's the motivation underlying the current discussion.) Did that help?, Dan On Mon, 1 Dec 2008, Ethan Merritt wrote: On Monday 01 December 2008 10:28:34 Edward A. Berry wrote: Ethan A Merritt wrote: On Friday 28 November 2008, Mueller, Juergen-Joachim wrote: Dear all, does anybody know a program to fill an unit cell a,b,c randomly by an arbitrary number of spheres (atoms)? First you would need to define random. Uniform density throughout the lattice? Uniform distribution of neighbor-neighbor distances? Uniform fractional coodinates? Must the placement conform to space group symmetry? Although I am sure it was not intended, this might suggest to some that uniform is equivalent to random- actually they are the opposite: a random distribution would have large areas with nothing and other places where two or three spheres are almost on top of each other. A uniform distribution is, well, uniform. I fear you are muddying the waters rather than clarifying. What you refer to as random distribution is better described as random sampling from a uniform distribution. Most programming languages have a function to generate a random number evenly distributed between 0 and 1. My point was that simple random sampling is not correct in the context of crystallographic symmetry. If you use this procedure to fill the unit cell, as originally requested, you will violate the crystal symmetry. If you use it to fill the asymmetric unit, then the distribution that describes placement within the full unit cell is no longer the same distribution as you sampled from, since it is now perturbed by the additional placements generated by crystallographic symmetric rather than by random sampling. That may be acceptable, or it may not, depending on the intended application. Decide how many atoms you want, get three random numbers for each atom, and those are your fractional coordinates of your random spheres. Coordconv will convert to orthogonal angstroms given your cell parameters. That was the uniform fractional coordinates case that I listed. It is unlikely to be the correct choice (although as always it depends on the question). This problem is that since it is based on fractional coordinates rather than the true cartesian coordinates, the resulting density of atomic centers will be strongly anisotropic. The density along each axis will be inversely proportional to the cell edge. You would do better to define a cartesian coordinate grid that fills the region of interest, and then assign an atom to each grid point with probability 1/N. This produces artifacts of its own, of course, since the distribution of interatomic distances is now discrete rather than continuous. The question what is random? is very deep, and the answer depends strongly on the intended application. -- *** Daniel Anderson, Ph.D. Email: [EMAIL PROTECTED] Phone: 310-206-3642 Fax: 310-206-3914 Howard Hughes Medical Institute at University of California Los Angeles Lab: Paul Boyer Hall Room 219 For US Postal Service and 2-dimensional, use: Box 951662 MRL5-748 Los Angeles, CA 90095-1662 For UPS, FedEx, DHL, or 2.5-3-dimensional, use: Boyer 219 611 Charles Young Drive East Los Angeles, CA 90095-1570 USA
[ccp4bb] Offtopic: FAD enzymatic assay
was working to set up an FAD enzymatic assay. I wished to be able to use 450nM to continuously monitor the progress of the reaction. The substrate I used is the natural substrate of the enzyme and the protein is recombinant protein and I assume it's active since I do see changes in TLC plate. But no signal was observed at all using a spectrometer. Does anyone here have any suggestions on how to correctly carry out FAD enzymatic assay? Also I heard from one postdoc here that FADH2 is quickly reoxidized by O2. Is that right? Should I do the assay under anaerobic condition? If so, how? Your help is highly appreciated! Thanks! Best regards, Mike
Re: [ccp4bb] Offtopic: FAD enzymatic assay
Mike: The trick may be doing the assay under anaerobic condition, especially if the FAD cofactor is sensitive to oxygen. You need an anaerobic train and tanometers for the experiment. Good refs: Hille, R. Biochemistry 1991 Sep 3;30(35):8522-9. Electron transfer within xanthine oxidase: a solvent kinetic isotope effect study. Hunt, J., Massey, V. J. Biol. Chem. 1994 Jul 22;269(29):18904-14 Studies of the reductive half-reaction of milk xanthine dehydrogenase. Cheers, Hongnan Cao UC Riverside Date: Mon, 1 Dec 2008 18:07:57 -0800From: [EMAIL PROTECTED]: [ccp4bb] Offtopic: FAD enzymatic assayTo: CCP4BB@JISCMAIL.AC.UK was working to set up an FAD enzymatic assay. I wished to be able to use 450nM to continuously monitor the progress of the reaction. The substrate I used is the natural substrate of the enzyme and the protein is recombinant protein and I assume it's active since I do see changes in TLC plate. But no signal was observed at all using a spectrometer. Does anyone here have any suggestions on how to correctly carry out FAD enzymatic assay? Also I heard from one postdoc here that FADH2 is quickly reoxidized by O2. Is that right? Should I do the assay under anaerobic condition? If so, how? Your help is highly appreciated!Thanks!Best regards,Mike _ MSN安全保护中心，免费修复系统漏洞，保护MSN安全！ http://im.live.cn/safe/center/
