[ccp4bb] off topic question BIAcore problem
Dear all, Recently I have measure a set of kinetic data of a receptor ligand interaction using BIAcore 3000. To my surprise, the dissociation rate is very low (~ 10e-6). During the measurement, I use a long dissociation time (2 hours) .I repeat several time which give very similar results. So I am wondering if the BIAcore can measure such a low off-rate kinetic. What is the limitation of BIAcore? Any review about that? Thanks in advance. Xianchi Dong Research Fellow Children's Hospital Boston Harvard Medical School
Re: [ccp4bb] off topic question BIAcore problem
I assume you use CM5 chips ? I further assume you run at pH 7.5 perhaps ? What's the pI of your analyte ? 7.5 ? Do you get significant binding to your reference cell under the conditions you are running ? You might get rebinding to your negatively charged surface and the dissociation you are measuring might not really be correct (masked through rebinding) Check that first I would say. You can measure low picomolar dissociations. I recently had one with ~80 pM. Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu On Feb 20, 2013, at 12:03 PM, xianchi dong wrote: Dear all, Recently I have measure a set of kinetic data of a receptor ligand interaction using BIAcore 3000. To my surprise, the dissociation rate is very low (~ 10e-6). During the measurement, I use a long dissociation time (2 hours) .I repeat several time which give very similar results. So I am wondering if the BIAcore can measure such a low off-rate kinetic. What is the limitation of BIAcore? Any review about that? Thanks in advance. Xianchi Dong Research Fellow Children's Hospital Boston Harvard Medical School
Re: [ccp4bb] off topic question BIAcore problem
Dear Xianchi, unfortunately, dissociation rate constant kd 10^-6 s^-1 was just beyond the limit of Biacore in 1999 (e.g. see Fig. 1 in http://www.ncbi.nlm.nih.gov/pubmed/10556876). I am not sure about these days. As Juergen noted, you may have a problem with rebinding (you could try to reduce amount of the ligand on the chip, see other tricks here http://users.path.ox.ac.uk/~vdmerwe/internal/spr.pdf). Regarding reviews, David Myszka writes enjoyable reviews on SPR. Hope that helps, Tomas On Wed, Feb 20, 2013 at 5:03 PM, xianchi dong dongxian...@gmail.com wrote: Dear all, Recently I have measure a set of kinetic data of a receptor ligand interaction using BIAcore 3000. To my surprise, the dissociation rate is very low (~ 10e-6). During the measurement, I use a long dissociation time (2 hours) .I repeat several time which give very similar results. So I am wondering if the BIAcore can measure such a low off-rate kinetic. What is the limitation of BIAcore? Any review about that? Thanks in advance. Xianchi Dong Research Fellow Children's Hospital Boston Harvard Medical School Dea
Re: [ccp4bb] off topic question BIAcore problem
Hi Xianchi, First of all: a very slow dissociation rate can also be an artifact: the analyte can be simply precipitating on the surface. You do need to rule this out by proper controls. But there is no rule saying that 10e-6 s-1 off rate is not realistic. Even with protein-small molecule binding, one can get extremely slow dissociation if the interaction is very strong. For example, the dissociation of biotin from streptavidin has a rate constant of the 10e-6 s(-1) order. A very slow dissociation rate is often (if not always) correlated with very tight binding (for example KD in picomolar range or even smaller). What is your calculated KD? The binding phase of the BIAcore curves should also reflect the fact that the off rate is low (the Kon-obs has an off rate term). We have measured some pico molar bindings with BIAcore in the past. It is doable, but difficult. You may also want to consider in-solution methods (so that you do not need to worry about artifacts caused by the surface), such as ITC (by competition method), or fluorescence-based methods. For BIAcore, when working with very strong bindings (KD 100pM), there are a few things to consider: 1) You may find great difficulties regenerating the chip - probably the biggest concern with BIAcore (considering the ridiculous price of chips). 2) How much RU should you conjugate to the chip? With strong interactions, as low as possible amount of your ligand should be labeled on the chip, for 4 purposes: a) to make regeneration easier; b) to reduce rebinding effect; c) to reduce mass transfer effect; d) to make sure you do not take away significant amount of analyte from the solution (discussed in 3)). 3) If you plan to span the KD range with the analyte, then if the KD is in picomolar molar range, you are supplying the surface with extremely dilute analyte solutions. In such case, you need to calculate if your flow rate is high enough, to compensate the loss of solute due to binding to surface, otherwise the real concentration of the analyte in the mobile phase will be much lower than the assumed analyte concentration. 4) The association phase of the BIAcore experiment is also affected by the dissociation rate. The observed binding rate Kon-obs contains a Koff term. When you are loading the analyte at near KD concentrations, the binding will take similar amount of time as the dissociation phase to reach plateau. The Kon-obs also contains a concentration terml, so the time required for reaching plateau will be shorter and shorter when you load with higher concentrations of the analytes. 5) The only proper way of labeling chip for slow dissociations is by covalent means. HTH, Zhijie From: xianchi dong Sent: Wednesday, February 20, 2013 12:03 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] off topic question BIAcore problem Dear all, Recently I have measure a set of kinetic data of a receptor ligand interaction using BIAcore 3000. To my surprise, the dissociation rate is very low (~ 10e-6). During the measurement, I use a long dissociation time (2 hours) .I repeat several time which give very similar results. So I am wondering if the BIAcore can measure such a low off-rate kinetic. What is the limitation of BIAcore? Any review about that? Thanks in advance. Xianchi Dong Research Fellow Children's Hospital Boston Harvard Medical School
Re: [ccp4bb] off topic question BIAcore problem
Thanks a lot for the kind reply. I used CM5 chip at pH 7.4. I am not quite sure about the PI of my protein but I have different truncations of my protein which behaved similarly. I can fit my data quite good with the Chi2 around 1 and Rmax 150. The kon is about 10e4 and the KD is around 0.2 nM. The concentration of analytes I used was from 100 nM to 5 nM. I used a control buffer which can disrupt the binding. In that buffer, no binding was observed. I also used fluorescence anisotropy to measure the Ki of the interaction which showed a 1 nM binding compared with the 0.2 nM binding in the SPR assay. On Wed, Feb 20, 2013 at 12:03 PM, xianchi dong dongxian...@gmail.comwrote: Dear all, Recently I have measure a set of kinetic data of a receptor ligand interaction using BIAcore 3000. To my surprise, the dissociation rate is very low (~ 10e-6). During the measurement, I use a long dissociation time (2 hours) .I repeat several time which give very similar results. So I am wondering if the BIAcore can measure such a low off-rate kinetic. What is the limitation of BIAcore? Any review about that? Thanks in advance. Xianchi Dong Research Fellow Children's Hospital Boston Harvard Medical School
Re: [ccp4bb] off topic question BIAcore problem
Well that looks pretty real then. You might have wrong concentrations in one or the other experiment perhaps hence the difference. Jürgen On Feb 20, 2013, at 5:45 PM, xianchi dong wrote: Thanks a lot for the kind reply. I used CM5 chip at pH 7.4. I am not quite sure about the PI of my protein but I have different truncations of my protein which behaved similarly. I can fit my data quite good with the Chi2 around 1 and Rmax 150. The kon is about 10e4 and the KD is around 0.2 nM. The concentration of analytes I used was from 100 nM to 5 nM. I used a control buffer which can disrupt the binding. In that buffer, no binding was observed. I also used fluorescence anisotropy to measure the Ki of the interaction which showed a 1 nM binding compared with the 0.2 nM binding in the SPR assay. On Wed, Feb 20, 2013 at 12:03 PM, xianchi dong dongxian...@gmail.commailto:dongxian...@gmail.com wrote: Dear all, Recently I have measure a set of kinetic data of a receptor ligand interaction using BIAcore 3000. To my surprise, the dissociation rate is very low (~ 10e-6). During the measurement, I use a long dissociation time (2 hours) .I repeat several time which give very similar results. So I am wondering if the BIAcore can measure such a low off-rate kinetic. What is the limitation of BIAcore? Any review about that? Thanks in advance. Xianchi Dong Research Fellow Children's Hospital Boston Harvard Medical School .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
Re: [ccp4bb] off topic question BIAcore problem
Hi Xianchi, How did you treat the control flow cell? And what is the composition of the control buffer that you use to disrupt binding? Is it denaturing or a mild condition? If the Rmax=150 and Koff=1e-6, then at 7200 s (2 hr), the signal will only drop 1RU from 150RU (assuming you can reach the Rmax), is that what you see on the dissociation curve? In reality, when loading analyte at 100nM, assuming you have Kon=1e4, Koff=1e-6, binding for 30min would only let you reach ~125 RU, and at the lower concentrations will be much worse. Consequently the total dissociation after 7200s would be less than 1 RU on most curves - I am not sure if the Biacore 3000 will have a stable enough baseline for you to confidently measure that. If you try to fit the dissociation phase alone to get the Koff, the reported Koff won't be too accurate due to the low S/N ratio. On the other hand the 5-100nM spanning would generate significant changes on the binding phase, thus the global fitting should be able to get a relatively accurate KD. 0.2nM from SPR and 1nM from in-solution experiment also sounds reasonable. Zhijie From: xianchi dong Sent: Wednesday, February 20, 2013 5:45 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] off topic question BIAcore problem Thanks a lot for the kind reply. I used CM5 chip at pH 7.4. I am not quite sure about the PI of my protein but I have different truncations of my protein which behaved similarly. I can fit my data quite good with the Chi2 around 1 and Rmax 150. The kon is about 10e4 and the KD is around 0.2 nM. The concentration of analytes I used was from 100 nM to 5 nM. I used a control buffer which can disrupt the binding. In that buffer, no binding was observed. I also used fluorescence anisotropy to measure the Ki of the interaction which showed a 1 nM binding compared with the 0.2 nM binding in the SPR assay. On Wed, Feb 20, 2013 at 12:03 PM, xianchi dong dongxian...@gmail.com wrote: Dear all, Recently I have measure a set of kinetic data of a receptor ligand interaction using BIAcore 3000. To my surprise, the dissociation rate is very low (~ 10e-6). During the measurement, I use a long dissociation time (2 hours) .I repeat several time which give very similar results. So I am wondering if the BIAcore can measure such a low off-rate kinetic. What is the limitation of BIAcore? Any review about that? Thanks in advance. Xianchi Dong Research Fellow Children's Hospital Boston Harvard Medical School
