Re: [ccp4bb] KD of dimerization, off topic
That's an extremely useful link - thanks to Will Stanley for posting that one. For a VP-ITC machine I'd guess that you need to load the injector with about 500ul of protein at a concentration of 80x the Kd or more. Notice that Alan Cooper was injecting 10 microliters of protein at 2mM with a 12 microMolar dissociation constant, per injection. You would probably want to maintain that approximate ratio - ~170 because it's mostly a question of measuring deltaH with a decent signal-to-noise per injection. I recall that it takes up to 500 microLiters to load the injection syringe on a VP-ITC without air gap between plunger tip and injection point - unless someone's got a nice trick to reduce that. The rule of thumb from the VP-ITC manual - and from practical experience on our machine here - for A+B <=> AB is using at least 10x the Kd in the sample chamber and about 80x the Kd in the injector. That's not exactly the same situation, but 80x vs 170x suggests the the considerations are much the same. Phil Jeffrey Princeton On 2/14/14 12:52 PM, Keller, Jacob wrote: What a nice idea this ITC dilution is--a great example of a wet lab technique learned en passant on the ccp4bb. I wonder what range of Kds could feasibly be measured with existing calorimeter sensitivities? JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Will Stanley Sent: Friday, February 14, 2014 12:18 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] KD of dimerization, off topic Hi Careina, Since alternative methods are being suggested... ITC can be good for quantitating a monomer-dimer equilibrium by diluting dimers out from a concentrated solution (which obviously favours the dimer) - and presuming a reasonable Kon/Koff. Alan Cooper has kindly figured out the data fitting for the rest of us: http://www.chem.gla.ac.uk/staff/alanc/itcdil.pdf I think Alan was using a VP-ITC when he was doing this stuff. Lower volumes - and presumably concentrations if the KD is small enough - are feasible in an ITC200. The protein is recoverable anyway. All the best, Will.
Re: [ccp4bb] KD of dimerization, off topic
What a nice idea this ITC dilution is--a great example of a wet lab technique learned en passant on the ccp4bb. I wonder what range of Kds could feasibly be measured with existing calorimeter sensitivities? JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Will Stanley Sent: Friday, February 14, 2014 12:18 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] KD of dimerization, off topic Hi Careina, Since alternative methods are being suggested... ITC can be good for quantitating a monomer-dimer equilibrium by diluting dimers out from a concentrated solution (which obviously favours the dimer) - and presuming a reasonable Kon/Koff. Alan Cooper has kindly figured out the data fitting for the rest of us: http://www.chem.gla.ac.uk/staff/alanc/itcdil.pdf I think Alan was using a VP-ITC when he was doing this stuff. Lower volumes - and presumably concentrations if the KD is small enough - are feasible in an ITC200. The protein is recoverable anyway. All the best, Will. On 14 February 2014 12:40, Williams, John Charles wrote: > Sedimentation equilibrium or sedimentation velocity experiments by analytical > centrifugation is the best method for this. > > From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of FOOS > Nicolas [nicolas.f...@synchrotron-soleil.fr] > Sent: Friday, February 14, 2014 12:25 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] KD of dimerization, off topic > > I agree with Dave, and i suggest one more method to estimate Kd, The > intrinsic fluorescence of proteins thanks to the aromatic chain side. > Maybe it's also possible to have an estimation with native gels if you use > prot A concentration as fixed and B protein concentration as variable. I am > not sure. > > > De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de David > Briggs [drdavidcbri...@gmail.com] Envoyé : vendredi 14 février 2014 > 09:13 À : CCP4BB@JISCMAIL.AC.UK Objet : Re: [ccp4bb] KD of > dimerization, off topic > > Hi Careina, > > I'm not sure you can assume that the ratio of monomer and dimer will stay > constant through the column - as you say, the protein is diluted during the > run, the ratio will change, unless you have a super tight dimer - which > clearly you do not. Also, as the mass and the molar extinction coefficient > will both double in the dimer, the relationship between absorbance and > concentration will be unchanged. > > Typically, such these sorts of questions are answered (at least me) by > equilibrium analytical centrifugation. > > Hth, > > Dave > > On 14 Feb 2014 08:03, "Careina Edgooms" > mailto:careinaedgo...@yahoo.com>> wrote: > Dear CCP4 board > > I have a protein that exists in equilibrium between monomer and dimer and I'm > trying to calculate KD using size exclusion. The problem is that the column > dilutes my sample so that if I put 20 uM on to the column, I only recover 0.5 > uM in dimer fraction and 2 uM in monomer fraction. I am getting confused as > to how to plot my KDs. Do I not regard my initial concentrations at all and > work only with the final concentrations that come off the column? > I would plot [monomer] squared vs [dimer] and I will assume that the > ratio of monomer to dimer will stay constant as the protein passes > through the column. (also I would calculate [dimer] using 2x monomer > extinction coefficient) > > Does this seem a reasonable way to calculate KDs and reasonable > argument? Also I am looking for good references for calculating Kds > when dealing with dimerization Thanks and sorry for off topic question > Careina > > > - > *SECURITY/CONFIDENTIALITY WARNING: > This message and any attachments are intended solely for the > individual or entity to which they are addressed. This communication > may contain information that is privileged, confidential, or exempt > from disclosure under applicable law (e.g., personal health > information, research data, financial information). Because this > e-mail has been sent without encryption, individuals other than the > intended recipient may be able to view the information, forward it to > others or tamper with the information without the knowledge or consent > of the sender. If you are not the intended recipient, or the employee > or person responsible for delivering the message to the intended > recipient, any dissemination, distribution or copying of the > communication is strictly prohibited. If you received the > communication in error, please notify the sender immediately by > replying to this m
Re: [ccp4bb] KD of dimerization, off topic
Hi Careina, Since alternative methods are being suggested... ITC can be good for quantitating a monomer-dimer equilibrium by diluting dimers out from a concentrated solution (which obviously favours the dimer) - and presuming a reasonable Kon/Koff. Alan Cooper has kindly figured out the data fitting for the rest of us: http://www.chem.gla.ac.uk/staff/alanc/itcdil.pdf I think Alan was using a VP-ITC when he was doing this stuff. Lower volumes - and presumably concentrations if the KD is small enough - are feasible in an ITC200. The protein is recoverable anyway. All the best, Will. On 14 February 2014 12:40, Williams, John Charles wrote: > Sedimentation equilibrium or sedimentation velocity experiments by analytical > centrifugation is the best method for this. > > From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of FOOS Nicolas > [nicolas.f...@synchrotron-soleil.fr] > Sent: Friday, February 14, 2014 12:25 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] KD of dimerization, off topic > > I agree with Dave, and i suggest one more method to estimate Kd, > The intrinsic fluorescence of proteins thanks to the aromatic chain side. > Maybe it's also possible to have an estimation with native gels if you use > prot A concentration as fixed and B protein concentration as variable. I am > not sure. > > > De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de David Briggs > [drdavidcbri...@gmail.com] > Envoyé : vendredi 14 février 2014 09:13 > À : CCP4BB@JISCMAIL.AC.UK > Objet : Re: [ccp4bb] KD of dimerization, off topic > > Hi Careina, > > I'm not sure you can assume that the ratio of monomer and dimer will stay > constant through the column - as you say, the protein is diluted during the > run, the ratio will change, unless you have a super tight dimer - which > clearly you do not. Also, as the mass and the molar extinction coefficient > will both double in the dimer, the relationship between absorbance and > concentration will be unchanged. > > Typically, such these sorts of questions are answered (at least me) by > equilibrium analytical centrifugation. > > Hth, > > Dave > > On 14 Feb 2014 08:03, "Careina Edgooms" > mailto:careinaedgo...@yahoo.com>> wrote: > Dear CCP4 board > > I have a protein that exists in equilibrium between monomer and dimer and I'm > trying to calculate KD using size exclusion. The problem is that the column > dilutes my sample so that if I put 20 uM on to the column, I only recover 0.5 > uM in dimer fraction and 2 uM in monomer fraction. I am getting confused as > to how to plot my KDs. Do I not regard my initial concentrations at all and > work only with the final concentrations that come off the column? > I would plot [monomer] squared vs [dimer] and I will assume that the ratio of > monomer to dimer will stay constant as the protein passes through the column. > (also I would calculate [dimer] using 2x monomer extinction coefficient) > > Does this seem a reasonable way to calculate KDs and reasonable argument? > Also I am looking for good references for calculating Kds when dealing with > dimerization > Thanks and sorry for off topic question > Careina > > > - > *SECURITY/CONFIDENTIALITY WARNING: > This message and any attachments are intended solely for the individual or > entity to which they are addressed. This communication may contain > information that is privileged, confidential, or exempt from disclosure under > applicable law (e.g., personal health information, research data, financial > information). Because this e-mail has been sent without encryption, > individuals other than the intended recipient may be able to view the > information, forward it to others or tamper with the information without the > knowledge or consent of the sender. If you are not the intended recipient, or > the employee or person responsible for delivering the message to the intended > recipient, any dissemination, distribution or copying of the communication is > strictly prohibited. If you received the communication in error, please > notify the sender immediately by replying to this message and deleting the > message and any accompanying files from your system. If, due to the security > risks, you do not wish to receive further communications via e-mail, please > reply to this message and inform the sender that you do not wish to receive > further e-mail from the sender. (fpc5p) > -
Re: [ccp4bb] KD of dimerization, off topic
Sedimentation equilibrium or sedimentation velocity experiments by analytical centrifugation is the best method for this. From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of FOOS Nicolas [nicolas.f...@synchrotron-soleil.fr] Sent: Friday, February 14, 2014 12:25 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] KD of dimerization, off topic I agree with Dave, and i suggest one more method to estimate Kd, The intrinsic fluorescence of proteins thanks to the aromatic chain side. Maybe it's also possible to have an estimation with native gels if you use prot A concentration as fixed and B protein concentration as variable. I am not sure. De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de David Briggs [drdavidcbri...@gmail.com] Envoyé : vendredi 14 février 2014 09:13 À : CCP4BB@JISCMAIL.AC.UK Objet : Re: [ccp4bb] KD of dimerization, off topic Hi Careina, I'm not sure you can assume that the ratio of monomer and dimer will stay constant through the column - as you say, the protein is diluted during the run, the ratio will change, unless you have a super tight dimer - which clearly you do not. Also, as the mass and the molar extinction coefficient will both double in the dimer, the relationship between absorbance and concentration will be unchanged. Typically, such these sorts of questions are answered (at least me) by equilibrium analytical centrifugation. Hth, Dave On 14 Feb 2014 08:03, "Careina Edgooms" mailto:careinaedgo...@yahoo.com>> wrote: Dear CCP4 board I have a protein that exists in equilibrium between monomer and dimer and I'm trying to calculate KD using size exclusion. The problem is that the column dilutes my sample so that if I put 20 uM on to the column, I only recover 0.5 uM in dimer fraction and 2 uM in monomer fraction. I am getting confused as to how to plot my KDs. Do I not regard my initial concentrations at all and work only with the final concentrations that come off the column? I would plot [monomer] squared vs [dimer] and I will assume that the ratio of monomer to dimer will stay constant as the protein passes through the column. (also I would calculate [dimer] using 2x monomer extinction coefficient) Does this seem a reasonable way to calculate KDs and reasonable argument? Also I am looking for good references for calculating Kds when dealing with dimerization Thanks and sorry for off topic question Careina - *SECURITY/CONFIDENTIALITY WARNING: This message and any attachments are intended solely for the individual or entity to which they are addressed. This communication may contain information that is privileged, confidential, or exempt from disclosure under applicable law (e.g., personal health information, research data, financial information). Because this e-mail has been sent without encryption, individuals other than the intended recipient may be able to view the information, forward it to others or tamper with the information without the knowledge or consent of the sender. If you are not the intended recipient, or the employee or person responsible for delivering the message to the intended recipient, any dissemination, distribution or copying of the communication is strictly prohibited. If you received the communication in error, please notify the sender immediately by replying to this message and deleting the message and any accompanying files from your system. If, due to the security risks, you do not wish to receive further communications via e-mail, please reply to this message and inform the sender that you do not wish to receive further e-mail from the sender. (fpc5p) -
[ccp4bb] AW: [ccp4bb] KD of dimerization, off topic
Dear Carina, An additional problem is that due to the dilution, but also due to the separation of monomers and dimers you get a reequilibration which is dependent on Kon/Koff of the interaction. Unless these are very slow, you cannot use size exclusion to determine the monomer/dimer ratio. Although not perfect, I would try dynamic light scattering. For calculating the Kd, I would just use the standard textbook formula, with A being identical to B. Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Careina Edgooms Gesendet: Freitag, 14. Februar 2014 09:04 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] KD of dimerization, off topic Dear CCP4 board I have a protein that exists in equilibrium between monomer and dimer and I'm trying to calculate KD using size exclusion. The problem is that the column dilutes my sample so that if I put 20 uM on to the column, I only recover 0.5 uM in dimer fraction and 2 uM in monomer fraction. I am getting confused as to how to plot my KDs. Do I not regard my initial concentrations at all and work only with the final concentrations that come off the column? I would plot [monomer] squared vs [dimer] and I will assume that the ratio of monomer to dimer will stay constant as the protein passes through the column. (also I would calculate [dimer] using 2x monomer extinction coefficient) Does this seem a reasonable way to calculate KDs and reasonable argument? Also I am looking for good references for calculating Kds when dealing with dimerization Thanks and sorry for off topic question Careina
Re: [ccp4bb] KD of dimerization, off topic
I agree with Dave, and i suggest one more method to estimate Kd, The intrinsic fluorescence of proteins thanks to the aromatic chain side. Maybe it's also possible to have an estimation with native gels if you use prot A concentration as fixed and B protein concentration as variable. I am not sure. De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de David Briggs [drdavidcbri...@gmail.com] Envoyé : vendredi 14 février 2014 09:13 À : CCP4BB@JISCMAIL.AC.UK Objet : Re: [ccp4bb] KD of dimerization, off topic Hi Careina, I'm not sure you can assume that the ratio of monomer and dimer will stay constant through the column - as you say, the protein is diluted during the run, the ratio will change, unless you have a super tight dimer - which clearly you do not. Also, as the mass and the molar extinction coefficient will both double in the dimer, the relationship between absorbance and concentration will be unchanged. Typically, such these sorts of questions are answered (at least me) by equilibrium analytical centrifugation. Hth, Dave On 14 Feb 2014 08:03, "Careina Edgooms" mailto:careinaedgo...@yahoo.com>> wrote: Dear CCP4 board I have a protein that exists in equilibrium between monomer and dimer and I'm trying to calculate KD using size exclusion. The problem is that the column dilutes my sample so that if I put 20 uM on to the column, I only recover 0.5 uM in dimer fraction and 2 uM in monomer fraction. I am getting confused as to how to plot my KDs. Do I not regard my initial concentrations at all and work only with the final concentrations that come off the column? I would plot [monomer] squared vs [dimer] and I will assume that the ratio of monomer to dimer will stay constant as the protein passes through the column. (also I would calculate [dimer] using 2x monomer extinction coefficient) Does this seem a reasonable way to calculate KDs and reasonable argument? Also I am looking for good references for calculating Kds when dealing with dimerization Thanks and sorry for off topic question Careina
Re: [ccp4bb] KD of dimerization, off topic
Hi Careina, I'm not sure you can assume that the ratio of monomer and dimer will stay constant through the column - as you say, the protein is diluted during the run, the ratio will change, unless you have a super tight dimer - which clearly you do not. Also, as the mass and the molar extinction coefficient will both double in the dimer, the relationship between absorbance and concentration will be unchanged. Typically, such these sorts of questions are answered (at least me) by equilibrium analytical centrifugation. Hth, Dave On 14 Feb 2014 08:03, "Careina Edgooms" wrote: > Dear CCP4 board > > I have a protein that exists in equilibrium between monomer and dimer and > I'm trying to calculate KD using size exclusion. The problem is that the > column dilutes my sample so that if I put 20 uM on to the column, I only > recover 0.5 uM in dimer fraction and 2 uM in monomer fraction. I am getting > confused as to how to plot my KDs. Do I not regard my initial > concentrations at all and work only with the final concentrations that come > off the column? > I would plot [monomer] squared vs [dimer] and I will assume that the ratio > of monomer to dimer will stay constant as the protein passes through the > column. (also I would calculate [dimer] using 2x monomer extinction > coefficient) > > Does this seem a reasonable way to calculate KDs and reasonable argument? > Also I am looking for good references for calculating Kds when dealing with > dimerization > Thanks and sorry for off topic question > Careina >
[ccp4bb] KD of dimerization, off topic
Dear CCP4 board I have a protein that exists in equilibrium between monomer and dimer and I'm trying to calculate KD using size exclusion. The problem is that the column dilutes my sample so that if I put 20 uM on to the column, I only recover 0.5 uM in dimer fraction and 2 uM in monomer fraction. I am getting confused as to how to plot my KDs. Do I not regard my initial concentrations at all and work only with the final concentrations that come off the column? I would plot [monomer] squared vs [dimer] and I will assume that the ratio of monomer to dimer will stay constant as the protein passes through the column. (also I would calculate [dimer] using 2x monomer extinction coefficient) Does this seem a reasonable way to calculate KDs and reasonable argument? Also I am looking for good references for calculating Kds when dealing with dimerization Thanks and sorry for off topic question Careina
