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 <jcwilli...@coh.org> 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" > <careinaedgo...@yahoo.com<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) > ---------------------------------------------------------------------
