Hello everyone!
Hope this email finds you well. I have an off-topic question regarding ITC binding studies, which was asked by a reviewer. We performed an ITC binding study (using Affinity ITC, TA Instruments) to evaluate protein-DNA interaction which resulted in a perfect sigmoidal curve. We used the ‘one set of sites’ binding algorithm (“independent” model) for curve fitting and to calculate binding and thermodynamic parameters. The study suggested two copies of the protein binding to a single duplex DNA, i.e., the stoichiometry of protein:DNA is 2:1 (N=2). The ITC calculated the KD (equilibrium molar dissociation constant) in μM (micromolar). But the reviewer is asking to report the KD in (micromolar)^2 instead of micromolar mentioning that the binding reaction is 2A + B <-> (A2)B and the complex is (A2)B and not AB. Though we're trying to explain to the reviewer that we couldn't find any software that can compute the KD in (micromolar)^2 for the stoichiometry of 2 but he is not agreeing to it. We have used the NanoAnalyze software from the TA instrument. This software does not have a model to measure the KD in (micromolar)^2. I would be grateful if you could help me to resolve this problem or at least let me know what explanation might be appropriate to answer the reviewer’s concern that it’s a general practice to report the KD in the Molar irrespective of stoichiometry. Thanks in advance. Regards Abhishek *Dr. Abhishek Suman* *Ph.D (Structural Biology)* Indian Institute of Technology Hyderabad Kandi 502 284 Sangareddy Telangana INDIA Contact: +91 91002 74548, +91 80843 11898 Email: [email protected] ---------------------------------------------------- P* Please don't print this e-mail or attachment unless necessary. Preserve trees on the planet.* -- Disclaimer:- This footer text is to convey that this email is sent by one of the users of IITH. So, do not mark it as SPAM. ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
