11 Questions 18 Answers 0 Followers
Questions related from Nathan Ponzar
Hi folks, I prep a lot of protein. I currently use B-PER for lysis, but it starts getting really expensive when prepping from large culture volume. For example, protein prep from 4-6 L worth of...
17 February 2023 5,922 1 View
Hello, Say you have a protein that is giving variable KD values for its natural ligand (for reasons not completely understood) from assay to assay, but very consistent results within a given...
28 August 2021 3,320 0 View
I have been doing a lot of thermofluor assays for determining binding affinities. This necessitates getting the raw fluorescence values organized into a different column for each sample....
31 July 2021 920 2 View
Say you have an enzyme with 3 ligands: a substrate, a cofactor, and an inhibitor. They all interact and effect one another. If one was to conduct a 3-dimensional titration of these ligands, are...
04 July 2021 1,137 2 View
I think this is a valid approach to determining Ki of competitive and non-competitive inhibition for mixed model inhibitors, but I would appreciate feedback. On this graph, the Y-values are...
28 November 2020 2,895 9 View
Hello, I have some data that looks like the line on the right, but I'm not sure what function to use. It seems like an exponential added to a line, or something of the like. I think it may be the...
19 November 2020 2,491 3 View
It is my understanding that single-turnover reactions isolate the chemical step, as opposed to being comprised of the chemical step + product dissociation as in kcat. Here's my issue though....
14 November 2020 2,649 7 View
Say you have an inhibitor who's binding is either dependent on or enhanced by a metal cation binding to either an allosteric site, or by forming part of the binding site of the inhibitor. So as...
18 October 2020 7,784 3 View
I express a recombinant protein in E. coli that requires Mg2+ and ATP to remove bound chaperones and activate the enzyme, but also requires TCEP. I currently achieve active protein, but with very...
01 October 2019 9,036 3 View
Hi folks, I have been doing some michaelis-menten analyses to determine mechanism of action for a few of our lab's RNase H inhibitors. Most of the time, the assay goes fine and I get nice data....
31 July 2019 666 5 View
In biochemistry, most work is done at room temperature. Yet, basic thermodynamics tells us that affinities often change with temperature, in positive or negative directions depending on the...
01 January 1970 4,634 8 View
