Date: Tue March 5th 2024
Time: 2pm UK time (other times)
Place:Zoom linkPrevious Meeting: issue #15
Who can come?: Anyone. No need to say anything unless you'd like to. If you'd like to contribute, please join Github.
Attendees: Dr Eve Carter (UCL), Dr Madison Edwards (Uni Toronto), Dr Claudia Tredup (Goethe-University Frankfurt), Prof Matilda Katan (UCL)
After visiting the Katan lab, Eve has removed the lag period she has previously observed at the start of Aldol 518 assays. The issue was with a bottle of CaCl2, which probably had a contaminant from the manufacturing process. She has been optimising this assay by varying the concentrations of different buffer components. First, she varied the concentration of free Ca2+ (using CaCl2 and EGTA) from 10-1000 uM, with 1000 uM shown to give the best results. Next, she varied the concentration of Aldol 518 between 25-100 uM with and without Aldol 355 enhancer. At these concentrations, Aldol 355 enhancer is required, and fluorescence increases as Aldol 518 concentration increases. She found that varying the Ca2+ concentrations between 0.5-2.0 mM without EGTA did not have a great effect on fluorescence. Finally, she varied KCl concentrations between 50-250 mM. Fluorescence was seen to increase as KCl concentration decreases, and Eve questioned whether these low, non-physiological concentrations of KCl could cause issues with the protein. Madison agreed that this could be the case and that concentration definitely shouldn't be below 50 mM and perhaps not below 100 mM. Matilda added that Aldol 518 is not very similar to PIP2, the natural substrate of PLCs, as, for example, it is not embedded in the membrane.
Following from the mass discrepancies discussed last month, Madison showed some results from a digest and MS of chicken PLCz1. She needs to discuss the results with a MS person in her lab as they are very complicated and imply many PTMs, although some of these are probably introduced during the digest.
Madison has received five compounds from Nuvisan to test in a thermal shift assay. She has tested two with chicken PLCz1 and saw slight inhibition with both of these. She plans to test the other three soon then will arrange a discussion with Nuvisan to see if the set up and results are similar to what they have seen.
To do:
[x] Eve to optimise the buffer of the Aldol assay.
[ ] Madison to review MS results to check for PTMs and compounds bound to chicken PLCz1.
[x] Madison to perform TSA with the compounds sent from Nuvisan.
L'esprit de l'escalier
If you'd like to follow up after the meeting, please comment below. You can also email, but please be clear if anything in the email should not be public domain - the default is open.
Date: Tue March 5th 2024 Time: 2pm UK time (other times) Place: Zoom link Previous Meeting: issue #15 Who can come?: Anyone. No need to say anything unless you'd like to. If you'd like to contribute, please join Github.
Attendees: Dr Eve Carter (UCL), Dr Madison Edwards (Uni Toronto), Dr Claudia Tredup (Goethe-University Frankfurt), Prof Matilda Katan (UCL)
Decks: Eve's presentation
Agenda:
In meeting:
After visiting the Katan lab, Eve has removed the lag period she has previously observed at the start of Aldol 518 assays. The issue was with a bottle of CaCl2, which probably had a contaminant from the manufacturing process. She has been optimising this assay by varying the concentrations of different buffer components. First, she varied the concentration of free Ca2+ (using CaCl2 and EGTA) from 10-1000 uM, with 1000 uM shown to give the best results. Next, she varied the concentration of Aldol 518 between 25-100 uM with and without Aldol 355 enhancer. At these concentrations, Aldol 355 enhancer is required, and fluorescence increases as Aldol 518 concentration increases. She found that varying the Ca2+ concentrations between 0.5-2.0 mM without EGTA did not have a great effect on fluorescence. Finally, she varied KCl concentrations between 50-250 mM. Fluorescence was seen to increase as KCl concentration decreases, and Eve questioned whether these low, non-physiological concentrations of KCl could cause issues with the protein. Madison agreed that this could be the case and that concentration definitely shouldn't be below 50 mM and perhaps not below 100 mM. Matilda added that Aldol 518 is not very similar to PIP2, the natural substrate of PLCs, as, for example, it is not embedded in the membrane.
Following from the mass discrepancies discussed last month, Madison showed some results from a digest and MS of chicken PLCz1. She needs to discuss the results with a MS person in her lab as they are very complicated and imply many PTMs, although some of these are probably introduced during the digest.
Madison has received five compounds from Nuvisan to test in a thermal shift assay. She has tested two with chicken PLCz1 and saw slight inhibition with both of these. She plans to test the other three soon then will arrange a discussion with Nuvisan to see if the set up and results are similar to what they have seen.
To do:
L'esprit de l'escalier If you'd like to follow up after the meeting, please comment below. You can also email, but please be clear if anything in the email should not be public domain - the default is open.
Next meeting: Tues 9th April