As a last step, to check if the assay qualifies for the screening purposes, Z’-factor was calculated. The dataset can be viewed on Zenodo.
For the purpose of compound library screenings, the compounds would be made available in DMSO and thus, when they would be incubated with the protein, the protein solution will now contain DMSO as well. It is important to determine the concentration of DMSO that could be tolerated by the protein such that the protein stays Read More …
The presence of detergent can prevent the sticking of the fluorophore to the sides of the wells of the plate and thus improve the signals. Hence, the effect of detergent was observed on the overall signals and the activity of HDAC11. The dataset can be viewed on Zenodo.
The linearity for HDAC11 activity for Boc-Lys-(TFA)-AMC substrate was monitored using the newly optimized buffer conditions. This was followed upon by Km calculation. The dataset can be viewed on zenodo.
An effect of adding reducing agent on the HDAC11 activity was monitored. Dataset can be found on Zenodo.
The pH range used earlier was expanded for further optimization to check if the activity could improve. The dataset is posted on Zenodo.
With previously optimized conditions, the linearity range for HDAC11 was obtained and the Km for the substrate was calculated. The data is posted on Zenodo.
Before performing the kinetic study for calculating the Km for HDAC11, it is important to know the duration of the stability of the protein under the assay conditions. This is being analyzed here.
A buffer screen at various pH was performed to pick the buffer that gives the best activity for HDAC11. The dataset is posted on Zenodo.
Since the activity of HDAC11 has been observed to be quite low as per the previous data, a preliminary test was performed to check any drastic increase in the activity in the presence of an additive. The data can be found on Zenodo.