HDACs

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.

The time of incubation and conc. of the developer to be used are important parameters during HDAC11 assay optimization and thus, have been considered here. The dataset is posted on Zenodo.

A follow-up to check the increased fluorescence signals at 0 min of the HDAC11 reaction with its substrate was performed. The data is uploaded on Zenodo.

Using Boc-Lys-(TFA)-AMC substrate, a preliminary time-course study was initiated to determine the appropriate HDAC11 concentration and the time to be used for the reaction (which are essential parameters to perform Km calculations later). Experimental details are on Zenodo.

In order to develop an assay for HDAC11, a kit-free method is being attempted wherein Boc-Lys-(TFA)-AMC, a previously reported substrate was used to check the activity of HDAC11 in a preliminary experiment. Dataset on Zenodo.

Preliminary tests to check if the Fluor-de-Lys-green HDAC activity kit could be used for developing a high-throughput HDAC11 assay have been performed here using controls and recombinantly purified HDAC11. Details on Zenodo.

HDAC 11 protein was purified from Sf9 cells using Ni-NTA chromatography. The protein was concentrated to 1.4 mg/ml (above which it tends to precipitate) and flash frozen. The details on the experimental procedures can be found on Zenodo.