I was in a search of a convenient and efficient protocol for the evaluation of inhibiton of HDAC1 (histone deacetylase 1). I am working on phytochemicals in the area of cancer research and I wanted to analyze the effect of a combined treatment on HDAC1. HDAC1 is a histone deacetlyase which plays a central role in the transcriptional regulation of genes involved in angiogenesis and apoptosis. Thus, inhibition of HDAC1 activity is one of the focuses of current anti-cancer strategies. In looking for an HDAC1 assay, I tested and evaluated various protocols. I rated them with respect to sensitivity and specificity as well as other aspects such as ease-of-use, hands-on time, the level of complexity of results interpretation and the required skill level of the user.
My research involves the use of tissue homogenates. Prior to dissection, lung tissues were perfused with Tris Buffer pH 7.4 to remove any red blood contamination. Tissue homogenates were prepared and then centrifuged. The supernatant was used for the assay.
The HDAC1 Inhibitor Screening Assay Kit includes the following reagents: HDAC assay buffer, HDAC substrate, HDAC developer and HDAC trichostatin A solution. The kit contains clear instructions for the preparation of reagents and also includes guidelines for positive and negative controls, as well as background wells. The assay involves two easy steps which are preformed in the same microplate. In the first step, the sample is incubated with HDAC1. This incubation takes place at 37°C for 30 minutes with constant shaking (and with the plate covered). After the incubation, the plate cover is removed and developer is added to each well and incubated at room temperature for 15 min. The deacetlyaton that occurs in the first step of the assay sensitizes the substrate such that treatment with the HDAC developer in the second step generates a fluorescent product. The fluorescent intensity of each well is then read using a spectroflourimeter with excitation and emission wavelengths of 340-360nm and 440-465nm, respectively.
I found the Cayman HDAC1 Inhibitor Screening Assay Kit instructions to be clear, concise and detailed enough so that anyone with basic laboratory skills could perform the assay. Also, the specific inhibitor that is included in the kit made verification of the specificity of the substrate straightforward; thus increasing the authenticity of the results. In addition, the use of a highly sensitive fluorescent technique means the assay is very sensitive. The drawbacks of the assay are its cost and that it must be stored at -80°C. Also, a plate reader with the appropriate fluorescence filters must in the lab. Overall, I recommend the HDAC1 Inhibitor Screening Assay Kit for its technology, reproducibility, sensitivity, and specificity. The instructions were clear, precise and accurate. In addition, there was very little clean up and preparation time.
HDAC 1 Inhibitor Screening Assay Kit from Cayman Chemical
The kit is user friendly, fast and self-explanatory.
Expensive. Cannot store above -80°C. Spectroflourimeter is required. Assay product is stable for 30 minutes only.
The Bottom Line
I recommend the HDAC1 Inhibitor Screening Assay Kit.