Tips for a Successful Biochemical Assay

Enzo Life Science provides over 40 years of experience in supplying research kits and biochemical and biological resources for researchers. With “Scientists Enabling Healthcare” as Enzo’s core value, we realize the importance in providing relevant information to our clients who are working in the Life Sciences and Drug Discovery fields. Our vast experience in the design and manufacturing of active enzymes and their substrates supports development of an ever-expanding portfolio of biochemical assays. These assays exemplify the synergy of Enzo’s capabilities, such as assembling small peptides, active recombinant protein synthesis, fluorescent labeling and detection technologies, and antibody development into robust reproducible assay kits.

Based on solutions we offer to researchers on a daily basis, we wanted to provide a list of suggestions to assist you in obtaining the most accurate, consistent, and reproducible data.


First, start by making sure the product manual is understood – reading over the protocol and storage information is the key to success. If stored properly, most kits can be stored up to a year, unless otherwise stated. Reconstituted components can typically be stored for two or three months. The components of each kit have been carefully formulated and validated, using the protocol provided. Enzymes must be handled correctly in order to retain maximum enzymatic activity. To achieve this, defrost the enzyme quickly in a room temperature water bath or by rubbing between fingers, then immediately store in an ice bath. The remaining unused enzyme should be refrozen quickly, by placing it at -70 °C. If possible, snap freeze in liquid nitrogen or dry ice/ethanol bath. To minimize the number of freeze/thaw cycles, aliquot into separate tubes and store at -70 °C.


With over a hundred assays to choose from, we provide a complete assay system to help you measure the enzyme inhibitor of your choice. Regardless if you are looking to do chemiluminescent, fluorometric, or colorimetric, Enzo has what you need. Our assays can be completed in two steps, one with a substrate and one with the developer, as well as an inhibitor that stops the enzyme and produces the fluorescent signal. In many cases, a NBS 1/2 –volume 96 well plate will also be provided in either clear or black with our manual stating the preferred plate. If required, some assays provide flexibility for change in the reaction volumes. The wells of the microplates provided can readily accommodate up to 150 µL. When changing the volume of the developer, it is important to keep two factors constant, the concentration of the inhibitor and the amount of developer concentrate. Do keep in mind that it is very important to compare your experimental samples to “time zero” which in most cases is your sample plus developer before the substrate is added and or a negative control- no enzyme.

Our FLUOR DE LYS® deacetylase assay platform has freed researchers from cumbersome protocols for screening HDAC & Sirtuin activity. Our high-quality chemiluminescent, fluorescent, and colorimetric assays deliver more high-quality hits, and are backed by a broad panel of characterized inhibitors and PTM-specific antibodies for ubiquitinylation, sumoylation, methylation, acetylation, and phosphorylation. The FLUOR DE LYS® SIRT1 fluorometric drug discovery assay kit is a complete assay system designed to measure the lysyl deacetylase activity of the recombinant human SIRT1 included in the kit. The kit is ideal for chemical library screening for candidate inhibitors or activators or kinetic assay of the enzyme under varying conditions. The FLUOR DE LYS® SIRT1 assay is based on the FLUOR DE LYS® SIRT1 Substrate and FLUOR DE LYS® Developer II combination. The assay procedure has two steps. First, the FLUOR DE LYS® SIRT1 Substrate, which contains a peptide comprising amino acids 379-382 of human p53 (Arg-His-Lys-Lys(Ac)), undergoes deacetylation of the substrate. This sensitizes the substrate so that, in the second step, treatment with the FLUOR DE LYS® Developer II produces a fluorophore.



The use of enzyme with colorimetric assays are a highly used application as well. The versatility to test the presence of enzyme, specific compounds and other analytes. Our BIOMOL® Green provides a simple and convenient method for colorimetric phosphate quantitation (abs. 600-680 nm). Unlike other molybdate/malachite green-based assays, BIOMOL® Green doesn’t require multiple solutions or reagents prepared fresh on the day of the assay. BIOMOL® Green is extremely stable (>6 mos. at 4°C) and is simply mixed, at room temperature, with any enzymatic reaction that has released free phosphate. Reported applications include assays for phospholipid phosphatases, tyrosyl-tRNA synthetase (coupled with pyrophosphatase) and a viral RNA triphosphatase. BIOMOL® Green has perhaps been most widely applied to protein phosphatase assays. Typically, a protein phosphatase (e.g. PTP1B or LAR) is simply incubated with an appropriate phosphopeptide (e.g. IR5,9,10). Addition of BIOMOL® Green stops the reaction and begins color development, which is read 20-30 minutes later. BIOMOL® Green may be used in cuvette or microplate-based assays and is ideal for high-throughput applications. Each kit comes with a standardized phosphate solution for assay calibration.


We hope you find these suggestions helpful in ensuring a successful drug discovery screening. At Enzo Life Sciences, we are able to offer you a wide range of Enzyme modulators that will enable your success. From modified Substrate-based, FRET-Based, and antibody-based, we offer a wide range of Proteins that are high in quality and flexible in all post-screen needs. Please check out our Enzyme Activity Assay platform to learn more. To learn more about different approaches on drug screening, see 10 Tips for Successful High Content Screening (HSC) Assay. Additionally, contact our Technical Support Team for further assistance.

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