Online Purchasing Account You are logged on as Guest. LoginRegister a New AccountShopping cart (Empty)
United States 

Tips for a Successful Biochemical Assay

Posted By Albert Wong
Tags: Successful Research Tips

Enzo Life Science provides over 40 years of experience in supplying research kits and biochemical and biological resources for researchers. With “Scientists Enabling Scientists” 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.


Figure 1: Convenient non-radioactive screening formats.



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.


Figure 2. Time Courses of FLUOR DE LYS® SIRT1 Deacetylation by Recombinant SIRT1. SIRT1 (2 U/well) was incubated (37°C) with the indicated concentrations of peptide substrate and 500 µM NAD+. Reactions were stopped at indicated times with FLUOR DE LYS® Developer II/2 mM nicotinamide and fluorescence measured (Ex. 360 nm, Em. 460 nm, gain=85).



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.


Figure 3. Reagent stability. Shelf life of Biomol green reagent.



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.

Share this TechNote

Never miss a new TechNote!

Receive our TechNotes as soon as they are published.


Follow Us!

Featured Literature

Brochures
Cancer Research Products
Brochures
Innovative Products to Analyze Immunological Disorders
 
comments powered by Disqus

Related Products

NF-ATc1 monoclonal antibody (7A6) 

ICC, IF, IHC, IP, WB, ChIP, Gel Shift | Print as PDF
 
ALX-804-022-R100 100 µl 573.00 USD
Do you need bulk/larger quantities?
 

CD80 (human) monoclonal antibody (mAb 104) 

Flow Cytometry, FUNC | Print as PDF
 
ENZ-ABS156-0100 100 µg 411.00 USD
Do you need bulk/larger quantities?
 

CD80 (human), (recombinant) 

Produced in HEK 293 cells., ≥95% (SDS-PAGE) | Print as PDF
 
ENZ-PRT171-0010 10 µg 183.00 USD
Do you need bulk/larger quantities?
 

Calcineurin Phosphatase assay kit 

Designed for inhibitor screening or kinetic assay of recombinant Calcineurin
Colorimetric detection, HTS, Activity assay | Print as PDF
 
BML-AK804-0001 96 wells 404.00 USD
Do you need bulk/larger quantities?
 

Calcineurin cellular activity assay kit 

Provides everything needed to assay calcineurin cellular activity, including recombinant calcineurin control
Colorimetric detection | Print as PDF
 
BML-AK816-0001 96 wells 492.00 USD
Do you need bulk/larger quantities?
 

Calcineurin inhibitory peptide 

148067-21-4 | Print as PDF
 
BML-PR104-0500 0.5 mg 118.00 USD
Do you need bulk/larger quantities?
 

PD-L1 (human), (recombinant) 

Produced in HEK cells. Human PD-L1 (aa 19-239) is fused at the N-terminus to a His-tag and FLAG®-tag., ≥90% (SDS-PAGE) | Print as PDF
 
ENZ-PRT223-0100 100 µg 480.00 USD
Do you need bulk/larger quantities?
 

PD-1 (human), (recombinant) 

Produced in HEK cells. Extracellular domain of PD-1 (aa 25-167) containing a 5’-His-tag, V5 epitope tag spacer, and a FLAG-tag., ≥90% (SDS-PAGE) | Print as PDF
 
ENZ-PRT190-0050 50 µg 307.00 USD
Do you need bulk/larger quantities?
 

PD-1 Recombinant monoclonal antibody (PDCD1/1410R) 

Flow Cytometry, IF, IHC (PS) | Print as PDF
 
ENZ-ABS348-0100 100 µg 440.00 USD
Do you need bulk/larger quantities?
 

PD-1 Recombinant monoclonal antibody (RMPD1-1) 

IF, IHC (PS) | Print as PDF
 
ENZ-ABS337-0100 100 µg 440.00 USD
Do you need bulk/larger quantities?
 

FasL, soluble (human) ELISA kit 

ELISA | Print as PDF
 
ALX-850-246-KI01 96 wells 601.00 USD
Do you need bulk/larger quantities?
 

SUPERFASLIGAND® Protein (soluble) (human), (recombinant) 

Fas ligand with improved stability providing significantly enhanced immune activation.
Produced in HEK 293 cells. The extracellular domain of human FasL (APO-1L; CD95L; CD178) (aa 103-281) is fused at the N-terminus to a linker peptide (26 aa) and a FLAG®-tag. Glycosylation of rhs SUPERFASLIGAND® is similar to natural human FasL., ≥95% (SDS-PAGE), ELISA | Print as PDF
 
ALX-522-020-C005 5 µg 284.00 USD
 
ALX-522-020-3005 3x5 µg SuperPack 657.00 USD
Do you need bulk/larger quantities?
 

FasL, soluble (human) (recombinant) set 

Produced in HEK 293 cells. The extracellular domain of human FasL (APO-1L; CD95L; CD178) (aa 103-281) is fused at the N-terminus to a linker peptide (26 aa) and a FLAG®-tag. Glycosylation of FasL, Soluble (human) (recombinant) is similar to natural human FasL., ≥95% (SDS-PAGE), FUNC | Print as PDF
 
ALX-850-014-KI02 1 Set 603.00 USD
Do you need bulk/larger quantities?
 

LAG-3 (human) monoclonal antibody (17B4) 

ICC, IHC (FS), IP, WB | Print as PDF
 
ALX-804-806-C100 100 µg 407.00 USD
Do you need bulk/larger quantities?
 

LAG-3 (human):Fc (human), (recombinant) 

Produced in CHO cells. The sequence coding for the 4 extracellular Ig-like domains of human LAG-3 (D1-D4) is fused to the Fc portion of human IgG1., ≥99% (SDS-PAGE), ELISA, Flow Cytometry | Print as PDF
 
ALX-522-078-C050 50 µg 508.00 USD
Do you need bulk/larger quantities?
 

LAG-3 (mouse):Fc (mouse), (recombinant) 

Produced in CHO cells. The sequence coding for the 4 extracellular Ig-like domains of mouse LAG-3 (D1-D4) is fused to the Fc portion of mouse IgG2a (hinge-CH2-CH3)., ≥95% (SDS-PAGE), Flow Cytometry | Print as PDF
 
ALX-522-099-C050 50 µg 508.00 USD
Do you need bulk/larger quantities?
 

IFN-γ (human), (recombinant) 

Produced in E.coli., ≥98% (SDS-PAGE gel & HPLC) | Print as PDF
 
ENZ-PRT141-0100 100 µg 206.00 USD
Do you need bulk/larger quantities?
 

IFN-γ (human), ELISA kit 

ELISA, Colorimetric detection | Print as PDF
 
ADI-900-136 96 wells 552.00 USD
Do you need bulk/larger quantities?
 

IFN-γ (mouse), ELISA kit 

ELISA, Colorimetric detection | Print as PDF
 
ADI-900-137 96 wells 530.00 USD
Do you need bulk/larger quantities?
 

GITR (human) monoclonal antibody (Elo-1) 

Purified from concentrated hybridoma tissue culture supernatant., Flow Cytometry | Print as PDF
 
ALX-804-825-C100 100 µg 341.00 USD
Do you need bulk/larger quantities?
 

GITR (human):Fc (human), (recombinant) 

Produced in HEK 293 cells. The cysteine-rich region of human GITR (AITR) (aa 26-161) is fused to the Fc portion of human IgG1., ≥90% (SDS-PAGE), ELISA | Print as PDF
 
ALX-522-061-C050 50 µg 416.00 USD
Do you need bulk/larger quantities?
 

GITRL (mouse) monoclonal antibody (MGTL 10) 

ELISA, Flow Cytometry, WB | Print as PDF
 
ALX-804-518-C050 50 µg 240.00 USD
Do you need bulk/larger quantities?
 

CD134 (human):Fc (human), (recombinant) 

Produced in HEK 293 cells. The cysteine-rich region of human CD134 (OX40) (aa 29-214) is fused to the Fc portion of human IgG1., ≥95% (SDS-PAGE), ELISA | Print as PDF
 
ALX-522-022-C050 50 µg 417.00 USD
Do you need bulk/larger quantities?
 

CD134L (soluble) (human), (recombinant) 

Produced in HEK 293 cells. The extracellular domain of human CD134L (OX40L) (aa 52-183) is fused at the N-terminus to a linker peptide (8 aa) and a FLAG®-tag., ELISA | Print as PDF
 
ALX-522-023-C010 10 µg 278.00 USD
 
ALX-522-023-3010 3x10 µg SuperPack 522.00 USD
Do you need bulk/larger quantities?
 

OX40L Recombinant monoclonal antibody (R4930) (Rabbit IgGκ) 

Flow Cytometry, FUNC | Print as PDF
 
ENZ-ABS555-0200 200 µg 440.00 USD
Do you need bulk/larger quantities?
 

CD137 (human):Fc (human), (recombinant) 

Produced in HEK 293 cells. The cysteine-rich region of human CD137 (4-1BB) (aa 1-186) is fused to the Fc portion of human IgG1., ≥95% (SDS-PAGE), ELISA, FUNC | Print as PDF
 
ALX-522-031-C050 50 µg 401.00 USD
Do you need bulk/larger quantities?
 

CD137L (soluble) (human), (recombinant) 

Produced in HEK 293 cells. The extracellular domain of human CD137L (aa 49-254) is fused at the N-terminus to a linker peptide (10 aa) and a FLAG®-tag., ≥95% (SDS-PAGE) | Print as PDF
 
ALX-522-111-C010 10 µg 190.00 USD
 
ALX-522-111-3010 3x10 µg SuperPack 392.00 USD
Do you need bulk/larger quantities?
 

MEGACD40L® Protein (soluble) (human), (recombinant) 

High activity, high purity CD40L protein for co-stimulatory activation of an immune response
Produced in CHO cells. The extracellular domain of human CD40L (CD154) (aa 116-261) is fused at the N-terminus to mouse ACRP30headless (aa 18-111) and a FLAG®-tag., ≥90% (SDS-PAGE) | Print as PDF
 
ALX-522-110-C010 10 µg 499.00 USD
Do you need bulk/larger quantities?
 

MEGACD40L® Protein (soluble) (mouse), (recombinant) 

Highly active
Produced in CHO cells. The extracellular domain of mouse CD40L (CD154) (aa 115-260) is fused at the N-terminus to mouse ACRP30headless (aa 18-111) and a FLAG®-tag., ≥95% (SDS-PAGE) | Print as PDF
 
ALX-522-120-C010 10 µg 536.00 USD
Do you need bulk/larger quantities?
 

CD40L, soluble (human) (recombinant) set 

Ligand plus enhancer for improved stability and enhanced immune activation.
Produced in E. coli. The extracellular domain of human CD40L (CD154) (aa 116-261) is fused at the N-terminus to a linker peptide (6 aa) and a FLAG®-tag., ≥95% (SDS-PAGE)., FUNC | Print as PDF
 
ALX-850-064-KI01 1 Set 515.00 USD
Do you need bulk/larger quantities?
 

CD40L, soluble (mouse) (recombinant) set 

Produced in HEK 293 cells. The extracellular domain of mouse CD40L (CD154) (aa 115-260) is fused at the N-terminus to a linker peptide (8 aa) and a FLAG®-tag., ≥90% (SDS-PAGE)., ELISA, FUNC | Print as PDF
 
ALX-850-075-KI01 1 Set 626.00 USD
Do you need bulk/larger quantities?
 

CD40 (human) monoclonal antibody (G28-5) 

Flow Cytometry, IHC (PS), FUNC | Print as PDF
 
ENZ-ABS130-0100 100 µg 431.00 USD
Do you need bulk/larger quantities?
 

CD40, soluble (human) ELISA kit 

ELISA | Print as PDF
 
ALX-850-262-KI01 96 wells 703.00 USD
Do you need bulk/larger quantities?
 

Recommend this page

 
For Research Use Only. Not for use in diagnostic procedures.
Keep in touch

©2018 Enzo Life Sciences, Inc.,