QuantiFluo™ Pepsin Inhibitor Assay Kit

Protocol SDS

Application

• For evaluation of drugs and screening of potential inhibitors to pepsin proteases.

Key Features

• Safe. Non-radioactive assay.
• Homogeneous and convenient. Homogenous “Mix-incubate-measure” type assay. No wash and reagent transfer steps are involved.
• Robust and High-throughput. Can be readily automated to assay thousands of samples per day. Robust assay with a Z’ factor of > 0.7.

Method

• FL485/530nm or FP485/530nm

Samples

• Compounds that affect pepsin activity

Species

• All

Procedures

• 45 min

Size

• 200 tests in 96-well plate

Detection Limit

• NA

Shelf Life

• 6 months

More Details

PEPSIN (EC 3.4.23.1) is a digestive, serine protease that hydrolyzes dietary proteins in many eukaryotic and prokaryotic organisms. Pepsin predominantly cleaves peptide chains at the amino side of the aromatic phenylalanine, tryptophan, and tyrosine amino acids. BioAssay Systems’ QuantiFluo™ Pepsin Inhibitor Assay Kit uses a fluorescein isothiocyanate (FITC)-labeled synthetic substrate. The fluorescein label is highly quenched. Upon digestion by pepsin present in the sample, the substrate is cleaved into smaller peptides, which abolishes the quenching of the fluorescence label. The fluorescence or fluorescence polarization (FP) of the FITC-labeled fragments is measured at λ_ex/em = 485/530 nm. Inhibition is determined by the decrease in fluorescence.

Is pepsin enzyme included in the kit?

The customer will need to provide their own enzyme. Porcine pepsin (e.g. MP Biomedicals, cat# 102598) was used for the development of this kit.

Do I need to prepare my test compounds under acidic conditions or can I prepare them in dH2O or Assay Buffer?

While it is likely fine to prepare test compounds in a more neutral solvent, we recommend preparing compounds under acidic conditions since pepsin shows maximal activity at ~pH 2.

Why does the reaction need to be neutralized with Assay Buffer after the reaction has finished?

While pepsin requires acidic conditions for maximum activity, the fluorescent detection dye used in this kit will not demonstrate fluorescence unless conditions are closer to neutral. Assay Buffer will effectively work as a stop reagent for the enzyme reaction and allow detection of the product.

For more detailed product information and questions, please feel free to email or call us at 1-510-782-9988 x 2. For more general information regarding our assays, please refer to Technical Support.

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If you or your labs do not have the equipment or scientists necessary to run this assay, BioAssay Systems can perform the service for you.

– Fast turnaround
– Quality data
– Low cost

Please email or call 1-510-782-9988 x 2 to discuss your projects.