How to Label Protease Substrate Products

Proteins are a component of every cell in the body. Proteases (also referred to as proteolytic enzymes or proteinases) are digestive enzymes whose function is to breakdown proteins. In addition to digestion, proteases are also essential for blood-clotting, metabolic control and immune regulation. During the biochemical process (catalysis), substrates bind to the enzyme's active site and are transformed into one or more products that are then released from the site. This process can be augmented or inhibited to increase or reduce reactions in the body, depending on the desired outcome. Labeling substrate products allows scientists to measure this enzymatic activity. Fluorescent labeling is a technique used to color particular molecules, making them easier to track.

Things You'll Need

• Target molecule cocktail
• Fluorescent dye labeling kit
• Fluorescence reading instrument

Show More

Instructions

How to Label Protease Substrate Products

• 1

  Choose your fluorescent dye. Common dyes include rhodamine and fluorescein. These can usually be purchased from manufacturers.

• 2

  Run the reaction and remove unused fluorophore from target molecules by using size exclusion chromatography (a process that separates molecules based on their size). Dye Removal Columns are also commonly used. These are often advertised with labeling kits.

• 3

  Detect the molecules. Use a fluorescence reading instrument, such as a fluorescence microscope. Light of a specific wavelength is passed through the molecule (indirectly for some techniques), illuminating the fluorophore.

• 4

  Take your measurements. Because assays measure either how much substrate was consumed or the amount of product released over a period of time, enzyme amounts can be measured in moles (physical quantity) or in enzyme units (activity).