Protease Testing

A Protease Assay is a quality control measure employed to identify and quantify the presence of proteases in various biological buffers, reagents, and solutions. Proteases are found ubiquitously in organisms like plants, animals, and microbes and are responsible for degrading protein aggregates, managing genome translation, and adhering to cellular structural guidelines [1]. Proteases are essential in ending the lifecycles of cellular proteins and have a very high affinity for specific protein types [2].

History of Protease Testing

This assay type was developed to address the pressing need for accurate and uncontaminated protein studies, and has continuously evolved to meet the demands of scientific inquiry. Over the years, protease assay methodologies have become more sophisticated, enabling scientists to detect and mitigate protease interference with high precision and sensitivity.


Image 1: The mechanism of a protease. As shown, Proteases have affinities for specific amino acids, and utilize their active cites for cleavage, resulting in broken down amino acid components [1]. Image 1 is inspired by Rao et. al.

Protease Testing for Buffers, Media, and Reagents

Proteases have the potential to impact the outcomes of in vitro protein studies by causing unintended degradation of protein material within select buffers, reagents and media types [3]. Any contamination by proteases in these tools can substantially compromise the scientific validity of experiments and research findings in their future applications. It is important to test for protease levels prior to performing any protein-related research, and to source materials from verified vendors.

Protease Testing at Boston BioProducts

Boston BioProducts relies on an ultrasensitive colorimetric method for the Protease Assay. This assay employs succinylated casein and Trinitrobenzene sulfonic acid (TNBSA). Casein is succinylated to block primary amine groups (I.e.., e-amino group of lysine and N-terminal a-amino group of protein) enhancing its suitability as a protease substrate. In the presence of proteases, succinylated casein is hydrolyzed, exposing primary amines, which subsequently react with TNBSA to generate a distinctive yellow-orange product. A Microplate Reader is then used to measure the absorbance of yellow-orange product, and data may be analyzed.


Images 2 and 3: The results of a quantitative protease assay. Upon analysis of the absorbance of the 96-well plate (image 2), a standard curve may be developed to assess the performance of the assay via it's R-squared value (image 3). An R-squared value close to or at 1 demonstrates the experimental values match the desired outcome with minimal error.

Considerations and Limitations of Protease Testing

Presence of Primary Amines in samples Be aware of primary amines in buffers, particularly compounds like Tris, which can produce false-positive results by generating color when reacting with TNBSA. Utilize a "Control Blank" devoid of succinylated casein for each sample to identify and mitigate any color development not related to true protease activity.
Cleanliness of the Environment and Containers Prepare buffers in a environment using clean containers to minimize the risk of contamination. Immediately filter solutions post-preparation to prevent microbial contamination and maintain solution integrity.
pH of the Buffer Solutions The optimal protease activity is at pH 8.0.
Presence of Reducing Agents The presence of reducing agents such as glutathione (GSH), dithiothreitol and Beta-mercaptoethanol results in increase in protease activity.
Ionic Concentration of the Buffer Solutions Protease activity decreases with the increasing salt concentration. Sample with higher salt concentration should be diluted (5-20 mM) before conducting protease activity.
Presence of Metal Ions Ca2+, Mg2+, Ba2+, NH2+, Li+, and Mn2+ activate protease activity. Whereas K+, Na+ Zn2+, Ni2+, and Sr2+ inhibit protease activity.
Presence of Detergents Non-ionic detergents (e.g., 1% Tween-20, 1% Tween-80 and 1% Triton X-114, and) have been shown to have no effect on protease activity. Anionic detergents such as SDS and cationic detergents, sodium deoxycholate and cetyltrimethylammonium bromide (CTAB) inhibit protease activity, even at a low concentration (e.g.,0.1%)
Presence of Chelators EDTA inhibits protease activity, and the inhibitory effect of EDTA increases with increasing concentration of EDTA.
Presence of Denaturants Denaturants such SDS and Urea result in decrease in Protease activity.
Presences of Organic Solvents Organic solvents such as ethyl acetate, methanol, ethanol and dimethyl sulfoxide interfere with the protease activity. Hence, the samples should be diluted appropriately

In addition to Protease Testing, Boston BioProducts provides a comprehensive set of QC tests for custom reagents. Learn more about custom reagent development services.