Protease Testing

Protease enzymes break down proteins, which can ruin lab analysis. Testing confirms your custom reagents are protease-free. Learn more about protease testing.

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 an 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₊Zn_2+, 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

References

Rao, M. B., Tanksale, A. M., Ghatge, M.S., and Deshpande, V. V. (1998). Molecular and Biotechnological Aspects of Microbial Proteases. Microbiology and Molecular Biology Reviews. 62 (3): 597-635
Razzaq, A., Shamsi, S., Ali, A., Ali, Q., Sajjad, M., Malik, A., & Ashraf, M. (2019). Microbial Proteases Applications. Frontiers in Bioengineering and Biotechnology
Lopez-Otín, C and Bond, J. S. (2008) Proteases: Multifunctional Enzymes in Life and Disease. J. Biol. Chem. 283 (45): 30433-30437.

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 an 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₊Zn_2+, 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