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Application
This product has been tested for cell culture applications. Trypsin has been used in a study to assess the potential application in animal cell culture of an alkaline protease from a ton-toxigenic mangrove isolate of Vibri sp. V26. Trypsin has also been used in a study to improve the detection of fungi in eosinophilic mucin.
Biochem/physiol Actions
This inhibitor acts against trypsin, and chymotrypsin and plasmin to a lesser extent. It will also inhibit proteases with mechanisms similar to trypsin, plasma kallikrein and coagulation Factor X. The trypsin inhibitor will not act against metalloproteases, tissue-baseed kallikrein, acid proteases, or thio proteases. This inhibitor acts by forming a 1:1 stoichiometric complex with the protease active site, and then cleaving a single arginine-isoleucine bond on the inhibitor. The inhibition is both reversible and pH dependent.
Preparation Note
The trypsin inhibitor is soluble in water and phosphate buffers at 10 mg/mL. It is soluble in balanced salt solutions at 1 mg/mL and in serum-free media. Concentrated solutions greater than 10 mg/mL may be hazy and have a yellow to amber color. After trypsinizing cells, resuspend in 1 mL trypsin inhibitor solution at 1 mg/mL for every mL of trypsin solution used for dissociation. The cell suspension should then be centrifuged at 1000 rpm, forming a cell pellet.
Solutions can retain activity when stored short-term at 2-8° C. Solutions are stable in frozen aliquots at -20°C.
Analysis Note
One mg of trypsin inhibitor will inhibit 1.0-3.0 mg trypsin of activity ~10,000 BAEE units per mg protein.
Other Notes
One trypsin unit = A253 of 0.001 per minute with N-alpha-benzoyl-L-arginine ethyl ester (BAEE) as substrate at pH 7.6 at 25 °C.
One trypsin unit will produce a ΔA253 of 0.001 per min with BAEE as substrate at pH 7.6 at 25 °C; reaction volume 3.2 ml, 1 cm light path.
The soybean trypsin inhibitor is a monomeric protein containing 181 amino acid residues in a single polypeptide chain crosslinked by two disulfide bridges.
