Trypsin Activity Colorimetric Assay Kit Kit
- Detection Method
- Detection Range
- 10-100 mU
- Minimum Detection Limit
- 10 mU
- Activity Assay (AcA)
- Sample Type
- Cell Culture Supernatant, Plasma, Serum, Tissue Samples
- In the assay, trypsin cleaves a substrate to generate p -nitroaniline (p -NA) which is detected at lambda=450nm. Since the color intensity is proportional to p -NA content, trypsin activity can be accurately measured. The kit detects 10-100 mU (p -NA unit) trypsin in sample.
- Trypsin Activity Assay Kit: Colorimetric Assay for detecting Trypsin Activity in Tissues, Cells, Serum etc. Rapid, Convenient & Sensitive.
Trypsin Assay Buffer
Trypsin Substrate (in DMSO)
Positive Control (lyophilized)
p-NA Standard (2 mM)
Trypsin Inhibitor (TLCK, 20 mM)
Chymotrypsin Inhibitor (TPCK,10 mM)
- Application Notes
- The kit detects 10-100 mU (p-NA unit) trypsin in various samples.
Further details regarding sample type: Cell and tissue culture supernatants, urine, plasma and serum, as well as many other biological fluids, etc.
1. Standard Curve Preparations: Add 0, 2, 4, 6, 8, 10 µL p -NA standard into a series of standards wells. Adjust volume to 50 µL/well with Trypsin Assay Buffer to generate 0, 4, 8, 12, 16, and 20 nM/well of the p -NA standard.
2. Sample and Positive Control Preparations: Prepare test samples at 50 µL/well with Assay Buffer in a 96-well plate. Serum can be directly added into sample wells, and the volume adjusted to 50 µL/well with Assay Buffer. Tissues or cells can be extracted with 4 volumes of the Trypsin Assay Buffer, centrifuge to get a clear extract. We suggest using several doses of your sample to ensure the readings are within the linear range. Treat with 1 µL of 50X chymotrypsin inhibitor (TPCK) solution and incubate for 10 minutes at room temperature. For the positive control, add 5 µL positive control solution to wells, adjust volume to 50 µL/well with Assay Buffer. If desired, set a trypsin inhibitor sample group as a control by adding 1 µL of 50X trypsin inhibitor(TLCK) solution to trypsin inhibitor sample control and incubate for 5 minutes.
3. Reaction Mix Preparation: Mix enough reagents for the number of assays to be performed. For each well, prepare a total 50 µL Reaction Mix containing: 48 µL Assay Buffer 2 µL Trypsin Substrate Mix then add 50 µL of the reaction mix to each well containing the p -NA standards, positive controls, test samples or test samples trypsin inhibitor control, mix well, incubate at 25 °C, protected from light.
4. Initially measure absorbance at 405 nm as absorbance (A 1 and A 1C for trypsin inhibitor control) at time T
1. After incubating the reaction for 1-2 hours (or incubate longer time if the trypsin activity is low) measure the absorbance (A 2 and A 2C at T 2 ). The color generated by cleavage of substrate is deltaA 405nm = (A 2 - A 2C ) - (A 1 - A 1C ) or (A2 - A1), if no trypsin inhibitor control was run. Note: It is essential to read A 1 and A 2 in the reaction linear range. It will be more accurate if you read the reaction kinetics. Then choose A 1 and A 2 in the reaction linear range.
- Calculation of Results
Calculation: Plot the p -NA standard Curve. Apply the deltaA 405nm to the standard curve to get the nmol of p -NA (amount generated between T 1 and T 2 in the reaction wells). Trypsin Activity = (see image 2) Where: B is the p -NA calculated form the Standard Curve (in nmol). T 1 and T 2 are the times of the first and second readings (in min). V is the pretreated sample volume added into the reaction well (in ml). One unit is defined as the amount of trypsin cleaves the substrate, yielding 1.0 nmol of p -NA per minute at 25 °C.
- For Research Use only
- -20 °C
- Expiry Date
- 12 months
Mitchell, Samulski: "Mechanistic insights into the enhancement of adeno-associated virus transduction by proteasome inhibitors." in: Journal of virology, Vol. 87, Issue 23, pp. 13035-41, 2013 (PubMed).
Hayashi, Kohno, Yasui, Murota, Kimura, Duncan, Nakashima, Yamamoto, Katayama, Ma, Chua, Suematsu, Shimokawa, Akira, Kubo, Mak, Matsuyama: "Characterization of dsRNA-induced pancreatitis model reveals the regulatory role of IFN regulatory factor 2 (Irf2) in trypsinogen5 gene transcription." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 108, Issue 46, pp. 18766-71, 2011 (PubMed).
- Mitchell, Samulski: "Mechanistic insights into the enhancement of adeno-associated virus transduction by proteasome inhibitors." in: Journal of virology, Vol. 87, Issue 23, pp. 13035-41, 2013 (PubMed).
- wu:fb57g08, zgc:109701, LOC397853, Trypsin, trp1, try1, trypsin, protease, serine 1, protease, serine 1 L homeolog, protease, serine, 1, trypsin, prss1, prss1.L, Deipr_2253
- Trypsin (EC 22.214.171.124) is a serine protease found in the digestive system of many vertebrates, where it hydrolyses proteins. Trypsin is produced in the pancreas as the inactive proenzyme trypsinogen. Active trypsin predominantly cleaves peptide chains at the carboxyl side of the amino acids lysine or arginine, except when either is followed by proline. It is used for numerous biotechnological processes. In the assay, trypsin cleaves a substrate to generate p-nitroaniline (p-NA) which is detected at λ=405 nm. Since the color intensity is proportional to p-NA content, trypsin activity can be accurately measured. The kit detects 10-100 mU (p-NA unit) trypsin in various samples.