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Trypsin Activity Colorimetric Assay Kit

based on 7 citations in multiple journalsTrypsin Activity Colorimetric Assay Kit74.2 4
Catalog #: K771
$370.00

Product Details

Cat # +Size K771-100
Size 100 assays
Detection Method Absorbance (405 nm)
Species Reactivity Mammalian
Applications The kit detects 10-100 mU (p-NA unit) trypsin in various samples.
Features & Benefits • Simple procedure; takes ~ 1-2 hours based on the levels of trypsin
Kit Components • 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)
Storage Conditions -20°C
Shipping Conditions Gel Pack
USAGE For Research Use Only! Not For Use in Humans.

Details

Trypsin (EC 3.4.21.4) 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.


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Should protease inhibitors be added to the buffer?
Trypsin itself is a Serine protease and hence adding protease inhihitors will affect its activity too.
Can a one time point measurement be done with this assay for the Trypsin activity in the sample?
We suggest doing measurements over time intervals because the rate of enzyme activity changes with respect to time. Once the customer has chosen a certain end time point for their experiments, they can do a cumulative one-time point measurement. However, Trypsin activity can be more accurately measured when you do kinetic measurements versus an end-point assay.
What is the detection range for Trypsin activity for this kit?
Our detection range is between 10-100mU.
Can milk be used as a sample in this kit?
Milk can be used with this kit. It is essential to run a pilot experiment using a broad range of dilutions so that the final dilution can be chosen such that the reading is within the linear range of the standard curve. It is essential that samples not be cloudy due to addition of too much milk for accurate OD measurements.
Will phenol red or any other component in media interfere in this assay?
Phenol red might change color at the pH which is optimum for Trypsin activity. Also any serum in the medium inhibits trypsin activity.
Can the activity of the positive control decrease over time? Can Trypsin with known acitivy from another source be used to validate this assay?
It is critical to remember that the purified Trypsin is dissolved, aliquoted into vials, then lyophilized to send to the customer. Then the customer reconstitutes it and uses it in the assay. Trypsin is notoriously well-known for digesting itself in solution leading to decrease in activity over time. Also if the customer purchases Trypsin from a vendor and tries to get back the exact activity numbers specified by the vendor using our assay, that is not going to be possible since the assay buffer composition and pH differences (between our Assay buffer and the conditions used by the vendor to define their units of activity) significantly affect Trypsin activity.
For the inhibitor treatments, should the inhibitor and reaction mix e added and then incubated?
Treat with 1 ul of 50X chymotrypsin inhibitor (TPCK) solution and incubate for 10 minutes at room temperature. Add 1 ul of 50X trypsin inhibitor (TLCK) solution to trypsin inhibitor sample control and incubate for 5 min. These incubations are before adding the reaction mix.
Can a negative control be set up by adding Trypsin inhibitor to the positiev control?
Yes, it can be done but we do not suggest inhibiting the purified Trypsin (positive control) to get a negative control. The negative control is based on the inhibition of the trypsin in the sample. A trypsin inhibitor sample group can be setup as a control by adding 1 µl of 50X trypsin inhibitor (TLCK) solution to trypsin inhibitor sample control. Once Trypsin is inhibited, any residual protease activity can be accounted for. Also, there is a Chymotrypsin inhibitor to account for any endogenous chymotrypsin like activity in the sample.
Why do we see the same OD for 25 ul and 50 ul sample?
It is possible that the customer does not see a dose-dependent relationship if they are adding too much of the enzyme and the readings are very high/saturated. The substrate becomes limiting if there is too much enzyme and can cause such issues. I would suggest diluting in the assay buffer or simply water and then optimize the sample amount needed in the assay to get values int he linear range of the std. curve.
What is the shelf-life of the kit? What about the positive control is stored unthawed at -20C or -80C for 2-3 years?
The shelf-life of the unopened kit is 1 year from the date of shipping from us when stored at -20C. Trypsin is notoriously well known for its self-destructive protease activity when stored for long periods of time. Once reconstituted, the positive control is to be used within 2months for best results. Hence we do not expect the positive control enzyme to retain normal activity over 2-3 years even at -20C or -80C.
Are trial sizes of this kit available?
Unfortunately, we do not have trial sizes of this kit available. However,for customers based in the US or Canada, a 10% off list price introductory discount can be offered on the list price. For all other customers based out of this area our regional BioVision distributor should be contacted
Can individual components of this kit be purchased separately?
Yes, any of the kits components can be purchased separately without having to buy the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Chen, X. et al. (2016) Interactive effects of dietary protein concentration and aflatoxin B1 on performance, nutrient digestibility, and gut health in broiler chicks Poult Sci. 2016 Jun 1;95(6):1312-25.
Bansemer, Matthew S. et al. (2016) Age-dependent response of digestive enzyme activities to dietary protein level and water temperature in greenlip abalone (Haliotis laevigata), Aquaculture. 2016 Jan; 451: 451-456
King et al., Isolation of Positive Modulator of Glucagon-like Peptide-1 Signaling from Trigonella foenum-graecum (Fenugreek) Seed.  J. Biol. Chem., Oct 2015; 290: 26235 - 26248.
Pruszynska-Oszmalek et al., In ovo injection of prebiotics and synbiotics affects the digestive potency of the pancreas in growing chickens. Poultry Science, Aug 2015; 94: 1909 - 1916.
Cheng et al., Modulation of Glucagon-like Peptide-1 (GLP-1) Potency by Endocannabinoid-like Lipids Represents a Novel Mode of Regulating GLP-1 Receptor Signaling. J. Biol. Chem., Jun 2015; 290: 14302 - 14313.
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