1Trypsin Activity Assay Kit
Catalog # 3043

For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:

Assay kit to measure trypsin activity

FORMAT:

96-well ELISA Plate with removeable strips

ASSAY TYPE:

Chromogen Assay

ASSAY TIME:

15 to 120 minutes

STANDARD RANGE:

N/A

NUMBER OF SAMPLES:

Up to 42 (duplicate) colorless samples/plate and up to 21 (duplicate) colored samples/plate

SAMPLE TYPES:

Tissue homogenate, cell homogenate, culture media, or purified enzymes

RECOMMENDED SAMPLE DILUTIONS:

1:1 (at least)

CHROMOGEN:

Uses a Boc-Gln-Ala-Arg-pNA substrate (read at 405 nm)

STORAGE:-20°C

VALIDATION DATA:N/ANOTES:

N/A© 2023 Chondrex, Inc. All Rights Reserved, 3043 3.0
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

2

Trypsin Activity Assay Kit
Catalog # 3043

For Research Use Only - Not Human or Therapeutic Use

INTRODUCTION
Trypsin is a mammalian serine protease and a member of the PA Clan (proteases of a mixed nucleophile, superfamily A), the largest of thecysteine and serine protease families. The PA Clan, which also includes trypsin-like proteases, can hydrolyze positively charged amino acidpeptide bonds in polypeptide chains, specifically the carbonyl group on Arg or Lys (1), effectively degrading peptides and proteins. As such,
trypsin produced by the pancreas plays a key role in facilitating protein digestion and absorbance of foods in the small intestine. It also
regulates the gastrointestinal immune response by controlling microbicide concentrations in the intestinal lumen and maintaining the integrityof the epithelial barrier (2-3). Due to these capabilities, trypsin is widely used in protein or peptide-related research for synthesizing andsequencing peptides, maintaining cultured cells, and digesting proteins (4). Recently, it was reported that proteinases in house dust mites
promoted allergenicity to proteins, resulting in allergic reactions (5). This suggests that proteinases may play roles in the development ofType I allergies. In order to validate the study results, the ability to measure enzyme activity is critical.
Chondrex, Inc. introduces a chromogenic Trypsin Activity Assay Kit (Cat # 3043) which uses a Boc-Gln-Ala-Arg-pNA substrate to measuretrypsin-like enzyme activity in 15 minutes or more (up to 120 minutes, depending on the enzyme activity in samples). The trypsin cleaves
the carbonyl group in the Arg of the substrate, liberating p-nitroanilide (pNA) which produces a yellow color and can be quantified using
optical density (6). This kit works for trypsin and any proteinase/peptidase which cleaves the substrate. Therefore, to accurately analyze aspecific proteinase’s activity, it may be necessary to add proteinase inhibitors to inactivate other proteinases in samples. This kit, whichemploys a short assay time and a microplate format, is ideal for assaying many samples, as compared to traditional cuvette readings or gel
assays (7). For more applications, please contact support@chondrex.com for published research references.

KIT COMPONENTS
Item

Quantity

AmountStorage

Trypsin Reference (30431)
Boc-Gln-Ala-Arg-pNA Substrate (30432)

1 vial1 vial200 mUnits, Lyophilized
50 µl, 100 mM in DMSO

-20°C
-20°C

Solution B (67015)
96-Well ELISA Plate

1 bottle
1 each50 ml
8-well strips x 12-20°C-20°C

ASSAY OUTLINE
Add 50 ml of diluted references and samples into wells

Add 50 ml of diluted Substrate into wells

Read plate at 405 nm every 3 minute to 15 minutes or longer© 2023 Chondrex, Inc. All Rights Reserved, 3043 3.0
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

3NOTES BEFORE USING ASSAY
NOTE 1: It is recommended that the standard and samples be run in duplicate.
NOTE 2: Warm up all buffers to room temperature before use.
NOTE 3: Measure exact volume of buffers using a serological pipet, as extra buffer is provided.
NOTE 4: For partial reagent use, please see the assay protocol’s corresponding step for the appropriate dilution ratio. For example, if theprotocol dilutes 50 µl of a stock solution in 10 ml of buffer for 12 strips, then for 6 strips, dilute 25 µl of the stock solution in 5 ml of buffer.
Partially used stock reagents may be kept in their original vials and stored at -20⁰C for use in a future assay.
NOTE 5: This kit contains animal components from non-infectious animals and should be treated as potential biohazards in use and fordisposal.

PLATE LAYOUT
Standard Assay Layout (colorless samples)
12345678

Standard Assay Layout (colored samples)

910111212345678910

1112

C

C

S2

S2

S6

S6

S10

S10

S14

S14

S18

S18

A

CC

S3

S3

S11

S11

S19

S19

S27

S27

S35

S35

AB

B

B

S4

S4

S12

S12

S20

S20

S28

S28

S36

S36BBBSB2SB2SB6SB6SB10SB10SB14SB14SB18SB18C

R1R1S5S5S13

S13

S21

S21

S29

S29

S37

S37

C

R1R1S3S3S7S7S11S11S15S15

S19

S19D

R2R2S6S6S14S14S22

S22

S30

S30

S38

S38

D

R2R2SB3SB3SB7SB7SB11SB11SB15

SB15

SB19SB19E

R3R3S7S7S15

S15

S23

S23

S31

S31

S39

S39

E

R3

R3S4S4S8

S8

S12

S12

S16

S16

S20

S20F

R4R4S8S8S16

S16S24

S24

S32

S32

S40

S40F

R4R4SB4SB4SB8SB8SB12SB12SB16SB16SB20SB20GS1S1S9S9S17S17S25

S25S33

S33

S41

S41

GS1S1S5S5S9S9S13S13S17S17S21S21H

S2S2S10S10S18S18S26

S26

S34

S34

S42S42H

SB1SB1SB5SB5SB9SB9SB13SB13SB17SB17SB21SB21ASSAY PROCEDURE
1.

Prepare Trypsin References: Four levels of reference standards are recommended. Dissolve 1 vial of Trypsin Reference Standard in
1 ml of Solution B (200 mUnits/ml) and keep it as a reference stock and 100% control. Add 100 µl of this standard stock solution to 150µl of Solution B to make a 67 mUnits/ml solution (Reference 1). Then serially dilute it with Solution B. For example, mix 125 µl of the67 mUnits/ml solution with an equal volume of Solution B to make a 33 mUnits/ml solution (Reference 2), and then repeat it two moretimes for 18 and 9 mUnits/ml solutions (Reference 3 and 4, respectively). The remaining 200 mUnits/ml reference stock may be stored
at -20°C for use in a second assay. Chondrex, Inc. recommends making fresh serial dilutions for each assay.
1000 ml
125 ml100 ml125ml125ml

R1

R2

R3R4B

1000 ml

150 ml

125ml

125ml

125ml

125ml

200 mUnits/ml

67 mUnits/ml

33 mUnits/ml

17 mUnits/ml

9 mUnits/ml

BlankC© 2023 Chondrex, Inc. All Rights Reserved, 3043 3.0
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

42.

Prepare Samples: Samples can be tissue homogenates, cell homogenates, culture media, or purified enzymes. Centrifuge at 10,000rpm for 5 minutes, then use the supernatant if the samples include insoluble materials. Dilute samples at least 1:1 with Solution B
depending on the estimated trypsin levels in the samples. Two to three different sample dilutions are recommended if the trypsin levelsin the samples are unknown.
NOTE 1: Samples must be diluted with Solution B to maintain optimal assay conditions.
NOTE 2: If the sample solution has color such as culture media, Chondrex, Inc. recommends using sample blank wells for the assays.
Please refer to Step 4.
NOTE 3: Depending on the needs of the experiment, please consider appropriate protease inhibitors, especially trypsin inhibitors
because they will affect the assay’s outcome.
NOTE 4: Freshly prepared samples are recommended for this assay. Samples can be immediately stored at -20°C after preparationand still be used for the assay. However, the enzyme activities will depend on the types of samples.

3.

Prepare Substrate Dilutions: Prepare the substrate solution with Solution B as shown in the following table (16 wells = 2 strips).
Strip #246810124.

Substrate (µl)
81725334250

Solution B (ml)0.8
1.7
2.5
3.3
4.2
5.0

Add 100% Control, References, and Samples: Choose 4-1 or 4-2 depending on samples
4-1. Colorless Samples: Use the plate layout for colorless samples. Add 50 µl of the following into their respective wells: 200 mUnits/ml
trypsin reference standard stock into the 100% control (C) wells, Solution B into the (B) wells, 67 – 9 mUnits/ml References into the(R1-R4) wells, and samples into the orange wells in duplicate. For example, add 50 µl of Sample 1 into the S1 wells, 50 µl of Sample
2 into the S2 wells, etc. Proceed to Step 5-1.
4-2. Colored Samples: Use the plate layout for colored samples. Add 50 µl of the following into their respective wells: 200 mUnits/ml
trypsin reference standard stock into the 100% control (C) wells, Solution B into the (B) wells, 67 – 9 mUnits/ml References into the(R1-R4) wells, and samples into the orange and gray wells in duplicate. For example, add 50 µl of Sample 1 into the S1 and SB1 wells,50 µl of Sample 2 into the S2 and SB2 wells, etc. Proceed to Step 5-2.

5.

Add Substrate:
5-1. Colorless samples: Add 50 µl of substrate solution to all wells at room temperature (25°C).
5-2. Colored samples: Add 50 µl of substrate solution into the purple and orange wells and add 50 µl of Solution B into the gray wells
at room temperature (25°C).

6.

Read: If it is possible, keep the plate reader temperature at 25°C. Read the plate at 405 nm every 3 minutes for 15 minutes. If thetrypsin activity is low, keep reading the plate every 10 minutes, up to 120 minutes.
NOTE: If OD values of samples are the same as the OD values of the 100% control, dilute samples for another assay or adjust using
kinetics analysis.

© 2023 Chondrex, Inc. All Rights Reserved, 3043 3.0
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

5

CALCULATING RESULTS
1.

Average the duplicated OD values for the standards, blanks (B),100% control (C), references (R1-R4), and samples (S).
NOTE: If using sample blanks, the averaged OD values of the sample blanks (SB) are subtracted from the averaged OD values of thesamples (S).

2.

One mUnit of trypsin activity (1 pNA mUnit) is defined as the cleavage of 1 nmole of substrate per minute (1 mUnit = 1 nmole/minute).
Because this kit uses 50 nmole of substrate per well, trypsin activity is calculated using the following equation:
NOTE: one pNA mUnit = 0.615 TAME mUnit = 35 BAEE mUnit.

ODBlank= OD in Blank (at 15 minutes)

Trypsin Activity at 15 Minutes (pNA mUnits/ml) =ODControl= OD in 100% Control (at 15 minutes)(ODSample - ODBlank) x 50 nmole

ODSample= OD in Test Samples (at 15 minutes)(ODControl - ODBlank) x 15 (minutes) x Sample Volume (ml)NOTE: The trypsin reference standard is used to check assay accuracy. Trypsin activity in individual samples must be calculated using theequation above.

Optional: If samples have high trypsin activity, then at the 15-minute point, the OD values may be the same as the 100% reference control.
In this case, calculate enzyme activity using the following equation:
ODBlank= OD in Blank (at 15 minutes)

Trypsin Activity (pNA mUnits/ml) =ODControl= OD in 100% Control (at 15 minutes)(ODSample time B - ODSample time A) x 50 nmole

ODSample Time A = OD in Test Samples at Time A (Ex: 6 minutes)(ODControl - ODBlank) x ∆Time (minutes) x Sample Volume (ml)ODSample Time B = OD in Test Samples at Time B (Ex: 9 minutes)∆Time = Time B – Time A

Figure 1 - Trypsin Activity Assay Kit
Dose Response Curve

Time Course
Time Course

15 Minute Incubation
60
R² = 0.9933

50

100% Control40

Reference 140Degradation (nmole)Degradation (nmole)

503020Reference 230Reference 320Reference 41010Blank0000102030405060369121570

Time (minutes)

Trypsin (mUnits/ml)© 2023 Chondrex, Inc. All Rights Reserved, 3043 3.0
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

6TROUBLESHOOTING
For frequently asked questions about assays and ELISAs, please see Chondrex, Inc.’s Assay FAQ for more information.

REFERENCES
1.

E. Di Cera, Serine Proteases. IUBMB Life 61, 510-5 (2009).

2.

M. Bajaj-Elliott, Trypsin and Host Defence: A New Role for an Old Enzyme. Gut 52, 166-7 (2003).

3.

D. Ghosh, E. Porter, B. Shen, S. Lee, D. Wilk, et al., Paneth Cell Trypsin Is the Processing Enzyme for Human defensin-5. NatImmunol 3, 583-90 (2002).

4.

J. Mótyán, F. Tóth, J. Tőzsér, Research Applications of Proteolytic Enzymes in Molecular Biology. Biomolecules 3, 923-42 (2013).

5.

M. Reithofer, B. Jahn-Schmid, Allergens With Protease Activity From House Dust Mites. Int J Mol Sci 18, 1368 (2017).

6.

B. Erlanger, N. Kokowsky, W. Cohen, The Preparation and Properties of Two New Chromogenic Substrates of Trypsin. Arch BiochemBiophys 95, 271-8 (1961).

7.

Z. Zhao, P. Russell, Trypsin Activity Assay in Substrate-Specific One- And Two-Dimensional Gels: A Powerful Method to Separateand Characterize Novel Proteases in Active Form in Biological Samples. Electrophoresis 24, 3284-8 (2003).

© 2023 Chondrex, Inc. All Rights Reserved, 3043 3.0
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com