Enteropeptidase/Enterokinase Activity Assay Kit

Catalog Number MAK204 Storage Temperature –20 C

TECHNICAL BULLETIN

Product Description Reagents and Equipment Required but Not Enteropeptidase (Enterokinase; EC 3.4.21.9) is an Provided that converts to , the activity  96 well flat-bottom plate – It is recommended to use of which is required for the activation of black plates with clear bottoms for fluorescence and procarboxypeptidases. It has an important role in assays. food and may serve as a marker of  Fluorescence multiwell plate reader differentiated and goblet cells.1,2 Precautions and Disclaimer The Enteropeptidase/Enterokinase Activity Assay Kit is For R&D use only. Not for drug, household, or other a rapid method to measure enteropeptidase activity in uses. Please consult the Safety Data Sheet for biological samples. Enteropeptidase activity is information regarding hazards and safe handling measured by cleaving a synthetic 7-amino- practices. 4-trifluoromethylcoumarin (AFC)-tagged substrate containing the recognition sequence for Preparation Instructions enteropeptidase. This yields AFC, a fluorescent product Briefly centrifuge vials at low speed before opening. To (Ex = 380/ Em = 500 nm), proportional to the enzymatic maintain reagent integrity, avoid repeated freeze/thaw activity present. One unit of enteropeptidase is the cycles. amount of enzyme that generates 1.0 nmole of AFC per minute at room temperature. Enteropeptidase Assay Buffer, Enteropeptidase Substrate, and AFC Standard – Warm each to Components room temperature before use. The kit is sufficient for 100 assays in 96 well plates. Human Enteropeptidase (Positive Control) – Enteropeptidase Assay Buffer 20 mL Reconstitute with 83 L of Enteropeptidase Assay Catalog Number MAK204A Buffer. Mix well by pipetting. Aliquot and store at –20 C. Use within 2 months of reconstitution. Enteropeptidase Substrate, 10 mM in DMSO 0.2 mL Catalog Number MAK204B Storage/Stability The kit is shipped on wet ice and storage at –20 C, Human Enteropeptidase 100 L protected from light, is recommended. Catalog Number MAK204C

AFC Standard, 1 mM 100 L Catalog Number MAK204D

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Procedure Assay Reaction AFC Standards for Fluorometric Detection 1. Set up the Reaction Mixes according to the scheme Dilute 10 L of the 1 mM (1 nmole/L) AFC Standard in Table 1. 50 L of the appropriate Reaction Mix is Solution with 90 L of Enteropeptidase Assay Buffer to required for each reaction (well). prepare a 100 M (100 pmole/L) AFC Standard Solution. Add 0, 2, 4, 6, 8, and 10 L of the 100 M Table 1. (100 pmole/L) AFC Standard Solution into 96 well Reaction Mixes plate, generating 0 (blank), 200, 400, 600, 800, and Standards, 1,000 pmole/well standards. Add Enteropeptidase Sample Reagent Controls, and Assay Buffer to each well to bring the total volume to Blank 100 L. Samples Enteropeptidase 48 L 50 L Sample Preparation Assay Buffer Enteropeptidase All samples and standards should be run in duplicate. 2 L – Substrate Add 5–50 L of the samples into duplicate wells. Bring samples to a final volume of 50 L using 2. Add 50 L of the appropriate Reaction Mix to each Enteropeptidase Assay Buffer. of the wells. Mix well using a horizontal shaker or by pipetting. Note: For unknown samples, it is suggested to test several sample dilutions to ensure the readings are 3. Measure the fluorescence (FLU, Ex = 380/ within the linear range of the standard curve. Em = 500 nm) in a microplate reader in kinetic mode for 30–60 minutes at room temperature. For samples exhibiting significant background, include Protect the plate from light during the incubation. It a Sample Blank for each sample by omitting the is recommended to take fluorescent readings every Enteropeptidase Substrate. The Sample Blank readings minute. can then be subtracted from the sample readings. Note: Incubation time depends on the activity of enteropeptidase in the samples. For a positive control (optional), add 5–10 L of the Enteropeptidase Positive Control solution to the desired 4. Continue taking measurements until the value of wells. Adjust the final volume to 50 L with the most active sample is greater than the value of Enteropeptidase Assay Buffer. the highest standard (1,000 pmole/well). At this time the most active sample is near or exceeds the end of the linear range of the standard curve.

Note: The AFC Standards can be read at the end of the incubation time.

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Results Enteropeptidase activity: Calculations Plot the fluorescence (FLU) for each well versus time. Enteropeptidase Activity = Sa (Reaction Time)  Sv Choose two time points (T1 and T2) in the linear range of the plot and determine the FLU at each time (FLU1 where: and FLU2). Sa = Amount of AFC (pmole) generated in unknown Note: It is essential that FLU1 and FLU2 fall within the sample well between T1 and T2 from standard linear range of the standard curve. curve Reaction Time = T2 – T1 (minutes) Correct for the background by subtracting the Sv = sample volume (mL) added to well measurement obtained for the blank AFC standard from that of the standards, controls, and samples. Enteropeptidase activity is reported as Background values can be significant and must be pmole/min/mL = milliunit/mL. subtracted from all readings. Use the values obtained from the appropriate AFC Standards to plot a standard One unit of enteropeptidase is the amount of enzyme curve. that generates 1.0 nmole of AFC per minute at room Note: A new standard curve must be set up each time temperature. the assay is run. Sample Calculation: Calculate the change in fluorescence measurement from T1 to T2 for the samples. Amount of AFC (Sa) = 584 pmole (from standard curve) FLU = FLU2 – FLU1 (T1) = 3 minutes Subtract the Sample Blank FLU value from the Sample FLU reading to obtain the corrected (T2) = 32 minutes measurement. Using the corrected measurement, determine the amount of AFC (pmole/well) generated Sample volume (Sv) = 0.050 mL by the enteropeptidase assay between T1 and T2 (Sa). Enteropeptidase activity in sample well:

pmole/min/mL = 584 pmole/well = 403 (milliunits/ mL) (32 min – 3 min)  0.050 mL/well

References 1. Imamura, T., and Kitamoto, Y., Expression of enteropeptidase in differentiated enterocytes, goblet cells, and the tumor cells in human . Am. J. Physiol. Gastrointest. Liver Physiol., 285, G1235–1241 (2003). 2. Braud, S. et al., Enteropeptidase: a associated with a starvation human phenotype and a novel target for obesity treatment. PLoS One., 7, e49612 (2012). doi: 10.1371/journal.pone.0049612.

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Troubleshooting Guide Problem Possible Cause Suggested Solution Cold assay buffer Assay Buffer must be at room temperature Omission of step in procedure Refer and follow Technical Bulletin precisely Assay not working Plate reader at incorrect wavelength Check filter settings of instrument For Fluorometric assays, use black plates Type of 96 well plate used with clear bottoms Use the Assay Buffer provided or refer to Samples prepared in different buffer Technical Bulletin for instructions Repeat the sample homogenization, Cell/Tissue culture samples were increasing the length and extent of incompletely homogenized homogenization step. Samples with erratic Samples used after multiple freeze-thaw Aliquot and freeze samples if needed to use readings cycles multiple times Presence of interfering substance in the If possible, dilute sample further sample Use of old or inappropriately stored Use fresh samples and store correctly until samples use Thaw all components completely and mix Improperly thawed components gently before use Check the storage requirements and store Use of improperly stored reagents Lower/higher the components appropriately readings in samples Allowing the reagents to sit for extended Prepare fresh Reaction Mixes before each and standards times on ice use Refer to Technical Bulletin and verify correct Incorrect incubation times or temperatures incubation times and temperatures Incorrect volumes used Use calibrated pipettes and aliquot correctly Thaw and resuspend all components before Use of partially thawed components preparing the Reaction Mixes Pipetting errors in preparation of standards Avoid pipetting small volumes Pipetting errors in the Reaction Mix Prepare Reaction Mixes whenever possible Pipette gently against the wall of the plate Non-linear standard Air bubbles formed in well curve well Standard stock is at incorrect Refer to the standard dilution instructions in concentration the Technical Bulletin Recheck calculations after referring to Calculation errors Technical Bulletin Substituting reagents from older kits/lots Use fresh components from the same kit Samples measured at incorrect Check the equipment and filter settings wavelength Unanticipated results Samples contain interfering substances If possible, dilute sample further Sample readings above/below the linear Concentrate or dilute samples so readings range are in the linear range

SS,GD,KVG,LS,MAM 08/20-1

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