Protease Activity Assay Kit

Protease Activity Assay Kit ab111750 is designed for the quantitative determination of proteases present in protein samples.

Protease assay principle

This protease assay uses fluorescein isothiocyanate (FITC)-labeled casein as a general protease substrate.

- The fluorescein label is highly quenched when present as a label on FITC-labeled casein.

- Upon digestion by proteases present in the sample, the FITC-Casein substrate is cleaved into smaller peptides which abolishes the quenching of the fluorescence label.

- The fluorescence of the FITC-labeled peptide fragments is then measured at Ex/Em = 485/530 nm on a plate reader.

The kit is supplied with Trypsin for use as a general protease control. However, other protease standard controls can also be used. This kit can detect < 500 pg/well Trypsin present in the sample.Replace text to the left with the text below:

- add samples and standards to wells

- add reaction mix and incubate at room temperature for 30 min

- analyze with a plate reader (Ex/Em 483/530 nm)

Other notes

This product was previously called K781 Biovision Protease Activity Fluorometric Assay Kit. Biovision was acquired by Abcam in 2021.

Proteases are naturally present in all organisms. These enzymes are involved in a multitude of physiological reactions from simple digestion of food proteins to highly regulated cascades. Proteases can either break specific peptide bonds (limited proteolysis), depending on the amino acid sequence of a protein, or break down a complete peptide to amino acids (unlimited proteolysis). The activity can be a destructive change (abolishing a protein's function), an activation of a function (preform to mature form) or it can be a signal in a signaling pathway.

Protease Assay Protocol

- When using the assay please use the PDF protocol download link to access the complete protocol which includes instructions for preparation of the components for use in the assay.

1. Sample Preparation:

a. For serum samples: Serum samples can be directly diluted in the Assay Buffer I/Assay Buffer.

b. For tissue or cell samples: Tissues or cells can be extracted with 4 volumes of the Assay Buffer I/Assay Buffer, centrifuge to remove insoluble material and get a clear extract.

Prepare test samples up to 50 μl/well with Assay Buffer I/Assay Buffer in a 96-well plate.

For positive control use 5μl of the reconstituted Protease Positive Control/Positive Control solution into wells and adjust volume to 50 μl with Assay Buffer I/Assay Buffer.

Include a reagent background control which only contains 50 μl of Assay Buffer I/Assay Buffer.

Note: This product detects proteolytic activity. Do not use protease inhibitors in the sample preparation step as it might interfere with the assay.

We suggest testing several doses of your sample to make sure readings are within the standard curve.

2. Standard Curve Preparation:

Add 0, 2, 4, 6, 8, 10 μl Fluoresence Standard VI/FITC Standard into a series of standards wells. Adjust the final volume to 100 μl/well with Assay Buffer I/Assay Buffer to generate 0, 0.05, 0.1, 0.15 0.2, and 0.25 nmol/well of the Fluoresence Standard VI/FITC Standard.

3. Reaction Mix: Mix enough reagents for the number of assays to be performed. For each well, prepare a total 50 μl Reaction Mix:

Assay Buffer I/Assay Buffer: 48 μl

Protease Substrate Solution: 2 μl

Add 50 μl of the Reaction Mix to each well containing the Positive Controls, Reagent Background Control and Samples. Mix well. (DO NOT ADD TO STANDARDS)

4. Measurement: Read Ex/Em = 485/530 nm R1 at T1. Read R2 again at T2 after incubating the reaction at room 25°C for 30 min (or longer time if the sample activity is low); protect from light. The fluorescence of the unquenched FITC generated by proteolytic digestion of the substrate is:

ΔRFU = R2 – R1

Notes:

a) It is essential to read R1 and R2 in the reaction linear range. It will be more accurate if you read the reaction kinetics, and then choose R1 and R2 in the reaction linear range.

b) Since the assay is a fluorescence quenching assay, the background reading is high, but sample reading are consistent.

5. Data Analysis

Subtract the zero standard from all standard readings.

Plot the Fluoresence Standard VI/FITC Standard Curve and apply the ΔRFU to the Standard Curve to get B nmol of FITC generated between T1 and T2 in the reaction wells.

Protease activity can then be calculated:

Protease Activity = (B x Dilution Factor) / ((T2 – T1) x V) = nmol/min/ml = mU/ml

Where:

B is the FITC amount (nmol) from the Standard Curve

T₁is the time (min) of the first reading (R₁)

T₂is the time (min) of the second reading (R₂)

V is the pretreated sample volume (ml) added into the reaction well

Unit Definition: One unit is defined as the amount of protease that cleaves the substrate, to yield an amount of fluorescence equivalent to 1.0 µmol of unquenched FITC per minute at 25°C.

REACH authorisation
Abcam has not and does not intend to apply for the REACH Authorisation of customers' uses of products that contain European Authorisation list (Annex XIV) substances.
It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.