Description
Key facts
Detection method:Fluorescent,
Sample types:Plasma, Tissue Extracts, Cell culture supernatant, Milk, Serum, Other biological fluids,
Assay type:Enzyme activity,
Sensitivity:< 500 pg/well,
Assay time:1h,
Assay Platform:Microplate reader
Product details:
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:
Protease assay protocol summary
- 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.
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:
is the FITC amount (nmol) from the Standard Curve
is the time (min) of the first reading (R
is the time (min) of the second reading (R
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.
Properties and Storage Information:
Shipped at conditions-Blue Ice, Appropriate short-term storage conditions--20°C, Appropriate long-term storage conditions--20°C, Storage information--20°C
Supplementary Information:
This supplementary information is collated from multiple sources and compiled automatically.
Proteases also known as peptidases or proteinases are enzymes that catalyze the breakdown of proteins into smaller peptides or amino acids by cleaving peptide bonds. These enzymes vary in mass typically ranging from 20 to 90 kDa depending on the specific type and its functional domain. They are expressed across different tissues and cellular compartments with high concentrations in digestive organs lysosomes and blood plasma. The mechanical process of protease activity is central to many physiological processes making their precise function an important focus of study in biology.
Biological function summary
Proteases play critical roles in maintaining cellular functions including protein catabolism cell signaling and the regulation of the cell cycle. They are often components of large complexes such as the proteasome where they help degrade ubiquitinated proteins ensuring protein homeostasis. Proteases also assist in activating precursor molecules into biologically active forms involving themselves in processes like blood coagulation immune responses and apoptosis. Their activity is essential in remodeling extracellular matrix and processing bioactive molecules further influencing diverse physiological pathways.
Pathways
Proteases significantly impact both the proteolytic and the ubiquitin-proteasome pathways. Within the proteolytic pathway enzymes break down proteins into peptides and amino acids for cellular recycling and energy production. The ubiquitin-proteasome pathway involving ubiquitin-related proteins is important for regulating protein degradation and turnover impacting cellular functions and stress responses. Proteases also interact with other proteins such as kinases and phosphatases which facilitate cellular signaling cascades reflecting their participation in broader biological networks.
Many proteases have associations with cancer and neurodegenerative diseases. Aberrant protease activity can lead to uncontrolled cell proliferation or faulty cell death contributing to carcinogenesis. In neurodegenerative disorders such as Alzheimer's disease improper protease activity results in the accumulation of misfolded proteins like amyloid-beta peptides disrupting neural function. Proteases also have connections with proteins such as tau and APP in Alzheimer's disease indicating their complex role in pathogenesis and providing potential targets for therapeutic interventions.
