A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl

The overall goal of the following experiment is to measure the proteolytic activity of the ine protease granzyme B.This is achieved by first preparing a lysate of the cells of interest and then quickly adding substrate to the samples. The granzyme B activity is then measured by a colormetric reaction. Ultimately, the highly specific cleavage of alla alla as s benzo by granzyme B can be assessed based on its unique substrate specificity for a spart acid residues.

The main advantage of this technique over existing methods, such as the detection of grand zone B expression using PCR or Western blot, is that hydrolysis of benzyl is specific for grand zone B and also indicates that the protease is active. To prepare the cell, lysates first isolate primary NK cells from the single cell suspensions of spleens from wild type C 57 black six, or control granzyme B gene null mice by negative selection using commercially available kits according to the manufacturer's instructions after the cells have been activated. Wash the culture three times in PBS at maximum speed for two to three seconds, and then resuspend the pellet containing a minimum of 5 million NK cells in 100 microliters of Nont P 40 lysis buffer on ice for 20 minutes.

Then spin down the lysate in a micro centrifuge for 10 minutes at 15, 000 Gs and transfer the supine agent to a fresh tube. After determining the protein concentration, according to the appropriate commercially available protocol, store the sate at a minimum approximately two milligrams per milliliter concentration at negative 20 degrees Celsius to perform a grand zyme B activity assay. Next dilute a minimum of 100 micrograms of protein lysate per sample in freshly prepared heaps buffer to a final volume of 160 microliters.

Then using a multi-channel pipette load 50 microliters of sample per well in triplicate into a 96 well vinyl U bottom plate. Taking care to include three buffer only control wells, add 100 microliters of diluted DTNB to each. Well then quickly add 50 microliters of freshly diluted alala asks s benzoyl substrate to each of the heaps buffer alone wells, followed by any negative controls, and finishing with the expected positive samples.

Measure the aase activity by the hydrolysis of the alala as benzyl in a microplate reader at an optical density of 405 nanometers using a kinetic assay protocol taking 25 readings every 15 seconds. Finally, use the plate reader software to calculate the maximum rate of substrate cleavage for each well, and then transfer the raw data to the appropriate statistical analysis software using granzyme gene knockout mice. It was discovered that the synthetic peptide substrate alala as benzyl is specifically hydrolyzed by granzyme B.Further robust cleavage of the synthetic substrate can be measured in cell lysates from wildtype primary NK cells, but not in NK cells from granzyme B knockout animals as a control.

In this experiment, the proteolytic activity of granzyme A, which has tripsin like substrate specificity and is present in lysates from both types of cells, was equal as measured by the hydrolysis of BLT benzyl, I-E-P-D-P-N-A has been used generically to test for granzyme B activity across species. In this representative experiment analysis of the tetra peptide sequences revealed that human but not mouse granzyme B can cleave efficiently after the sequence IEPD, which matches the recognition sequence in both human and mouse bid. In contrast, decreasing the amount of human NK leukemia cell lysate from 30 micrograms to 10 micrograms still results in sufficient gram enzyme B to hydrolyze I-E-P-D-P-N-A substrate.

After watching this video, you should have a good understanding of how to choose the appropriate substrate and experimental controls for the in vitro analysis of red human and mouse enzyme B.