Product Details
| abID | ab157405 |
|---|---|
| Cat # +Size | K169-100 |
| Size | 100 assays |
| Detection Method | Fluorescence (Ex/Em = 380/500 nm) |
| Applications | Screening Granzyme B inhibitors, characterize/Study Granzyme B inhibitors. |
| Features & Benefits | • Simple, sensitive and high-throughput • Rapid and convenient • Includes Inhibitor control, Ac-IEPD-CHO. |
| Kit Components | • Granzyme B Assay Buffer • Granzyme B Substrate • Granzyme B Enzyme (human recombinant) • Inhibitor Control (Ac-IEPD-CHO, 250 μM) |
| Storage Conditions | -20°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
Granzyme B (GZMB, EC number 3.4.21.79) also known as Granzyme-2 is a serine protease stored in the granules of activated cytotoxic T cells and NK cells. Upon target cell contact, Granzyme B is directionally exocytosed and with the assistance of perforin enters the target cell. With its unique substrate specificity (Granzyme B prefers an aspartic acid residue at the P1 site of its substrates), Granzyme B processes and activates various pro-caspases thereby inducing apoptosis in the target cell. In BioVision’s Granzyme B Inhibitor Screening Kit, active human Granzyme B enzyme hydrolyzes the specific substrate to release the quench of fluorescent group, which can be detected fluorometrically at Ex/Em = 380/500 nm. In the presence of potent Granzyme B Inhibitor, the hydrolyzation of substrate will be impeded. The Kit provides a rapid, simple, sensitive, and reliable test suitable for high throughput screening of Granzyme B inhibitors or characterize/study Granzyme B inhibitors. Inhibitor control, Ac-IEPD-CHO is included to compare the efficacy of test inhibitors.
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Can the kit be used with EGFP or ECFP?
For K168 requires excitation at 380nm. This should not theoretically excite the GFP(Ex/Em = 488/507 nm) and CFP(Ex./Em. = 458/480 nm). There could be some fluorescence contribution from these proteins at 500nm emission. Hence subtracting background should work.
What lysis buffer do you recommend for sample preparation?
For cell samples: Start with ˜2X10⁶ cells, suspend the cell pellet 500 µl (or ˜4 volumes) of the assay buffer on ice, homogenize using a Dounce homogenizer (10-50 passes) on ice, until efficient lysis is confirmed by viewing the cells under the microscope. Spin down the sample and collect the supernatant. Use the supernatant for your subsequent assays. Appropriate dilutions of the sample must be tested in order to ensure the readings will fall within the linear range of the standard curve.
What is the substrate?
The proprietary substrate is Granzyme B specific.
Could you let me know whether you have used baculovirus to produce this product?
None of the kit components have been made using baculovirus
How many tests can be run with this kit?
Each kit lets you run 100 reactions, including all controls
Can the kit work on bacteria or yeast cells?
We have optimized the kit with mammalian samples. However, theoretically these kits should work with samples from multiple species/sources. Since the optimal conditions depend on the sample type, the protocol has to be be adapted to fit the samples for efficient results. Please refer to this kits citations to see what kind of samples have been used with this kit other than mammalian samples. The kit has been standardized for mammalian cells only.
What is the exact volume of sample required for this assay?
There is no specific volume we can recommend for the amount any sample to be used since it is completely sample concentration and quality based. You have to do a pilot expt with multiple sample volumes to determine the optimal volume which gives a reading within the linear range of the standard curve. Please refer to the citations for this product to see what other clients have used with similar sample types.
Do you have trial sizes of this kit?
Unfortunately, we do not have trial sizes of this kit available. However, if you are based in the US or Canada, we will give you a 10% off list price introductory discount on its purchase price. If you are based out of this area please contact yopur regional BioVision distributor.
What is the shelf life of this kit?
The expiry date is typically 1 year from the date of shipment
Can we purchase individual components of this kit?
Yes, you can purchase any of the kit's components without the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Can we use an alternate buffer for sample preparation (cell lysis, sample dilutions etc)?
Our assay buffers are optimized for the reactions they are designed for. They not only contain some detergents for efficient lysis of your cells/tissue, but also contain some proprietary components required for the further reactions. Therefore, we highly recommend using the buffers provided in the kit for the best results.
