Description
Granzyme B Colorimetric Cell-Based ELISA Kit
The Granzyme B Colorimetric Cell-Based ELISA Kit offered by Assay Genie is a cutting-edge tool for detecting Granzyme B levels in cell lysates and tissue homogenates. This kit boasts exceptional sensitivity and specificity, ensuring accurate and reproducible results for various research applications.Granzyme B is a key enzyme involved in immune responses, particularly in the cytotoxic activity of T cells and NK cells. It plays a crucial role in immune surveillance against viral infections, tumors, and autoimmune diseases.
Detection of Granzyme B levels can provide valuable insights into immune function and disease progression.With the Granzyme B Colorimetric Cell-Based ELISA Kit, researchers can delve into the intricate mechanisms of immune responses and potential therapeutic targets for immune-mediated diseases. Trust Assay Genie to deliver reliable and high-quality tools for your research needs.
| Product Name: | Granzyme B Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB00679 |
| ELISA Type: | Cell-Based |
| Target: | Granzyme B |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The Granzyme B Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Granzyme B protein expression profile in cells. The kit can be used for measuring the relative amounts of Granzyme B in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Granzyme B.
Qualitative determination of Granzyme B concentration is achieved by an indirect ELISA format. In essence, Granzyme B is captured by Granzyme B-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:
| 1. | A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values. |
| 2. | Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted. |
| Database Information: | Gene ID: 3002, UniProt ID: P10144/P20718, OMIM: 123910, Unigene: Hs.1051 |
| Gene Symbol: | GZMB |
| Sub Type: | None |
| UniProt Protein Function: | GZMB: This enzyme is necessary for target cell lysis in cell- mediated immune responses. It cleaves after Asp. Seems to be linked to an activation cascade of caspases (aspartate-specific cysteine proteases) responsible for apoptosis execution. Cleaves caspase-3, -7, -9 and 10 to give rise to active enzymes mediating apoptosis. By staphylococcal enterotoxin A (SEA) in peripheral blood leukocytes. Inactivated by the serine protease inhibitor diisopropylfluorophosphate. Belongs to the peptidase S1 family. Granzyme subfamily. |
| UniProt Protein Details: | Protein type:Apoptosis; EC 3.4.21.79; Protease Chromosomal Location of Human Ortholog: 14q12 Cellular Component: cytoplasm; cytosol; immunological synapse; intracellular membrane-bound organelle; membrane; nucleus Molecular Function:protein binding; serine-type endopeptidase activity; serine-type peptidase activity Biological Process: apoptosis; natural killer cell mediated cytotoxicity; protein processing |
| NCBI Summary: | This gene encodes a member of the granzyme subfamily of proteins, part of the peptidase S1 family of serine proteases. The encoded preproprotein is secreted by natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) and proteolytically processed to generate the active protease, which induces target cell apoptosis. This protein also processes cytokines and degrades extracellular matrix proteins, and these roles are implicated in chronic inflammation and wound healing. Expression of this gene may be elevated in human patients with cardiac fibrosis. [provided by RefSeq, Sep 2016] |
| UniProt Code: | P10144 |
| NCBI GenInfo Identifier: | 317373361 |
| NCBI Gene ID: | 3002 |
| NCBI Accession: | P10144.2 |
| UniProt Secondary Accession: | P10144,Q8N1D2, Q9UCC1, |
| UniProt Related Accession: | P10144 |
| Molecular Weight: | |
| NCBI Full Name: | Granzyme B |
| NCBI Synonym Full Names: | granzyme B |
| NCBI Official Symbol: | GZMB |
| NCBI Official Synonym Symbols: | C11; HLP; CCPI; CGL1; CSPB; SECT; CGL-1; CSP-B; CTLA1; CTSGL1 |
| NCBI Protein Information: | granzyme B |
| UniProt Protein Name: | Granzyme B |
| UniProt Synonym Protein Names: | C11; CTLA-1; Cathepsin G-like 1; CTSGL1; Cytotoxic T-lymphocyte proteinase 2; Lymphocyte protease; Fragmentin-2; Granzyme-2; Human lymphocyte protein; HLP; SECT; T-cell serine protease 1-3E |
| Protein Family: | Granzyme |
| UniProt Gene Name: | GZMB |
| Component | Quantity |
| 96-Well Cell Culture Clear-Bottom Microplate | 2 plates |
| 10X TBS | 24 mL |
| Quenching Buffer | 24 mL |
| Blocking Buffer | 50 mL |
| 15X Wash Buffer | 50 mL |
| Primary Antibody Diluent | 12 mL |
| 100x Anti-Phospho Target Antibody | 60 µL |
| 100x Anti-Target Antibody | 60 µL |
| Anti-GAPDH Antibody | 60 µL |
| HRP-Conjugated Anti-Rabbit IgG Antibody | 12 mL |
| HRP-Conjugated Anti-Mouse IgG Antibody | 12 mL |
| SDS Solution | 12 mL |
| Stop Solution | 24 mL |
| Ready-to-Use Substrate | 12 mL |
| Crystal Violet Solution | 12 mL |
| Adhesive Plate Seals | 2 seals |
The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:
- Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
- Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
- 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
- Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
- Graph paper or computer software capable of generating or displaying logarithmic functions
- Absorbent papers or vacuum aspirator
- Test tubes or microfuge tubes capable of storing ≥1 ml
- Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
- Orbital shaker (optional)
- Deionized or sterile water
*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1. | Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells. |
| 2. | Incubate the cells for overnight at 37°C, 5% CO2. |
| 3. | Treat the cells as desired. |
| 4. | Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice. |
| 5. | Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells. |
| 6. | Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4°C for a week. |
| 7. | Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature. |
| 8. | Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time. |
| 9. | Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature. |
| 10. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 11. | Add 50 µL of 1x primary antibodies (Anti-Granzyme B Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature. |
| 12. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 13. | Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature. |
| 14. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 15. | Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark. |
| 16. | Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader. |
(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)
