Caspase 1 (Phospho-Ser376) Colorimetric Cell-Based ELISA Kit
- SKU:
- CBCAB01443
- Product Type:
- ELISA Kit
- ELISA Type:
- Cell Based Phospho Specific
- Research Area:
- Cell Death
- Reactivity:
- Human
- Reactivity:
- Mouse
- Reactivity:
- Rat
- Detection Method:
- Colorimetric
Description
| Product Name: | Caspase 1 (Phospho-Ser376) Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB01443 |
| ELISA Type: | Cell-Based |
| Target: | Caspase 1 (Phospho-Ser376) |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nm |
| Format: | 2 x 96-Well Microplates |
The Caspase 1 (Phospho-Ser376) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Caspase 1 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Caspase 1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Caspase 1 phosphorylation.
Qualitative determination of Caspase 1 (Phospho-Ser376) concentration is achieved by an indirect ELISA format. In essence, Caspase 1 (Phospho-Ser376) is captured by Caspase 1 (Phospho-Ser376)-specific primary (1ø) antibodies while the HRP-conjugated secondary (2ø) antibodies bind the Fc region of the 1ø antibody. Through this binding, the HRP enzyme conjugated to the 2ø 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: 834, UniProt ID: P29466, OMIM: 147678, Unigene: Hs.2490 |
| Gene Symbol: | CASP1 |
| Sub Type: | Phospho |
| UniProt Protein Function: | CASP1: Thiol protease that cleaves IL-1 beta between an Asp and an Ala, releasing the mature cytokine which is involved in a variety of inflammatory processes. Important for defense against pathogens. Cleaves and activates sterol regulatory element binding proteins (SREBPs). Can also promote apoptosis. Heterotetramer that consists of two anti-parallel arranged heterodimers, each one formed by a 20 kDa (p20) and a 10 kDa (p10) subunit. The p20 subunit can also form a heterodimer with the epsilon isoform which then has an inhibitory effect. May be a component of the inflammasome, a protein complex which also includes PYCARD, CARD8 and NALP2 and whose function would be the activation of proinflammatory caspases. Interacts with CARD17/INCA and CARD18. Expressed in larger amounts in spleen and lung. Detected in liver, heart, small intestine, colon, thymus, prostate, skeletal muscle, peripheral blood leukocytes, kidney and testis. No expression in the brain. Specifically inhibited by the cowpox virus Crma protein. Belongs to the peptidase C14A family. 5 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Apoptosis; Protease; EC 3.4.22.36 Chromosomal Location of Human Ortholog: 11q23 Cellular Component: cytoplasm; extracellular region; cytosol Molecular Function:protein binding; cysteine-type endopeptidase activity; endopeptidase activity; caspase activator activity Biological Process: caspase activation; positive regulation of I-kappaB kinase/NF-kappaB cascade; apoptosis; membrane hyperpolarization; interleukin-1 beta production; response to lipopolysaccharide; positive regulation of interleukin-1 beta secretion; mitochondrial depolarization; proteolysis; signal transduction; response to ATP; positive regulation of interleukin-1 alpha secretion; regulation of inflammatory response; response to hypoxia; innate immune response; regulation of autophagy |
| NCBI Summary: | This gene encodes a protein which is a member of the cysteine-aspartic acid protease (caspase) family. Sequential activation of caspases plays a central role in the execution-phase of cell apoptosis. Caspases exist as inactive proenzymes which undergo proteolytic processing at conserved aspartic residues to produce 2 subunits, large and small, that dimerize to form the active enzyme. This gene was identified by its ability to proteolytically cleave and activate the inactive precursor of interleukin-1, a cytokine involved in the processes such as inflammation, septic shock, and wound healing. This gene has been shown to induce cell apoptosis and may function in various developmental stages. Studies of a similar gene in mouse suggest a role in the pathogenesis of Huntington disease. Alternative splicing results in transcript variants encoding distinct isoforms. [provided by RefSeq, Mar 2012] |
| UniProt Code: | P29466 |
| NCBI GenInfo Identifier: | 266321 |
| NCBI Gene ID: | 834 |
| NCBI Accession: | P29466.1 |
| UniProt Secondary Accession: | P29466,Q53EY6, Q6DMQ1, Q6GSS3, Q6PI75, Q9UCN3, B5MDZ1 |
| UniProt Related Accession: | P29466 |
| Molecular Weight: | 10,417 Da |
| NCBI Full Name: | Caspase-1 |
| NCBI Synonym Full Names: | caspase 1, apoptosis-related cysteine peptidase |
| NCBI Official Symbol: | CASP1 |
| NCBI Official Synonym Symbols: | ICE; P45; IL1BC |
| NCBI Protein Information: | caspase-1; IL1B-convertase; CASP1 nirs variant 1; IL-1 beta-converting enzyme; interleukin 1, beta, convertase; interleukin 1-B converting enzyme; caspase 1, apoptosis-related cysteine peptidase (interleukin 1, beta, convertase) |
| UniProt Protein Name: | Caspase-1 |
| UniProt Synonym Protein Names: | Interleukin-1 beta convertase; IL-1BC; Interleukin-1 beta-converting enzyme; ICE; IL-1 beta-converting enzyme; p45Cleaved into the following 2 chains:Caspase-1 subunit p20; Caspase-1 subunit p10 |
| UniProt Gene Name: | CASP1 |
| UniProt Entry Name: | CASP1_HUMAN |
| 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-Caspase 1 (Phospho-Ser376) Antibody, Anti-Caspase 1 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.)
