Description
MADD Colorimetric Cell-Based ELISA
The MADD Colorimetric Cell-Based ELISA Kit is a cutting-edge assay developed for the accurate detection of MADD levels in cell lysates and culture supernatants. This kit offers exceptional sensitivity and specificity, providing researchers with reliable and consistent results for their studies.MADD, also known as MAP-kinase activating death domain protein, plays a key role in cell signaling pathways and apoptosis regulation. Dysregulation of MADD has been linked to various diseases, including cancer and neurodegenerative disorders, making it a valuable biomarker for understanding disease pathogenesis and developing targeted therapies.
With its user-friendly protocol and high-performance capabilities, the MADD Colorimetric Cell-Based ELISA Kit is an indispensable tool for researchers in the fields of cell biology, molecular biology, and drug discovery. Trust AssayGenie for all your ELISA kit needs.
| Product Name: | MADD Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB01166 |
| ELISA Type: | Cell-Based |
| Target: | MADD |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The MADD Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect MADD protein expression profile in cells. The kit can be used for measuring the relative amounts of MADD in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on MADD.
Qualitative determination of MADD concentration is achieved by an indirect ELISA format. In essence, MADD is captured by MADD-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: 8567, UniProt ID: Q8WXG6, OMIM: 603584, Unigene: Hs.82548 |
| Gene Symbol: | MADD |
| Sub Type: | None |
| UniProt Protein Function: | MADD: a guanine nucleotide exchange factor (GEF) that activates RAB3A/C/D. A GEF for Rab27A/B in melanocytes and during isoproterenol (IPR)-stimulated amylase release from rat parotid acinar cells. Plays a significant role in regulating cell proliferation, survival and death through alternatively spliced isoforms. Component of the TNFRSF1A signaling complex: MADD links TNFRSF1A with MAP kinase activation. Plays an important regulatory role in physiological cell death (TNF-alpha-induced, caspase-mediated apoptosis). Interacts with the death domain of TNFRSF1A through its own death domain. Highly expressed in fetal brain and kidney, adult testis, ovary, brain and heart. Overexpression of MADD activates the mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase (ERK). Expression of the MADD death domain stimulates both the ERK and c-JUN N-terminal kinase MAP kinases and induces the phosphorylation of cytosolic phospholipase A2. Isoform 1 is pro-apoptotic, isoform 5 is anti-apoptotic and isoform 3 and isoform 4 have no effect. Isoform 5 is constitutively expressed in all tissues. Isoform 7 is expressed in fetal liver and in several cancer cell lines. Isoform 5 is associated with increased cell proliferation and isoform 2 with decreased proliferation. Belongs to the MADD family. Seven isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Adaptor/scaffold; GEFs, Rab; GEFs; Membrane protein, integral Chromosomal Location of Human Ortholog: 11p11.2 Cellular Component: cytoplasm; plasma membrane; integral to membrane Molecular Function:protein binding; death receptor binding; Rab guanyl-nucleotide exchange factor activity; protein kinase activator activity Biological Process: regulation of Rab protein signal transduction; regulation of apoptosis; cell surface receptor linked signal transduction; activation of MAPK activity; regulation of cell cycle |
| NCBI Summary: | Tumor necrosis factor alpha (TNF-alpha) is a signaling molecule that interacts with one of two receptors on cells targeted for apoptosis. The apoptotic signal is transduced inside these cells by cytoplasmic adaptor proteins. The protein encoded by this gene is a death domain-containing adaptor protein that interacts with the death domain of TNF-alpha receptor 1 to activate mitogen-activated protein kinase (MAPK) and propagate the apoptotic signal. It is membrane-bound and expressed at a higher level in neoplastic cells than in normal cells. Several transcript variants encoding different isoforms have been described for this gene. [provided by RefSeq, Jul 2008] |
| UniProt Code: | Q8WXG6 |
| NCBI GenInfo Identifier: | 209862998 |
| NCBI Gene ID: | 8567 |
| NCBI Accession: | NP_001129415.1 |
| UniProt Secondary Accession: | Q8WXG6,O15065, O15293, Q15732, Q15741, Q8IWD7, Q8WXG3 Q8WXG4, Q8WXG5, A8K8S7, B5MEE5, D3DQR4, |
| UniProt Related Accession: | Q8WXG6 |
| Molecular Weight: | 183,303 Da |
| NCBI Full Name: | MAP kinase-activating death domain protein isoform i |
| NCBI Synonym Full Names: | MAP-kinase activating death domain |
| NCBI Official Symbol: | MADD |
| NCBI Official Synonym Symbols: | DENN; IG20; RAB3GEP |
| NCBI Protein Information: | MAP kinase-activating death domain protein; Rab3 GDP/GTP exchange factor; insulinoma glucagonoma clone 20; differentially expressed in normal and neoplastic cells |
| UniProt Protein Name: | MAP kinase-activating death domain protein |
| UniProt Synonym Protein Names: | Differentially expressed in normal and neoplastic cells; Insulinoma glucagonoma clone 20; Rab3 GDP/GTP exchange factor |
| Protein Family: | Malonyl-S-ACP:biotin-protein carboxyltransferase |
| UniProt Gene Name: | MADD |
| UniProt Entry Name: | MADD_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-MADD 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.)
