Description
GATA6 Colorimetric Cell-Based ELISA Kit
The GATA6 Colorimetric Cell Based ELISA Kit is specifically designed for the precise measurement of GATA6 levels in cell lysates and cell culture supernatants. This innovative kit offers exceptional sensitivity and specificity, guaranteeing consistent and accurate results for a variety of research applications.GATA6 is a transcription factor that plays a critical role in cell differentiation and tissue development. It is involved in various physiological processes, including cardiac development, gastrointestinal development, and stem cell maintenance.
Dysregulation of GATA6 has been linked to a range of diseases, such as congenital heart defects and gastrointestinal disorders, making it an important target for research and therapeutic development.With its advanced technology and user-friendly protocols, the GATA6 Colorimetric Cell Based ELISA Kit is an invaluable tool for researchers studying GATA6 biology and its implications in disease pathology. Trust in this kit to deliver reliable and reproducible data for your experiments.
| Product Name: | GATA6 Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB00673 |
| ELISA Type: | Cell-Based |
| Target: | GATA6 |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The GATA6 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect GATA6 protein expression profile in cells. The kit can be used for measuring the relative amounts of GATA6 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on GATA6.
Qualitative determination of GATA6 concentration is achieved by an indirect ELISA format. In essence, GATA6 is captured by GATA6-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: 2627, UniProt ID: Q92908, OMIM: 601656, Unigene: Hs.514746 |
| Gene Symbol: | GATA6 |
| Sub Type: | None |
| UniProt Protein Function: | GATA6: Transcriptional activator that regulates SEMA3C and PLXNA2. Thought to be important for regulating terminal differentiation and/or proliferation. Interacts with LMCD1. Expressed in heart, gut and gut-derived tissues. 2 isoforms of the human protein are produced by alternative initiation. |
| UniProt Protein Details: | Protein type:Motility/polarity/chemotaxis; Transcription factor Chromosomal Location of Human Ortholog: 18q11.1-q11.2 Cellular Component: nucleoplasm; nucleus; transcription factor complex Molecular Function:chromatin binding; protein binding; protein kinase binding; transcription factor activity; transcription factor binding Biological Process: blood coagulation; cell development; cell fate commitment; cellular protein metabolic process; male gonad development; negative regulation of apoptosis; negative regulation of transcription from RNA polymerase II promoter; negative regulation of transcription, DNA-dependent; negative regulation of transforming growth factor-beta1 production; negative regulation of transforming growth factor-beta2 production; positive regulation of angiogenesis; positive regulation of BMP signaling pathway; response to drug; smooth muscle cell differentiation; transcription from RNA polymerase II promoter Disease: Atrial Septal Defect 9; Atrioventricular Septal Defect 5; Conotruncal Heart Malformations; Heart Defects, Congenital, And Other Congenital Anomalies; Tetralogy Of Fallot |
| NCBI Summary: | This gene is a member of a small family of zinc finger transcription factors that play an important role in the regulation of cellular differentiation and organogenesis during vertebrate development. This gene is expressed during early embryogenesis and localizes to endo- and mesodermally derived cells during later embryogenesis and thereby plays an important role in gut, lung, and heart development. Mutations in this gene are associated with several congenital defects. [provided by RefSeq, Mar 2012] |
| UniProt Code: | Q92908 |
| NCBI GenInfo Identifier: | 215273987 |
| NCBI Gene ID: | 2627 |
| NCBI Accession: | Q92908.2 |
| UniProt Secondary Accession: | Q92908,P78327, B0YJ17, |
| UniProt Related Accession: | Q92908 |
| Molecular Weight: | 45,386 Da |
| NCBI Full Name: | Transcription factor GATA-6 |
| NCBI Synonym Full Names: | GATA binding protein 6 |
| NCBI Official Symbol: | GATA6 |
| NCBI Protein Information: | transcription factor GATA-6 |
| UniProt Protein Name: | Transcription factor GATA-6 |
| UniProt Synonym Protein Names: | GATA-binding factor 6 |
| Protein Family: | GATA transcription factor |
| UniProt Gene Name: | GATA6 |
| UniProt Entry Name: | GATA6_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-GATA6 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.)
