Description
TF2A1 Colorimetric Cell-Based ELISA
The TF2A1 Colorimetric Cell-Based ELISA Kit is specifically designed for the precise and sensitive detection of TF2A1 (Transcription Factor AP-2 alpha) levels in cell lysates and cell culture samples. This kit offers high specificity and accuracy, providing researchers with reliable and reproducible results for a variety of experimental setups.TF2A1 is a key transcription factor that regulates gene expression and plays a crucial role in various cellular processes, including cell growth, differentiation, and development.
Dysregulation of TF2A1 has been implicated in multiple diseases, making it a valuable target for research in areas such as cancer, developmental biology, and neurobiology.With its advanced technology and user-friendly format, the TF2A1 Colorimetric Cell-Based ELISA Kit offers researchers a powerful tool for studying TF2A1 biology and its impact on health and disease. Order yours today and unlock new insights into TF2A1 function and regulation.
| Product Name: | TF2A1 Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB01033 |
| ELISA Type: | Cell-Based |
| Target: | TF2A1 |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The TF2A1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect TF2A1 protein expression profile in cells. The kit can be used for measuring the relative amounts of TF2A1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on TF2A1.
Qualitative determination of TF2A1 concentration is achieved by an indirect ELISA format. In essence, TF2A1 is captured by TF2A1-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: 2957, UniProt ID: P52655, OMIM: 600520, Unigene: Hs.592334/Hs.593630 |
| Gene Symbol: | GTF2A1 |
| Sub Type: | None |
| UniProt Protein Function: | GTF2A1: a general transcription factor that plays an important role in transcriptional activation. A component of the transcription machinery of RNA polymerase II. Phosphorylated by Taf(II) 250 on serines that regulate TBP binding. Interacts with TBP (the TATA-binding protein). Two isoforms, 42 kDa and 37 kDa, are produced by alternative initiation. |
| UniProt Protein Details: | Protein type:Transcription, coactivator/corepressor Chromosomal Location of Human Ortholog: 14q31.1 Cellular Component: cytoplasm; nucleoplasm; transcription factor TFIIA complex Molecular Function:DNA binding; protein binding; protein heterodimerization activity; TATA-binding protein binding; transcription coactivator activity; transcription factor binding Biological Process: RNA elongation from RNA polymerase II promoter; snRNA transcription from RNA polymerase II promoter; transcription from RNA polymerase II promoter; transcription initiation from RNA polymerase II promoter |
| NCBI Summary: | Accurate transcription initiation on TATA-containing class II genes involves the ordered assembly of RNA polymerase II (POLR2A; MIM 180660) and several general initiation factors (summarized by DeJong and Roeder, 1993 [PubMed 8224848]). One of these factors is TFIIA, which when purified from HeLa extracts consists of 35-, 19-, and 12-kD subunits.[supplied by OMIM, Jul 2010] |
| UniProt Code: | P52655 |
| NCBI GenInfo Identifier: | 1711663 |
| NCBI Gene ID: | 2957 |
| NCBI Accession: | P52655.1 |
| UniProt Secondary Accession: | P52655,Q3KNQ9, |
| UniProt Related Accession: | P52655 |
| Molecular Weight: | 37,152 Da |
| NCBI Full Name: | Transcription initiation factor IIA subunit 1 |
| NCBI Synonym Full Names: | general transcription factor IIA subunit 1 |
| NCBI Official Symbol: | GTF2A1 |
| NCBI Official Synonym Symbols: | TF2A1; TFIIA; TFIIAL; TFIIA-42 |
| NCBI Protein Information: | transcription initiation factor IIA subunit 1 |
| UniProt Protein Name: | Transcription initiation factor IIA subunit 1 |
| UniProt Synonym Protein Names: | General transcription factor IIA subunit 1; TFIIAL; Transcription initiation factor TFIIA 42 kDa subunit; TFIIA-42 |
| Protein Family: | Transcription initiation factor |
| UniProt Gene Name: | GTF2A1 |
| UniProt Entry Name: | TF2AA_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-TF2A1 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.)
