Description
OCT1 Colorimetric Cell-Based ELISA Kit
The OCT1 Colorimetric Cell-Based ELISA Kit is a cutting-edge tool designed for the accurate and reliable detection of OCT1 levels in cell samples. This kit boasts high sensitivity and specificity, ensuring precise and reproducible results for a variety of research applications.OCT1 is a key transporter protein that plays a crucial role in the uptake and elimination of various endogenous and exogenous substances in cells. Its function is essential in drug metabolism and response, making it a valuable target for studying drug interactions, toxicity, and efficacy.
Whether you are studying drug transport mechanisms, drug-drug interactions, or simply exploring the role of OCT1 in cellular physiology, the OCT1 Colorimetric Cell-Based ELISA Kit is a valuable asset for your research needs. Trust in this kit to provide you with accurate and reliable data to advance your scientific inquiries.
| Product Name: | OCT1 Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB00506 |
| ELISA Type: | Cell-Based |
| Target: | 44470 |
| Reactivity: | Human, Mouse |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The OCT1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Oct1 protein expression profile in cells. The kit can be used for measuring the relative amounts of Oct1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Oct1.
Qualitative determination of OCT1 concentration is achieved by an indirect ELISA format. In essence, OCT1 is captured by OCT1-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: 5451, UniProt ID: P14859, OMIM: 164175, Unigene: Hs.493649 |
| Gene Symbol: | POU2F1 |
| Sub Type: | None |
| UniProt Protein Function: | Transcription factor that binds to the octamer motif (5'-ATTTGCAT-3') and activates the promoters of the genes for some small nuclear RNAs (snRNA) and of genes such as those for histone H2B and immunoglobulins. Modulates transcription transactivation by NR3C1, AR and PGR. In case of human herpes simplex virus (HSV) infection, POU2F1 forms a multiprotein-DNA complex with the viral transactivator protein VP16 and HCFC1 thereby enabling the transcription of the viral immediate early genes. |
| NCBI Summary: | The OCT1 transcription factor was among the first identified members of the POU transcription factor family (summarized by Sturm et al., 1993 [PubMed 8314572]). Members of this family contain the POU domain, a 160-amino acid region necessary for DNA binding to the octameric sequence ATGCAAAT.[supplied by OMIM, Jul 2010] |
| UniProt Code: | P14859 |
| NCBI GenInfo Identifier: | 28202257 |
| NCBI Gene ID: | 5451 |
| NCBI Accession: | P14859.2 |
| UniProt Secondary Accession: | P14859,Q5TBT7, Q6PK46, Q8NEU9, Q9BPV1, B1AL91, B1AL93 B4E029, J3KP77, |
| UniProt Related Accession: | P14859 |
| Molecular Weight: | 72,082 Da |
| NCBI Full Name: | POU domain, class 2, transcription factor 1 |
| NCBI Synonym Full Names: | POU class 2 homeobox 1 |
| NCBI Official Symbol: | POU2F1 |
| NCBI Official Synonym Symbols: | OCT1; OTF1; oct-1B |
| NCBI Protein Information: | POU domain, class 2, transcription factor 1 |
| UniProt Protein Name: | POU domain, class 2, transcription factor 1 |
| UniProt Synonym Protein Names: | NF-A1; Octamer-binding protein 1; Oct-1; Octamer-binding transcription factor 1; OTF-1 |
| Protein Family: | POU domain, class 2, transcription factor |
| UniProt Gene Name: | POU2F1 |
| UniProt Entry Name: | PO2F1_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-OCT1 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.)
