Description
RIT1 Colorimetric Cell-Based ELISA Kit
The RIT1 Colorimetric Cell-Based ELISA Kit is a valuable tool for researchers looking to study the RIT1 signaling pathway. RIT1 is a critical protein involved in cell growth, differentiation, and survival, making it a key target in cancer research and drug development.This kit offers high sensitivity and specificity, allowing for accurate quantification of RIT1 levels in cell lysates and tissue samples. With reliable and reproducible results, researchers can confidently analyze the role of RIT1 in various cellular processes and disease mechanisms.
By understanding the signaling cascades involving RIT1, researchers can gain insights into diseases such as cancer, neurodevelopmental disorders, and metabolic syndromes. The RIT1 Colorimetric Cell-Based ELISA Kit provides a comprehensive and efficient solution for studying RIT1 biology and potential therapeutic interventions.
| Product Name: | RIT1 Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB00847 |
| ELISA Type: | Cell-Based |
| Target: | RIT1 |
| Reactivity: | Human, Mouse |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The RIT1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect RIT1 protein expression profile in cells. The kit can be used for measuring the relative amounts of RIT1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on RIT1.
Qualitative determination of RIT1 concentration is achieved by an indirect ELISA format. In essence, RIT1 is captured by RIT1-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: 6016, UniProt ID: Q92963, OMIM: 609591, Unigene: Hs.491234 |
| Gene Symbol: | RIT1 |
| Sub Type: | None |
| UniProt Protein Function: | RIT1: a small G protein of the Ras family. Plays a crucial role in coupling NGF stimulation to the activation of both EPHB2 and p38-alpha signaling pathways and in NGF- dependent neuronal differentiation. Stimulation of the NGF and EGF receptor signaling pathways results in rapid and prolonged activation. Interacts with afadin, the C-terminal domain of RalGDS and RLF, but not with RIN1 and PIK3CA. RLF binds exclusively to the active GTP-bound form. Strongly interacts with BRAF, but only weakly with c-Raf. BRAF and c-RAF association is dependent upon the GTP-bound state. Interacts with RGL3. Shows rapid uncatalyzed guanine nucleotide dissociation rates, which are much faster than those of most Ras subfamily members. |
| UniProt Protein Details: | Protein type:G protein, monomeric, Ras; G protein; G protein, monomeric Chromosomal Location of Human Ortholog: 1q22 Molecular Function:calmodulin binding; protein binding Biological Process: Ras protein signal transduction; signal transduction Disease: Noonan Syndrome 8 |
| NCBI Summary: | This gene encodes a member of a subfamily of Ras-related GTPases. The encoded protein is involved in regulating p38 MAPK-dependent signaling cascades related to cellular stress. This protein also cooperates with nerve growth factor to promote neuronal development and regeneration. Alternate splicing results in multiple transcript variants. [provided by RefSeq, Feb 2012] |
| UniProt Code: | Q92963 |
| NCBI GenInfo Identifier: | 38258628 |
| NCBI Gene ID: | 6016 |
| NCBI Accession: | Q92963.1 |
| UniProt Secondary Accession: | Q92963,O00646, O00720, Q5VY89, Q5VY90, B4DQE8, |
| UniProt Related Accession: | Q92963 |
| Molecular Weight: | 27,148 Da |
| NCBI Full Name: | GTP-binding protein Rit1 |
| NCBI Synonym Full Names: | Ras like without CAAX 1 |
| NCBI Official Symbol: | RIT1 |
| NCBI Official Synonym Symbols: | NS8; RIT; RIBB; ROC1 |
| NCBI Protein Information: | GTP-binding protein Rit1 |
| UniProt Protein Name: | GTP-binding protein Rit1 |
| UniProt Synonym Protein Names: | Ras-like protein expressed in many tissues; Ras-like without CAAX protein 1 |
| Protein Family: | GTP-binding protein |
| UniProt Gene Name: | RIT1 |
| UniProt Entry Name: | RIT1_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-RIT1 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.)
