Description
Akt1 S1 (Phospho-Thr246)Colorimetric Cell-Based ELISA Kit
The AKT1-S1 Phospho Thr246 Colorimetric Cell-Based ELISA Kit is a cutting-edge assay designed for the detection of phosphorylated AKT1-S1 at threonine 246 in cell lysates. This kit offers high sensitivity and specificity, ensuring accurate and reproducible results for your research needs.AKT1-S1 is a key signaling molecule in various cellular processes, including cell growth, survival, and metabolism. Phosphorylation of AKT1-S1 at threonine 246 plays a crucial role in its activation and downstream signaling pathways, making it a valuable target for studying cell signaling mechanisms and potential drug development.
With this ELISA kit, researchers can easily measure the levels of phosphorylated AKT1-S1 in cell lysates, providing valuable insights into signaling pathways and potential therapeutic targets. Ideal for a wide range of research applications, the AKT1-S1 Phospho Thr246 Colorimetric Cell-Based ELISA Kit is a powerful tool for advancing your research in cell biology and drug discovery.
| Product Name: | Akt1 S1 (Phospho-Thr246)Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB01282 |
| ELISA Type: | Cell-Based |
| Target: | Akt1 S1 (Phospho-Thr246) |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nm |
| Format: | 2 x 96-Well Microplates |
The Akt1 S1 (Phospho-Thr246) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Akt1 S1 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Akt1 S1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Akt1 S1 phosphorylation.
Qualitative determination of Akt1 S1 (Phospho-Thr246) concentration is achieved by an indirect ELISA format. In essence, Akt1 S1 (Phospho-Thr246) is captured by Akt1 S1 (Phospho-Thr246)-specific primary (1ø) antibodies while the HRP-conjugated secondary (2ø) antibodies bind the Fc region of the 1ø antibody. Through this binding, the HRP enzyme conjugated to the 2ø 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: 84335, UniProt ID: Q96B36, OMIM: 610221, Unigene: Hs.515542 |
| Gene Symbol: | AKT1S1 |
| Sub Type: | Phospho |
| UniProt Protein Function: | Subunit of mTORC1, which regulates cell growth and survival in response to nutrient and hormonal signals. mTORC1 is activated in response to growth factors or amino acids. Growth factor-stimulated mTORC1 activation involves a AKT1-mediated phosphorylation of TSC1-TSC2, which leads to the activation of the RHEB GTPase that potently activates the protein kinase activity of mTORC1. Amino acid-signaling to mTORC1 requires its relocalization to the lysosomes mediated by the Ragulator complex and the Rag GTPases. Activated mTORC1 up-regulates protein synthesis by phosphorylating key regulators of mRNA translation and ribosome synthesis. mTORC1 phosphorylates EIF4EBP1 and releases it from inhibiting the elongation initiation factor 4E (eiF4E). mTORC1 phosphorylates and activates S6K1 at 'Thr-389', which then promotes protein synthesis by phosphorylating PDCD4 and targeting it for degradation. Within mTORC1, AKT1S1 negatively regulates mTOR activity in a manner that is dependent on its phosphorylation state and binding to 14-3-3 proteins. Inhibits RHEB-GTP-dependent mTORC1 activation. Substrate for AKT1 phosphorylation, but can also be activated by AKT1-independent mechanisms. May also play a role in nerve growth factor-mediated neuroprotection. |
| NCBI Summary: | AKT1S1 is a proline-rich substrate of AKT (MIM 164730) that binds 14-3-3 protein (see YWHAH, MIM 113508) when phosphorylated (Kovacina et al., 2003 [PubMed 12524439]).[supplied by OMIM, Mar 2008] |
| UniProt Code: | Q96B36 |
| NCBI GenInfo Identifier: | 74731194 |
| NCBI Gene ID: | 84335 |
| NCBI Accession: | Q96B36.1 |
| UniProt Secondary Accession: | Q96B36,Q96BI4, Q96IK7, Q96NG2, Q9BWR5, A8MTQ1, B2RE93 J3KPM3, |
| UniProt Related Accession: | Q96B36 |
| Molecular Weight: | 29,262 Da |
| NCBI Full Name: | Proline-rich AKT1 substrate 1 |
| NCBI Synonym Full Names: | AKT1 substrate 1 |
| NCBI Official Symbol: | AKT1S1Â Â |
| NCBI Official Synonym Symbols: | Lobe; PRAS40Â Â |
| NCBI Protein Information: | proline-rich AKT1 substrate 1 |
| UniProt Protein Name: | Proline-rich AKT1 substrate 1 |
| UniProt Synonym Protein Names: | 40 kDa proline-rich AKT substrate |
| Protein Family: | Proline-rich AKT1 substrate |
| UniProt Gene Name: | AKT1S1Â Â |
| UniProt Entry Name: | AKTS1_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-Akt1 S1 (Phospho-Thr246) Antibody, Anti-Akt1 S1 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.)
