Fos (Phospho-Ser362) Colorimetric Cell-Based ELISA Kit
- SKU:
- CBCAB00229
- Product Type:
- ELISA Kit
- ELISA Type:
- Cell Based Phospho Specific
- Reactivity:
- Human
- Reactivity:
- Mouse
- Reactivity:
- Rat
- Detection Method:
- Colorimetric
Description
| Product Name: | Fos (Phospho-Ser362) Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB00229 |
| ELISA Type: | Cell-Based |
| Target: | Fos (Phospho-Ser362) |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nm |
| Format: | 2 x 96-Well Microplates |
The Fos (Phospho-Ser362) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Fos protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Fos in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Fos phosphorylation.
Qualitative determination of Fos (Phospho-Ser362) concentration is achieved by an indirect ELISA format. In essence, Fos (Phospho-Ser362) is captured by Fos (Phospho-Ser362)-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: 2353, UniProt ID: P01100, OMIM: 164810, Unigene: Hs.25647 |
| Gene Symbol: | FOS |
| Sub Type: | Phospho |
| UniProt Protein Function: | Fos: a proto-oncogenic transcription factor of the bZIP family. Dimerizes with proteins of the JUN family, thereby forming the transcription factor complex AP-1. FOS proteins function as regulators of cell proliferation, differentiation, and transformation. In some cases, expression of FOS has also been associated with apoptotic cell death. Expression increases upon a variety of stimuli, including growth factors, cytokines, neurotransmitters, polypeptide hormones, stress and cell injury. |
| UniProt Protein Details: | Protein type:Motility/polarity/chemotaxis; DNA-binding; Transcription factor; Oncoprotein Chromosomal Location of Human Ortholog: 14q24.3 Cellular Component: nucleoplasm; transcription factor complex; neuron projection; membrane; endoplasmic reticulum; nucleus; cytosol Molecular Function:protein binding; double-stranded DNA binding; transcription factor activity; transcription factor binding Biological Process: transcription from RNA polymerase II promoter; response to gravity; response to cAMP; positive regulation of osteoclast differentiation; positive regulation of transcription, DNA-dependent; response to toxin; stress-activated MAPK cascade; response to lipopolysaccharide; toll-like receptor 3 signaling pathway; female pregnancy; toll-like receptor 10 signaling pathway; toll-like receptor 5 signaling pathway; regulation of transcription factor activity; transforming growth factor beta receptor signaling pathway; conditioned taste aversion; DNA methylation; inflammatory response; toll-like receptor 4 signaling pathway; aging; response to corticosterone stimulus; response to drug; response to light stimulus; nervous system development; MyD88-independent toll-like receptor signaling pathway; sleep; toll-like receptor 2 signaling pathway; cellular response to hormone stimulus; regulation of transcription from RNA polymerase II promoter; MyD88-dependent toll-like receptor signaling pathway; response to mechanical stimulus; response to cytokine stimulus; cellular response to extracellular stimulus; toll-like receptor signaling pathway; innate immune response; positive regulation of transcription from RNA polymerase II promoter; response to cold; toll-like receptor 9 signaling pathway; response to progesterone stimulus |
| NCBI Summary: | The Fos gene family consists of 4 members: FOS, FOSB, FOSL1, and FOSL2. These genes encode leucine zipper proteins that can dimerize with proteins of the JUN family, thereby forming the transcription factor complex AP-1. As such, the FOS proteins have been implicated as regulators of cell proliferation, differentiation, and transformation. In some cases, expression of the FOS gene has also been associated with apoptotic cell death. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P01100 |
| NCBI GenInfo Identifier: | 120470 |
| NCBI Gene ID: | 2353 |
| NCBI Accession: | P01100.1 |
| UniProt Secondary Accession: | P01100,P18849, A8K4E2, B4DQ65, |
| UniProt Related Accession: | P01100 |
| Molecular Weight: | 380 |
| NCBI Full Name: | Proto-oncogene c-Fos |
| NCBI Synonym Full Names: | FBJ murine osteosarcoma viral oncogene homolog |
| NCBI Official Symbol: | FOS |
| NCBI Official Synonym Symbols: | p55; AP-1; C-FOS |
| NCBI Protein Information: | proto-oncogene c-Fos; activator protein 1; cellular oncogene c-fos; G0/G1 switch regulatory protein 7; FBJ murine osteosarcoma viral (v-fos) oncogene homolog (oncogene FOS) |
| UniProt Protein Name: | Proto-oncogene c-Fos |
| UniProt Synonym Protein Names: | Cellular oncogene fos; G0/G1 switch regulatory protein 7 |
| Protein Family: | Fos-related antigen |
| UniProt Gene Name: | FOS |
| UniProt Entry Name: | FOS_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-Fos (Phospho-Ser362) Antibody, Anti-Fos 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.)
