Description
c-Jun (Phospho-Thr91)Colorimetric Cell-Based ELISA Kit
The C-Jun Phospho-Thr91 Colorimetric Cell-Based ELISA Kit is a highly specific and sensitive assay designed for the detection of phosphorylated C-Jun at Thr91 in cell lysates. This kit provides researchers with reliable and reproducible results, making it ideal for studying signaling pathways involved in cell growth, differentiation, and apoptosis.C-Jun is a key transcription factor that plays a critical role in regulating gene expression in response to various cellular stimuli. Phosphorylation of C-Jun at Thr91 is known to affect its activity and stability, making it an important target for understanding the mechanisms underlying cellular processes such as proliferation, differentiation, and survival.
With its high sensitivity and specificity, the C-Jun Phospho-Thr91 Colorimetric Cell-Based ELISA Kit is an invaluable tool for researchers studying intracellular signaling pathways and looking to elucidate the role of phosphorylated C-Jun in various cellular processes.
| Product Name: | c-Jun (Phospho-Thr91)Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB01555 |
| ELISA Type: | Cell-Based |
| Target: | c-Jun (Phospho-Thr91) |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nm |
| Format: | 2 x 96-Well Microplates |
The c-Jun (Phospho-Thr91) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect c-Jun protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated c-Jun in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on c-Jun phosphorylation.
Qualitative determination of c-Jun (Phospho-Thr91) concentration is achieved by an indirect ELISA format. In essence, c-Jun (Phospho-Thr91) is captured by c-Jun (Phospho-Thr91)-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: 3725, UniProt ID: P05412, OMIM: 165160, Unigene: Hs.714791 |
| Gene Symbol: | JUN |
| Sub Type: | Phospho |
| UniProt Protein Function: | Transcription factor that recognizes and binds to the enhancer heptamer motif 5'-TGA[CG]TCA-3'. Promotes activity of NR5A1 when phosphorylated by HIPK3 leading to increased steroidogenic gene expression upon cAMP signaling pathway stimulation. Involved in activated KRAS-mediated transcriptional activation of USP28 in colorectal cancer (CRC) cells (PubMed:24623306). Binds to the USP28 promoter in colorectal cancer (CRC) cells (PubMed:24623306). |
| NCBI Summary: | This gene is the putative transforming gene of avian sarcoma virus 17. It encodes a protein which is highly similar to the viral protein, and which interacts directly with specific target DNA sequences to regulate gene expression. This gene is intronless and is mapped to 1p32-p31, a chromosomal region involved in both translocations and deletions in human malignancies. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P05412 |
| NCBI GenInfo Identifier: | 135298 |
| NCBI Gene ID: | 3725 |
| NCBI Accession: | P05412.2 |
| UniProt Secondary Accession: | P05412,Q6FHM7, Q96G93, |
| UniProt Related Accession: | P05412 |
| Molecular Weight: | |
| NCBI Full Name: | Transcription factor AP-1 |
| NCBI Synonym Full Names: | jun proto-oncogene |
| NCBI Official Symbol: | JUN |
| NCBI Official Synonym Symbols: | AP1; AP-1; c-Jun |
| NCBI Protein Information: | transcription factor AP-1 |
| UniProt Protein Name: | Transcription factor AP-1 |
| UniProt Synonym Protein Names: | Activator protein 1; AP1; Proto-oncogene c-Jun; V-jun avian sarcoma virus 17 oncogene homolog; p39 |
| Protein Family: | C-Jun-amino-terminal kinase-interacting protein |
| UniProt Gene Name: | JUN |
| UniProt Entry Name: | JUN_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-c-Jun (Phospho-Thr91) Antibody, Anti-c-Jun 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.)
