Description
CrkII (Phospho-Tyr221)Colorimetric Cell-Based ELISA Kit
The CRKII Phospho-Tyr221 Colorimetric Cell-Based ELISA Kit is specifically designed for the accurate and sensitive detection of phosphorylated CRKII protein at tyrosine 221 in cell lysates. This kit offers high specificity and reliability, allowing for precise quantification of CRKII activation in a variety of cell types and conditions.CRKII is a key signaling molecule involved in cell growth, differentiation, and migration, making it a critical player in various cellular processes. Phosphorylation at tyrosine 221 is known to enhance the activity of CRKII, leading to downstream signaling cascades that impact cell behavior.
By using the CRKII Phospho-Tyr221 Colorimetric Cell-Based ELISA Kit, researchers can gain valuable insights into the activation status of CRKII in their experimental systems, paving the way for a better understanding of cellular signaling pathways and potential therapeutic targets. Trust in the precision and accuracy of this kit for your research needs.
| Product Name: | CrkII (Phospho-Tyr221)Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB01239 |
| ELISA Type: | Cell-Based |
| Target: | CrkII (Phospho-Tyr221) |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nm |
| Format: | 2 x 96-Well Microplates |
The CrkII (Phospho-Tyr221) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect CrkII protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated CrkII in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on CrkII phosphorylation.
Qualitative determination of CrkII (Phospho-Tyr221) concentration is achieved by an indirect ELISA format. In essence, CrkII (Phospho-Tyr221) is captured by CrkII (Phospho-Tyr221)-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: 1398, UniProt ID: P46108, OMIM: 164762, Unigene: Hs.638121 |
| Gene Symbol: | CRK |
| Sub Type: | Phospho |
| UniProt Protein Function: | CRK: an adaptor protein with an SH2-SH3-SH3 domain structure. Recruits cytoplasmic proteins through SH2-phospho-tyrosine interaction. Phosphorylated by Abl, IGF-IR and EGFR. Phosphorylation induces a change in intramolecular folding and SH2 interactions, causing its rapid dissociation from the tyrosine kinase complex. |
| UniProt Protein Details: | Protein type:Motility/polarity/chemotaxis; Adaptor/scaffold; Oncoprotein Chromosomal Location of Human Ortholog: 17p13.3 Cellular Component: cytoplasm; plasma membrane; cytosol; nucleus; actin cytoskeleton Molecular Function:protein phosphorylated amino acid binding; protein binding; ephrin receptor binding; SH3/SH2 adaptor activity; SH2 domain binding Biological Process: regulation of transcription from RNA polymerase II promoter; regulation of Rac protein signal transduction; platelet activation; activation of MAPKK activity; nerve growth factor receptor signaling pathway; regulation of actin cytoskeleton organization and biogenesis; insulin receptor signaling pathway; ephrin receptor signaling pathway; innate immune response; vascular endothelial growth factor receptor signaling pathway; blood coagulation |
| NCBI Summary: | This gene encodes a member of an adapter protein family that binds to several tyrosine-phosphorylated proteins. The product of this gene has several SH2 and SH3 domains (src-homology domains) and is involved in several signaling pathways, recruiting cytoplasmic proteins in the vicinity of tyrosine kinase through SH2-phosphotyrosine interaction. The N-terminal SH2 domain of this protein functions as a positive regulator of transformation whereas the C-terminal SH3 domain functions as a negative regulator of transformation. Two alternative transcripts encoding different isoforms with distinct biological activity have been described. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P46108 |
| NCBI GenInfo Identifier: | 158939322 |
| NCBI Gene ID: | 1398 |
| NCBI Accession: | P46108.2 |
| UniProt Secondary Accession: | P46108,Q96GA9, Q96HJ0, A8MWE8, B0LPE8, D3DTH6, |
| UniProt Related Accession: | P46108 |
| Molecular Weight: | 304 |
| NCBI Full Name: | Adapter molecule crk |
| NCBI Synonym Full Names: | v-crk avian sarcoma virus CT10 oncogene homolog |
| NCBI Official Symbol: | CRK |
| NCBI Official Synonym Symbols: | p38; CRKII |
| NCBI Protein Information: | adapter molecule crk; proto-oncogene c-Crk |
| UniProt Protein Name: | Adapter molecule crk |
| UniProt Synonym Protein Names: | Proto-oncogene c-Crk; p38 |
| Protein Family: | Crk-like protein |
| UniProt Gene Name: | CRK |
| UniProt Entry Name: | CRK_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-CrkII (Phospho-Tyr221) Antibody, Anti-CrkII 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.)
