Description
CD71/TfR (Phospho-Ser24)Colorimetric Cell-Based ELISA Kit
The CD71/TfR (Phospho-Ser24) Colorimetric Cell-Based ELISA Kit is a cutting-edge tool designed for the accurate measurement of CD71/TfR (Transferrin Receptor) phosphorylated at Ser24 levels in cell samples. This kit offers high sensitivity and specificity, ensuring precise and reproducible results for a variety of research applications.CD71/TfR phosphorylated at Ser24 is an important protein involved in cell signaling and regulation, playing a key role in processes such as cell growth, differentiation, and iron metabolism. Dysregulation of CD71/TfR phosphorylation has been implicated in various diseases, including cancer, anemia, and neurodegenerative disorders, making it a valuable biomarker for understanding these conditions and potentially developing therapeutic interventions.
With the CD71/TfR (Phospho-Ser24) Colorimetric Cell-Based ELISA Kit, researchers can accurately quantify the levels of phosphorylated CD71/TfR in cell samples, advancing their understanding of cellular signaling pathways and disease mechanisms. This kit is a valuable tool for anyone studying cell biology, signal transduction, or disease pathogenesis.
| Product Name: | CD71/TfR (Phospho-Ser24)Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB00443 |
| ELISA Type: | Cell-Based |
| Target: | CD71/TfR (Phospho-Ser24) |
| Reactivity: | Human, Mouse |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nm |
| Format: | 2 x 96-Well Microplates |
The CD71/TfR (Phospho-Ser24) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect CD71/TfR protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated CD71/TfR in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on CD71/TfR phosphorylation.
Qualitative determination of CD71/TfR (Phospho-Ser24) concentration is achieved by an indirect ELISA format. In essence, CD71/TfR (Phospho-Ser24) is captured by CD71/TfR (Phospho-Ser24)-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: 4155, UniProt ID: P02786, OMIM: 159430, Unigene: Hs.551713 |
| Gene Symbol: | TFR1 |
| Sub Type: | Phospho |
| UniProt Protein Function: | TfR: the transferrin receptor. Regulates the cellular uptake of iron occurs via receptor-mediated endocytosis of ligand-occupied receptors into specialized endosomes. Endosomal acidification leads to iron release. The apotransferrin-receptor complex is then recycled to the cell surface with a return to neutral pH and the concomitant loss of affinity of apotransferrin for its receptor. Transferrin receptor is necessary for development of erythrocytes and the nervous system. |
| UniProt Protein Details: | Protein type:Receptor, misc.; Cell surface; Membrane protein, integral Chromosomal Location of Human Ortholog: 3q29 Cellular Component: basolateral plasma membrane; cell surface; coated pit; cytoplasmic membrane-bound vesicle; endosome; external side of plasma membrane; extracellular region; extracellular space; integral to plasma membrane; intracellular membrane-bound organelle; perinuclear region of cytoplasm; plasma membrane; recycling endosome Molecular Function:double-stranded RNA binding; glycoprotein binding; identical protein binding; protein binding; protein homodimerization activity; transferrin receptor activity Biological Process: regulation of cell growth; regulation of cell proliferation; transferrin transport |
| NCBI Summary: | This gene encodes a cell surface receptor necessary for cellular iron uptake by the process of receptor-mediated endocytosis. This receptor is required for erythropoiesis and neurologic development. Multiple alternatively spliced variants have been identified. [provided by RefSeq, Sep 2015] |
| UniProt Code: | P02786 |
| NCBI GenInfo Identifier: | 108935939 |
| NCBI Gene ID: | 7037 |
| NCBI Accession: | P02786.2 |
| UniProt Secondary Accession: | P02786,Q1HE24, Q59G55, Q9UCN0, Q9UCU5, Q9UDF9, Q9UK21 D3DXB0, |
| UniProt Related Accession: | P02786 |
| Molecular Weight: | 84,871 Da |
| NCBI Full Name: | Transferrin receptor protein 1 |
| NCBI Synonym Full Names: | transferrin receptor |
| NCBI Official Symbol: | TFRC |
| NCBI Official Synonym Symbols: | T9; TR; TFR; p90; CD71; TFR1; TRFR; IMD46 |
| NCBI Protein Information: | transferrin receptor protein 1 |
| UniProt Protein Name: | Transferrin receptor protein 1 |
| UniProt Synonym Protein Names: | T9; p90; CD_antigen: CD71 |
| Protein Family: | Transferrin receptor protein |
| UniProt Gene Name: | TFRC |
| UniProt Entry Name: | TFR1_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-CD71/TfR (Phospho-Ser24) Antibody, Anti-CD71/TfR 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.)
