Description
K6PP Colorimetric Cell-Based ELISA
The K6PP Colorimetric Cell-Based ELISA Kit is a cutting-edge assay designed for the detection of specific biomarkers in cell cultures. This kit provides high sensitivity and specificity, allowing researchers to accurately measure protein levels in cell culture supernatants.Utilizing a colorimetric assay, the K6PP kit allows for the quantitative analysis of target proteins, aiding in the study of various diseases and cellular processes.
With its user-friendly protocol and reliable results, this kit is essential for researchers looking to investigate protein expression in cell-based models.The K6PP Colorimetric Cell-Based ELISA Kit is a versatile tool for a wide range of research applications, offering researchers the ability to study protein dynamics and pathways in cell cultures with precision and accuracy.
| Product Name: | K6PP Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB01057 |
| ELISA Type: | Cell-Based |
| Target: | K6PP |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The K6PP Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect K6PP protein expression profile in cells. The kit can be used for measuring the relative amounts of K6PP in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on K6PP.
Qualitative determination of K6PP concentration is achieved by an indirect ELISA format. In essence, K6PP is captured by K6PP-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: 5214, UniProt ID: Q01813, OMIM: 171840, Unigene: Hs.26010 |
| Gene Symbol: | K6PP |
| Sub Type: | None |
| UniProt Protein Function: | PFKP: phosphofructokinase, platelet type. An ubiquitous metabolic enzyme involved in the synthesis and degradation of fructose 2,6-bisphosphate. Key control step of glycolysis. An allosteric enzyme activated by ADP, AMP, or fructose bisphosphate and inhibited by ATP or citrate. Activity: ATP D-fructose 6-phosphate = ADP D-fructose 1,6-bisphosphate. The holoenzyme consists of 4 subunits. The liver and muscle enzymes are homo-tetramers of four liver or muscle isoforms, respectively. The red blood cell enzyme consists hetero-tetramers of the muscle and liver isoforms. A subunit composition with a higher proportion of platelet type subunits is found in platelets, brain and fibroblasts. |
| UniProt Protein Details: | Protein type:Carbohydrate Metabolism - glycolysis and gluconeogenesis; Carbohydrate Metabolism - pentose phosphate pathway; EC 2.7.1.11; Carbohydrate Metabolism - galactose; Kinase, other; Carbohydrate Metabolism - fructose and mannose Chromosomal Location of Human Ortholog: 10p15.3-p15.2 Cellular Component: cytosol; membrane; nucleus Molecular Function:6-phosphofructokinase activity; protein complex binding |
| NCBI Summary: | The PFKP gene encodes the platelet isoform of phosphofructokinase (PFK) (ATP:D-fructose-6-phosphate-1-phosphotransferase, EC 2.7.1.11). PFK catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis. The PFKP gene, which maps to chromosome 10p, is also expressed in fibroblasts. See also the muscle (PFKM; MIM 610681) and liver (PFKL; MIM 171860) isoforms of phosphofructokinase, which map to chromosomes 12q13 and 21q22, respectively. Vora (1981) [PubMed 6451249] determined that full tetrameric phophofructokinase enzyme expressed in platelets can be composed of subunits P4, P3L, and P2L2.[supplied by OMIM, Mar 2008] |
| UniProt Code: | Q01813 |
| NCBI GenInfo Identifier: | 1346355 |
| NCBI Gene ID: | 5214 |
| NCBI Accession: | Q01813.2 |
| UniProt Secondary Accession: | Q01813,Q5VSR7, Q5VSR8, B3KS15, |
| UniProt Related Accession: | Q01813 |
| Molecular Weight: | 85596 Da |
| NCBI Full Name: | ATP-dependent 6-phosphofructokinase, platelet type |
| NCBI Synonym Full Names: | phosphofructokinase, platelet |
| NCBI Official Symbol: | PFKP |
| NCBI Official Synonym Symbols: | PFKF; PFK-C; PFK-P; ATP-PFK |
| NCBI Protein Information: | ATP-dependent 6-phosphofructokinase, platelet type |
| UniProt Protein Name: | ATP-dependent 6-phosphofructokinase, platelet type |
| UniProt Synonym Protein Names: | 6-phosphofructokinase type C; Phosphofructo-1-kinase isozyme C; PFK-C; Phosphohexokinase |
| Protein Family: | ATP-dependent 6-phosphofructokinase |
| UniProt Gene Name: | PFKP |
| UniProt Entry Name: | PFKAP_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-K6PP 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.)
