Description
GLU2B Colorimetric Cell-Based ELISA
The Glu2B Colorimetric Cell-based ELISA kit from Assay Genie is a highly sensitive and specific assay designed for the detection of Glucose-6-Phosphate Dehydrogenase (G6PD) activity in a cell-based system. This kit is ideal for researchers looking to accurately measure G6PD levels in various cell types, including cancer cells, to study its role in glucose metabolism and potential therapeutic targets.G6PD is a key enzyme involved in the pentose phosphate pathway and plays a crucial role in maintaining cellular redox balance and protecting cells from oxidative stress.
Dysregulation of G6PD activity has been implicated in various diseases, including cancer, diabetes, and neurodegenerative disorders, making it a valuable biomarker for understanding disease pathogenesis and identifying novel treatment strategies.With its high sensitivity and precision, the Glu2B Colorimetric Cell-based ELISA kit offers researchers a reliable tool for investigating G6PD activity in cell-based models, providing valuable insights into its biological functions and potential therapeutic implications.
| Product Name: | GLU2B Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB00975 |
| ELISA Type: | Cell-Based |
| Target: | GLU2B |
| Reactivity: | Human, Mouse |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The GLU2B Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect GLU2B protein expression profile in cells. The kit can be used for measuring the relative amounts of GLU2B in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on GLU2B.
Qualitative determination of GLU2B concentration is achieved by an indirect ELISA format. In essence, GLU2B is captured by GLU2B-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: 5589, UniProt ID: P14314, OMIM: 177060, Unigene: Hs.610830 |
| Gene Symbol: | PRKCSH |
| Sub Type: | None |
| UniProt Protein Function: | PRKCSH: Regulatory subunit of glucosidase II. Defects in PRKCSH are a cause of polycystic liver disease (PCLD). PCLD is an autosomal dominant disorder and is characterized by the presence of multiple liver cysts of biliary epithelial origin. PCLD is a distinct clinical and genetic entity that can occur independently from autosomal dominant polycystic kidney disease (ADPKD), which in a considerable but uncertain proportion of cases is associated with hepatic cysts. 2 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Adaptor/scaffold Chromosomal Location of Human Ortholog: 19p13.2 Cellular Component: endoplasmic reticulum; endoplasmic reticulum lumen; intracellular Molecular Function:protein kinase C binding; phosphoprotein binding; calcium ion binding Biological Process: cellular protein metabolic process; protein folding; innate immune response; protein amino acid N-linked glycosylation via asparagine; N-glycan processing; post-translational protein modification Disease: Polycystic Liver Disease |
| NCBI Summary: | This gene encodes the beta-subunit of glucosidase II, an N-linked glycan-processing enzyme in the endoplasmic reticulum. The encoded protein is an acidic phosphoprotein known to be a substrate for protein kinase C. Mutations in this gene have been associated with the autosomal dominant polycystic liver disease. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Jan 2014] |
| UniProt Code: | P14314 |
| NCBI GenInfo Identifier: | 116242499 |
| NCBI Gene ID: | 5589 |
| NCBI Accession: | P14314.2 |
| UniProt Secondary Accession: | P14314,Q96BU9, Q96D06, Q9P0W9, A8K318, |
| UniProt Related Accession: | P14314 |
| Molecular Weight: | 528 |
| NCBI Full Name: | Glucosidase 2 subunit beta |
| NCBI Synonym Full Names: | protein kinase C substrate 80K-H |
| NCBI Official Symbol: | PRKCSH |
| NCBI Official Synonym Symbols: | PCLD; PLD1; G19P1; PKCSH; AGE-R2 |
| NCBI Protein Information: | glucosidase 2 subunit beta; hepatocystin; AGE-binding receptor 2; glucosidase II subunit beta; protein kinase C substrate, 80 Kda protein; protein kinase C substrate 60.1 kDa protein heavy chain |
| UniProt Protein Name: | Glucosidase 2 subunit beta |
| UniProt Synonym Protein Names: | 80K-H protein; Glucosidase II subunit beta; Protein kinase C substrate 60.1 kDa protein heavy chain; PKCSH |
| Protein Family: | Glucosidase |
| UniProt Gene Name: | PRKCSH |
| UniProt Entry Name: | GLU2B_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-GLU2B 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.)
