Description
MBD3 Colorimetric Cell-Based ELISA
The MBD3 Colorimetric Cell-Based ELISA Kit is a cutting-edge tool designed for the detection of MBD3 protein levels in cell lysates. This kit offers high sensitivity and specificity, allowing for accurate and reliable measurements of MBD3 expression in a variety of cell types.MBD3 is a key component of the nucleosome remodeling and histone deacetylase complex, playing a crucial role in gene regulation and chromatin remodeling. Dysregulation of MBD3 has been implicated in various diseases, including cancer, neurological disorders, and developmental abnormalities, making it a valuable target for research and therapeutic development.
With the MBD3 Colorimetric Cell-Based ELISA Kit, researchers can easily study the function and expression of MBD3 in cellular processes, opening up new possibilities for understanding disease mechanisms and identifying potential therapeutic interventions. Trust in this advanced kit for accurate and reproducible results in your research endeavors.
| Product Name: | MBD3 Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB00967 |
| ELISA Type: | Cell-Based |
| Target: | MBD3 |
| Reactivity: | Human, Mouse |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The MBD3 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect MBD3 protein expression profile in cells. The kit can be used for measuring the relative amounts of MBD3 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on MBD3.
Qualitative determination of MBD3 concentration is achieved by an indirect ELISA format. In essence, MBD3 is captured by MBD3-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: 53615, UniProt ID: O95983, OMIM: 603573, Unigene: Hs.178728/Hs.714161 |
| Gene Symbol: | MBD3 |
| Sub Type: | None |
| UniProt Protein Function: | MBD3: methyl-CpG binding (MBD) proteins appear to bind differentially methylated domains (DMDs) of DNA, to recruit repression complexes, and to silence the locus. The methyl CpG binding domain of human MBD3 may not directly bind mCpG but does interact with the NuRD/Mi2 components HDAC1 and MTA2. Plays a role in histone deacetylation and nucleosome remodeling. Increased levels have been reported in lung cancer and glioblastomas and may be a therapeutic target. |
| UniProt Protein Details: | Protein type:Gene silencing; Transcription, coactivator/corepressor Chromosomal Location of Human Ortholog: 19p13.3 Cellular Component: nuclear chromatin; nucleoplasm; protein complex Molecular Function:DNA binding; histone deacetylase activity; methyl-CpG binding; nucleosomal DNA binding; protein binding Biological Process: ATP-dependent chromatin remodeling; methylation-dependent chromatin silencing; negative regulation of transcription from RNA polymerase II promoter |
| NCBI Summary: | DNA methylation is the major modification of eukaryotic genomes and plays an essential role in mammalian development. This gene belongs to a family of nuclear proteins which are characterized by the presence of a methyl-CpG binding domain (MBD). The encoded protein is a subunit of the NuRD, a multisubunit complex containing nucleosome remodeling and histone deacetylase activities. Unlike the other family members, the encoded protein is not capable of binding to methylated DNA. The protein mediates the association of metastasis-associated protein 2 with the core histone deacetylase complex. Alternative splicing results in multiple transcript variants of this gene. [provided by RefSeq, Jul 2013] |
| UniProt Code: | O95983 |
| NCBI GenInfo Identifier: | 50400820 |
| NCBI Gene ID: | 53615 |
| NCBI Accession: | O95983.1 |
| UniProt Secondary Accession: | O95983,Q6PIL9, Q6PJZ9, Q86XF4, A8K4B7, D6W5Z2, |
| UniProt Related Accession: | O95983 |
| Molecular Weight: | 29,014 Da |
| NCBI Full Name: | Methyl-CpG-binding domain protein 3 |
| NCBI Synonym Full Names: | methyl-CpG binding domain protein 3 |
| NCBI Official Symbol: | MBD3 |
| NCBI Protein Information: | methyl-CpG-binding domain protein 3 |
| UniProt Protein Name: | Methyl-CpG-binding domain protein 3 |
| UniProt Synonym Protein Names: | Methyl-CpG-binding protein MBD3 |
| Protein Family: | Methyl-CpG-binding domain protein |
| UniProt Gene Name: | MBD3 |
| UniProt Entry Name: | MBD3_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-MBD3 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.)
