Description
DJ-1 Colorimetric Cell-Based ELISA Kit
The DJ-1 Colorimetric Cell-Based ELISA Kit offered by Assay Genie is a reliable tool for detecting and quantifying levels of DJ-1 protein in cell lysates, serum, plasma, and other biological samples. DJ-1 is a key protein involved in oxidative stress response, mitochondrial function, and neuroprotection, making it a valuable biomarker for studying Parkinson's disease, cancer, and other neurodegenerative disorders.This ELISA kit provides high sensitivity and specificity, ensuring accurate and reproducible results for your research needs.
With its user-friendly protocol and quick assay time, this kit is suitable for a variety of applications in the fields of cell biology, neuroscience, and drug discovery.Don't miss out on the opportunity to enhance your research with the DJ-1 Colorimetric Cell-Based ELISA Kit from Assay Genie. Unlock new insights into DJ-1 protein expression and function to advance our understanding of disease mechanisms and pave the way for potential therapeutic interventions.
| Product Name: | DJ-1 Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB00293 |
| ELISA Type: | Cell-Based |
| Target: | DJ-1 |
| Reactivity: | Human, Mouse |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The DJ-1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect DJ-1 protein expression profile in cells. The kit can be used for measuring the relative amounts of DJ-1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on DJ-1.
Qualitative determination of DJ-1 concentration is achieved by an indirect ELISA format. In essence, DJ-1 is captured by DJ-1-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: 11315, UniProt ID: Q99497, OMIM: 602533, Unigene: Hs.419640 |
| Gene Symbol: | PARK7 |
| Sub Type: | None |
| UniProt Protein Function: | Protein and nucleotide deglycase that catalyzes the deglycation of the Maillard adducts formed between amino groups of proteins or nucleotides and reactive carbonyl groups of glyoxals (PubMed:25416785, PubMed:28596309). Thus, functions as a protein deglycase that repairs methylglyoxal- and glyoxal-glycated proteins, and releases repaired proteins and lactate or glycolate, respectively. Deglycates cysteine, arginine and lysine residues in proteins, and thus reactivates these proteins by reversing glycation by glyoxals. Acts on early glycation intermediates (hemithioacetals and aminocarbinols), preventing the formation of advanced glycation endproducts (AGE) that cause irreversible damage (PubMed:25416785, PubMed:28013050, PubMed:26995087). Also functions as a nucleotide deglycase able to repair glycated guanine in the free nucleotide pool (GTP, GDP, GMP, dGTP) and in DNA and RNA. Is thus involved in a major nucleotide repair system named guanine glycation repair (GG repair), dedicated to reversing methylglyoxal and glyoxal damage via nucleotide sanitization and direct nucleic acid repair (PubMed:28596309). Also displays an apparent glyoxalase activity that in fact reflects its deglycase activity (PubMed:22523093). Plays an important role in cell protection against oxidative stress and cell death acting as oxidative stress sensor and redox-sensitive chaperone and protease; functions probably related to its primary function (PubMed:17015834, PubMed:20304780, PubMed:18711745, PubMed:12796482, PubMed:19229105, PubMed:25416785, PubMed:26995087). It is involved in neuroprotective mechanisms like the stabilization of NFE2L2 and PINK1 proteins, male fertility as a positive regulator of androgen signaling pathway as well as cell growth and transformation through, for instance, the modulation of NF-kappa-B signaling pathway (PubMed:12612053, PubMed:15502874, PubMed:14749723, PubMed:17015834, PubMed:21097510, PubMed:18711745). Eliminates hydrogen peroxide and protects cells against hydrogen peroxide-induced cell death (PubMed:16390825). Required for correct mitochondrial morphology and function as well as for autophagy of dysfunctional mitochondria (PubMed:19229105, PubMed:16632486). Plays a role in regulating expression or stability of the mitochondrial uncoupling proteins SLC25A14 and SLC25A27 in dopaminergic neurons of the substantia nigra pars compacta and attenuates the oxidative stress induced by calcium entry into the neurons via L-type channels during pacemaking (PubMed:18711745). Regulates astrocyte inflammatory responses, may modulate lipid rafts-dependent endocytosis in astrocytes and neuronal cells (PubMed:23847046). In pancreatic islets, involved in the maintenance of mitochondrial reactive oxygen species (ROS) levels and glucose homeostasis in an age- and diet dependent manner. Protects pancreatic beta cells from cell death induced by inflammatory and cytotoxic setting. Binds to a number of mRNAs containing multiple copies of GG or CC motifs and partially inhibits their translation but dissociates following oxidative stress (PubMed:18626009). Metal-binding protein able to bind copper as well as toxic mercury ions, enhances the cell protection mechanism against induced metal toxicity (PubMed:23792957). In macrophages, interacts with the NADPH oxidase subunit NCF1 to direct NADPH oxidase-dependent ROS production, and protects against sepsis. |
| NCBI Summary: | The product of this gene belongs to the peptidase C56 family of proteins. It acts as a positive regulator of androgen receptor-dependent transcription. It may also function as a redox-sensitive chaperone, as a sensor for oxidative stress, and it apparently protects neurons against oxidative stress and cell death. Defects in this gene are the cause of autosomal recessive early-onset Parkinson disease 7. Two transcript variants encoding the same protein have been identified for this gene. [provided by RefSeq, Jul 2008] |
| UniProt Code: | Q99497 |
| NCBI GenInfo Identifier: | 56404943 |
| NCBI Gene ID: | 11315 |
| NCBI Accession: | Q99497.2 |
| UniProt Secondary Accession: | Q99497,O14805, Q6DR95, Q7LFU2, B2R4Z1, |
| UniProt Related Accession: | Q99497 |
| Molecular Weight: | 19,891 Da |
| NCBI Full Name: | Protein/nucleic acid deglycase DJ-1 |
| NCBI Synonym Full Names: | Parkinsonism associated deglycase |
| NCBI Official Symbol: | PARK7 |
| NCBI Official Synonym Symbols: | DJ1; DJ-1; GATD2; HEL-S-67p |
| NCBI Protein Information: | protein/nucleic acid deglycase DJ-1 |
| UniProt Protein Name: | Protein/nucleic acid deglycase DJ-1 |
| UniProt Synonym Protein Names: | Maillard deglycase |
| Protein Family: | DJ-1 protein |
| UniProt Gene Name: | PARK7 |
| 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-DJ-1 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.)
