Description
ERCC5 Colorimetric Cell-Based ELISA
The ERCC5 Colorimetric Cell-Based ELISA Kit is a cutting-edge assay designed for the precise measurement of ERCC5 levels in cell lysates, tissue homogenates, and biological samples. With its advanced technology, this kit offers unparalleled sensitivity and accuracy, ensuring trustworthy and consistent results for various research applications.ERCC5, also known as Xeroderma Pigmentosum group G (XPG) protein, plays a crucial role in DNA repair mechanisms and maintaining genomic stability. Dysregulation of ERCC5 has been linked to a range of genetic disorders and diseases, including cancer and neurological disorders.
Therefore, this kit serves as a valuable tool for studying the function of ERCC5 and its implications in disease pathology, as well as for identifying potential therapeutic targets and interventions.The ERCC5 Colorimetric Cell-Based ELISA Kit is a must-have for researchers seeking to unravel the intricate mechanisms of DNA repair and genomic maintenance, offering accurate and reliable results that pave the way for groundbreaking discoveries in the field of molecular biology and genetics.
| Product Name: | ERCC5 Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB00996 |
| ELISA Type: | Cell-Based |
| Target: | ERCC5 |
| Reactivity: | Human |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The ERCC5 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect ERCC5 protein expression profile in cells. The kit can be used for measuring the relative amounts of ERCC5 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on ERCC5.
Qualitative determination of ERCC5 concentration is achieved by an indirect ELISA format. In essence, ERCC5 is captured by ERCC5-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: 2073, UniProt ID: P28715, OMIM: 133520, Unigene: Hs.258429 |
| Gene Symbol: | ERCC5 |
| Sub Type: | None |
| UniProt Protein Function: | XPG: Single-stranded structure-specific DNA endonuclease involved in DNA excision repair. Makes the 3'incision in DNA nucleotide excision repair (NER). Acts as a cofactor for a DNA glycosylase that removes oxidized pyrimidines from DNA. May also be involved in transcription-coupled repair of this kind of damage, in transcription by RNA polymerase II, and perhaps in other processes too. Defects in ERCC5 are the cause of xeroderma pigmentosum complementation group G (XP-G); also known as xeroderma pigmentosum VII (XP7). Xeroderma pigmentosum is an autosomal recessive pigmentary skin disorder characterized by solar hypersensitivity of the skin, high predisposition for developing cancers on areas exposed to sunlight and, in some cases, neurological abnormalities. Some XP-G patients present features of Cockayne syndrome, including dwarfism, sensorineural deafness, microcephaly, mental retardation, pigmentary retinopathy, ataxia, decreased nerve conduction velocities. Belongs to the XPG/RAD2 endonuclease family. XPG subfamily. 2 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Deoxyribonuclease; EC 3.1.-.-; DNA repair, damage Chromosomal Location of Human Ortholog: 13q33 Cellular Component: DNA replication factor A complex; DNA-directed RNA polymerase II, holoenzyme; holo TFIIH complex; nucleoplasm; nucleus Molecular Function:bubble DNA binding; double-stranded DNA binding; endodeoxyribonuclease activity; protein binding; protein homodimerization activity; protein N-terminus binding; single-stranded DNA binding Biological Process: negative regulation of apoptosis; nucleotide-excision repair, DNA incision; nucleotide-excision repair, DNA incision, 3'-to lesion; nucleotide-excision repair, DNA incision, 5'-to lesion; nucleotide-excision repair, preincision complex assembly; nucleotide-excision repair, preincision complex stabilization; response to UV; response to UV-C; transcription-coupled nucleotide-excision repair; UV protection Disease: Xeroderma Pigmentosum, Complementation Group G |
| NCBI Summary: | This gene encodes a single-strand specific DNA endonuclease that makes the 3' incision in DNA excision repair following UV-induced damage. The protein may also function in other cellular processes, including RNA polymerase II transcription, and transcription-coupled DNA repair. Mutations in this gene cause xeroderma pigmentosum complementation group G (XP-G), which is also referred to as xeroderma pigmentosum VII (XP7), a skin disorder characterized by hypersensitivity to UV light and increased susceptibility for skin cancer development following UV exposure. Some patients also develop Cockayne syndrome, which is characterized by severe growth defects, mental retardation, and cachexia. Read-through transcription exists between this gene and the neighboring upstream BIVM (basic, immunoglobulin-like variable motif containing) gene. [provided by RefSeq, Feb 2011] |
| UniProt Code: | P28715 |
| NCBI GenInfo Identifier: | 205371791 |
| NCBI Gene ID: | 2073 |
| NCBI Accession: | P28715.3 |
| UniProt Secondary Accession: | P28715,Q5JUS4, Q5JUS5, Q7Z2V3, Q8IZL6, Q8N1B7, Q9HD59 Q9HD60, A6NGT4, |
| UniProt Related Accession: | P28715 |
| Molecular Weight: | 27,259 Da |
| NCBI Full Name: | DNA repair protein complementing XP-G cells |
| NCBI Synonym Full Names: | ERCC excision repair 5, endonuclease |
| NCBI Official Symbol: | ERCC5 |
| NCBI Official Synonym Symbols: | XPG; UVDR; XPGC; COFS3; ERCM2; ERCC5-201 |
| NCBI Protein Information: | DNA repair protein complementing XP-G cells |
| UniProt Protein Name: | DNA repair protein complementing XP-G cells |
| UniProt Synonym Protein Names: | DNA excision repair protein ERCC-5; Xeroderma pigmentosum group G-complementing protein |
| Protein Family: | DNA repair protein |
| UniProt Gene Name: | ERCC5 |
| UniProt Entry Name: | ERCC5_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-ERCC5 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.)
