Epigenetics and Nuclear Signaling
APEX1 Colorimetric Cell-Based ELISA
- SKU:
- CBCAB00091
- Product Type:
- ELISA Kit
- ELISA Type:
- Cell Based
- Research Area:
- Epigenetics and Nuclear Signaling
- Reactivity:
- Human
- Reactivity:
- Mouse
- Reactivity:
- Rat
- Detection Method:
- Colorimetric
Description
| Product Name: | APEX1 Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB00091 |
| ELISA Type: | Cell-Based |
| Target: | APEX1 |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The APEX1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect APEX1 protein expression profile in cells. The kit can be used for measuring the relative amounts of APEX1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on APEX1.
Qualitative determination of APEX1 concentration is achieved by an indirect ELISA format. In essence, APEX1 is captured by APEX1-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: 328, UniProt ID: P27695, OMIM: 107748, Unigene: Hs.73722 |
| Gene Symbol: | APEX1 |
| Sub Type: | None |
| UniProt Protein Function: | APE1: a multifunctional enzyme that plays a central role in the cellular response to oxidative stress including DNA repair and redox regulation of transcriptional factors. Binds DNA and RNA. Functions as an apurinic/apyrimidinic (AP) endodeoxyribonuclease in the DNA base excision repair (BER) pathway, a 3'-5' exoribonuclease for mismatched deoxyribonucleotides at the 3' termini of nicked or gapped DNA molecules, and a DNA 3' phosphodiesterase capable of removing lesions (such as phosphoglycolate) blocking the 3' side of DNA strand breaks. Is a loading factor for POLB onto non-incised AP sites in DNA, stimulates the 5'-terminal deoxyribose 5'- phosphate (dRp) excision activity of POLB, and involved in the DNA cleavage step of class switch recombination (CSR). Possesses reversible nuclear redox activity to regulate DNA binding affinity and transcriptional activity of transcriptional factors by controlling the redox status of their DNA-binding domain, such as the FOS/JUN AP-1 complex after exposure to IR. Binds to negative calcium response elements (nCaREs). Stimulates the YBX1-mediated MDR1 promoter activity, when acetylated at Lys-6 and Lys-7, leading to drug resistance. Is an endoribonuclease involved in the control of single-stranded RNA metabolism. Plays a role in regulating MYC mRNA turnover. In association with NMD1, plays a role in the rRNA quality control process during cell cycle progression. Interacts with SIRT1; the interaction is increased in the context of genotoxic stress. Interacts with HDAC1, HDAC2 and HDAC3; the interactions are not dependent on the APEX1 acetylation status. Up-regulated in presence of reactive oxygen species (ROS), like bleomycin, H2O2 and phenazine methosulfate. NPM1 stimulates endodeoxyribonuclease activity on double-stranded DNA with AP sites, but inhibits endoribonuclease activity on single-stranded RNA containing AP sites. Belongs to the DNA repair enzymes AP/ExoA family. |
| UniProt Protein Details: | Protein type:Endoplasmic reticulum; Deoxyribonuclease; Hydrolase; Transcription, coactivator/corepressor; Nuclear receptor co-regulator; Lyase; Nucleolus; EC 4.2.99.18; DNA-binding; DNA repair, damage Chromosomal Location of Human Ortholog: 14q11.2 Cellular Component: centrosome; cytoplasm; endoplasmic reticulum; mitochondrion; nuclear chromosome, telomeric region; nuclear speck; nucleolus; nucleoplasm; nucleus; perinuclear region of cytoplasm; ribosome Molecular Function:3'-5' exonuclease activity; chromatin DNA binding; damaged DNA binding; DNA binding; DNA-(apurinic or apyrimidinic site) lyase activity; double-stranded DNA specific 3'-5' exodeoxyribonuclease activity; double-stranded DNA specific exodeoxyribonuclease activity; double-stranded telomeric DNA binding; endodeoxyribonuclease activity; endonuclease activity; metal ion binding; oxidoreductase activity; phosphodiesterase I activity; phosphoric diester hydrolase activity; protein binding; ribonuclease H activity; site-specific endodeoxyribonuclease activity, specific for altered base; transcription coactivator activity; transcription corepressor activity; uracil DNA N-glycosylase activity Biological Process: base-excision repair; base-excision repair, base-free sugar-phosphate removal; DNA repair; positive regulation of DNA repair; regulation of mRNA stability; telomere maintenance |
| NCBI Summary: | Apurinic/apyrimidinic (AP) sites occur frequently in DNA molecules by spontaneous hydrolysis, by DNA damaging agents or by DNA glycosylases that remove specific abnormal bases. AP sites are pre-mutagenic lesions that can prevent normal DNA replication so the cell contains systems to identify and repair such sites. Class II AP endonucleases cleave the phosphodiester backbone 5' to the AP site. This gene encodes the major AP endonuclease in human cells. Splice variants have been found for this gene; all encode the same protein. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P27695 |
| NCBI GenInfo Identifier: | 113984 |
| NCBI Gene ID: | 328 |
| NCBI Accession: | P27695.2 |
| UniProt Secondary Accession: | P27695,Q969L5, Q99775, |
| UniProt Related Accession: | P27695 |
| Molecular Weight: | 35,555 Da |
| NCBI Full Name: | DNA-(apurinic or apyrimidinic site) lyase |
| NCBI Synonym Full Names: | apurinic/apyrimidinic endodeoxyribonuclease 1 |
| NCBI Official Symbol: | APEX1 |
| NCBI Official Synonym Symbols: | APE; APX; APE1; APEN; APEX; HAP1; REF1 |
| NCBI Protein Information: | DNA-(apurinic or apyrimidinic site) lyase |
| UniProt Protein Name: | DNA-(apurinic or apyrimidinic site) lyase |
| UniProt Synonym Protein Names: | APEX nuclease; APEN |
| Protein Family: | DNA-(apurinic or apyrimidinic site) lyase |
| UniProt Gene Name: | APEX1 |
| UniProt Entry Name: | APEX1_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-APEX1 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.)
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