Rat DNA-(apurinic or apyrimidinic site) lyase (Apex1) ELISA Kit
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- SKU:
- RTEB1285
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- P43138
- Range:
- 0.156-10 ng/ml
- ELISA Type:
- Sandwich
- Reactivity:
- Rat
Frequently bought together:
Description
| 商品名: | Rat DNA-(apurinic or apyrimidinic site) lyase (Apex1) ELISA Kit |
| 製品コード: | RTEB1285 |
| エイリアス: | DNA-(apurinic or apyrimidinic site) lyase, APEX nuclease, APEN, Apurinic-apyrimidinic endonuclease 1, AP endonuclease 1, REF-1, Redox factor-1, Apex1, Ape, Apex, Ref1, 3.1.-.- |
| Uniprot: | P43138 |
| 反応性: | Rat |
| 範囲: | 0.156-10 ng/ml |
| 検出方法: | Sandwich |
| サイズ: | 96 Assay |
| 保管所: | Please see kit components below for exact storage details |
| ノート: | For research use only |
| 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.Protein type: Deoxyribonuclease; Lyase; DNA repair, damage; Transcription, coactivator/corepressor; Nucleolus; Hydrolase; DNA-binding; Nuclear receptor co-regulator; EC 4.2.99.18; Endoplasmic reticulumCellular Component: centrosome; cytoplasm; endoplasmic reticulum; mitochondrion; nuclear speck; nucleolus; nucleoplasm; nucleus; perinuclear region of cytoplasm; transcription factor complexMolecular 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; endonuclease activity; endoribonuclease activity; metal ion binding; NF-kappaB binding; oxidoreductase activity; phosphoric diester hydrolase activity; protein binding; protein complex binding; site-specific endodeoxyribonuclease activity, specific for altered base; transcription coactivator activityBiological Process: aging; base-excision repair; cell redox homeostasis; DNA recombination; DNA repair; negative regulation of smooth muscle cell migration; positive regulation of DNA repair; regulation of mRNA stability; regulation of transcription, DNA-dependent; response to drug; response to organic nitrogen; telomere maintenance; transcription, DNA-dependent |
| UniProt Protein Details: | |
| NCBI Summary: | may play roles in DNA repair and in redox homeostasis; regulates Pax8 transcription [RGD, Feb 2006] |
| UniProt Code: | P43138 |
| NCBI GenInfo Identifier: | 13162337 |
| NCBI Gene ID: | 79116 |
| NCBI Accession: | NP_077062.1 |
| UniProt Secondary Accession: | P43138,Q548N9 |
| UniProt Related Accession: | P43138 |
| Molecular Weight: | 35,538 Da |
| NCBI Full Name: | DNA-(apurinic or apyrimidinic site) lyase |
| NCBI Synonym Full Names: | APEX nuclease (multifunctional DNA repair enzyme) 1 |
| NCBI Official Symbol: | Apex1 |
| NCBI Official Synonym Symbols: | APE; Apex; REF-1 |
| 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_RAT |
| 成分 | 額 | 保管所 |
| ELISA Microplate (Dismountable) | 8×12 strips | -20°C |
| Lyophilized Standard | 2 | -20°C |
| Sample Diluent | 20ml | -20°C |
| Assay Diluent A | 10mL | -20°C |
| Assay Diluent B | 10mL | -20°C |
| Detection Reagent A | 120µL | -20°C |
| Detection Reagent B | 120µL | -20°C |
| Wash Buffer | 30mL | 4°C |
| Substrate | 10mL | 4°C |
| Stop Solution | 10mL | 4°C |
| Plate Sealer | 5 | - |
その他の必要な材料と設備：
- Microplate reader with 450 nm wavelength filter
- Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
- Incubator
- Deionized or distilled water
- Absorbent paper
- Buffer resevoir
*ノート: プロトコルは、各バッチ/ロットに固有です。正しい手順については、キットに含まれているプロトコルに従ってください。
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.
| ステップ | プロトコル |
| 1. | Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C. |
| 2. | Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform. |
| 3. | Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper. |
| 4. | Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C. |
| 5. | Repeat the wash process for five times as conducted in step 3. |
| 6. | Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction. |
| 7. | Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. |
| 8. | Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters. |
| 9. | After experiment, store all reagents according to the specified storage temperature respectively until their expiry. |
ELISAアッセイを実施する場合、可能な限り最良の結果を達成するためにサンプルを準備することが重要です。以下に、さまざまなサンプルタイプのサンプルを準備するための手順のリストを示します。
| サンプルタイプ: | プロトコル: |
| 血清: | If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. |
| プラズマ: | Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit. |
| 尿および脳脊髄液: | Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid. |
| 細胞培養上清: | Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately. |
| 細胞溶解物: | Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| 組織ホモジネート: | The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C. |
| 組織溶解物: | Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| 母乳: | Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles. |