Description
Rat Aryl hydrocarbon receptor (Ahr) ELISA Kit
The Rat Aryl Hydrocarbon Receptor (AHR) ELISA Kit is specifically designed for the accurate measurement of AHR levels in rat serum, plasma, and cell culture supernatants. This kit offers high sensitivity and specificity, guaranteeing precise and consistent results for a variety of research purposes.The aryl hydrocarbon receptor is a key transcription factor that plays a critical role in mediating the biological effects of environmental toxins and endogenous ligands. Dysregulation of the AHR pathway has been linked to various diseases, such as cancer, autoimmune disorders, and metabolic conditions, making it a valuable biomarker for investigating these health issues and developing potential treatments.
Overall, the Rat AHR ELISA Kit provides researchers with a reliable tool to study the role of AHR in physiological and pathological processes, advancing our understanding of complex diseases and facilitating the development of innovative therapies.
| Product Name: | Rat Aryl hydrocarbon receptor (Ahr) ELISA Kit |
| SKU: | RTEB0903 |
| Size: | 96T |
| Target: | Rat Aryl hydrocarbon receptor (Ahr) |
| Synonyms: | Ah receptor |
| Assay Type: | Sandwich |
| Detection Method: | ELISA |
| Reactivity: | Rat |
| Detection Range: | 0.156-10ng/mL |
| Sensitivity: | 0.085ng/mL |
| Intra CV: | 5.7% | ||||||||||||||||||||
| Inter CV: | 8.4% | ||||||||||||||||||||
| Linearity: |
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| Recovery: |
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| Function: | Ligand-activated transcriptional activator. Binds to the XRE promoter region of genes it activates. Activates the expression of multiple phase I and II xenobiotic chemical metabolizing enzyme genes (such as the CYP1A1 gene). Mediates biochemical and toxic effects of halogenated aromatic hydrocarbons. Involved in cell-cycle regulation. Likely to play an important role in the development and maturation of many tissues. Regulates the circadian clock by inhibiting the basal and circadian expression of the core circadian component PER1. Inhibits PER1 by repressing the CLOCK-ARNTL/BMAL1 heterodimer mediated transcriptional activation of PER1. |
| Uniprot: | P41738 |
| Sample Type: | Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids |
| Specificity: | Natural and recombinant rat Aryl hydrocarbon receptor |
| Sub Unit: | Efficient DNA binding requires dimerization with another bHLH protein. In the nucleus, heterodimer of AHR and ARNT. Interacts with coactivators including SRC-1, RIP140 and NOCA7, and with the corepressor SMRT. Interacts with MYBBP1A, NEDD8 and IVNS1ABP. Interacts with ARNTL/BMAL1 (By similarity). Interacts with HSP90AB1. |
| Research Area: | Cancer |
| Subcellular Location: | Cytoplasm Nucleus Initially cytoplasmic; upon binding with ligand and interaction with a HSP90, it translocates to the nucleus. |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | AHR: a nuclear receptor for aryl hydrocarbons involved in xenobiotic metabolism, cell cycle regulation, and development in response to both endogenous and environmental signals. AhR was initially identified as a receptor for dioxins, which are environmental pollutants generated by waste incineration and other industrial processes. AhR ligands include polycyclic aromatic hydrocarbons, including the carcinogen benzo(a)pyrene and other components of cigarette smoke. Naturally occurring AhR ligands include flavonoids, which are aromatic plant secondary compounds commonly found in vegetables and fruits. Cytoplasmic aryl hydrocarbon receptors are found in protein complexes with heat shock proteins. Upon ligand binding, AhR dissociates from heat shock proteins and translocate to the nucleus where it dimerizes with AhR nuclear translocator (ARNT, HIF-1b). The AhR/ARNT heterodimer binds to nuclear xenobiotic response elements to control the expression of genes associated with xenobiotic metabolism, including several cytochrome P450 genes. AhR is ubiquitously expressed and is thought to play a role in regulation of cell proliferation and differentiation, cytokine expression, and xenobiotic metabolism. Research studies link AhR activity with the control of regulatory T-cell and T-helper 17 cell differentiation, regulation of the inflammatory response, and the onset of lung cancer. |
| UniProt Protein Details: | Protein type:Transcription factor; Nuclear receptor; DNA-binding Cellular Component: cytoplasm; cytosol; nucleoplasm; nucleus Molecular Function:aryl hydrocarbon receptor activity; DNA binding; Hsp90 protein binding; ligand-dependent nuclear receptor activity; protein binding; protein dimerization activity; protein heterodimerization activity; RNA polymerase II transcription factor activity, enhancer binding; sequence-specific DNA binding; transcription factor activity; transcription factor binding Biological Process: apoptosis; B cell homeostasis; blood circulation; blood vessel development; blood vessel morphogenesis; blood vessel remodeling; camera-type eye development; cell morphogenesis; circadian regulation of gene expression; embryonic hemopoiesis; gland development; immune system process; intracellular receptor-mediated signaling pathway; liver development; lymphocyte homeostasis; negative regulation of systemic arterial blood pressure; negative regulation of transcription from RNA polymerase II promoter; negative regulation of transcription, DNA-dependent; negative regulation of vasoconstriction; ovarian follicle development; patterning of blood vessels; positive regulation of cell size; positive regulation of growth rate; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; positive regulation of transcriptional preinitiation complex assembly; post-embryonic hemopoiesis; prostate gland development; regulation of B cell proliferation; regulation of blood vessel size; regulation of gene expression; regulation of transcription from RNA polymerase II promoter; regulation of transcription, DNA-dependent; reproductive structure development; response to estradiol stimulus; response to organic cyclic substance; response to toxin; response to xenobiotic stimulus; smooth muscle development; spleen development; T cell homeostasis; transcription from RNA polymerase II promoter |
| NCBI Summary: | binds 3-methylcholanthrene and other aromatic hydrocarbons; may play a role in bone formation [RGD, Feb 2006] |
| UniProt Code: | P41738 |
| NCBI GenInfo Identifier: | 29337195 |
| NCBI Gene ID: | 25690 |
| NCBI Accession: | P41738.2 |
| UniProt Secondary Accession: | P41738,O88930, O89105, |
| UniProt Related Accession: | P41738 |
| Molecular Weight: | 69,332 Da |
| NCBI Full Name: | Aryl hydrocarbon receptor |
| NCBI Synonym Full Names: | aryl hydrocarbon receptor |
| NCBI Official Symbol: | Ahr |
| NCBI Protein Information: | aryl hydrocarbon receptor |
| UniProt Protein Name: | Aryl hydrocarbon receptor |
| Protein Family: | Aldehyde reductase |
| UniProt Gene Name: | Ahr |
| UniProt Entry Name: | AHR_RAT |
| Component | Quantity (96 Assays) | Storage |
| 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 | - |
Other materials and equipment required:
- 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
*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
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.
| Step | |
| 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. |
When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.
| Sample Type | Protocol |
| Serum | 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. |
| Plasma | 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. |
| Urine & Cerebrospinal Fluid | 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. |
| Cell culture supernatant | 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. |
| Cell lysates | 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. |
| Tissue homogenates | 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. |
| Tissue lysates | 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. |
| Breast Milk | 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. |
