The Mouse Corticotropin-Releasing Factor Receptor 1 (CRHR1) ELISA Kit offered by AssayGenie is a valuable tool for the precise measurement of CRHR1 levels in mouse serum, plasma, and cell culture supernatants. With its exceptional sensitivity and specificity, this kit ensures dependable and consistent results, making it an excellent choice for a variety of research applications.CRHR1 is a key receptor involved in the stress response and regulation of the hypothalamic-pituitary-adrenal axis.
Dysregulation of CRHR1 has been implicated in various disorders, including anxiety, depression, and addiction. Therefore, the accurate quantification of CRHR1 levels is essential for investigating these conditions and developing potential therapeutic interventions.Overall, the Mouse CRHR1 ELISA Kit from AssayGenie is a reliable tool for researchers looking to explore the role of CRHR1 in health and disease.
G-protein coupled receptor for CRH (corticotropin-releasing factor) and UCN (urocortin). Has high affinity for CRH and UCN. Ligand binding causes a conformation change that triggers signaling via guanine nucleotide-binding proteins (G proteins) and down-stream effectors, such as adenylate cyclase. Promotes the activation of adenylate cyclase, leading to increased intracellular cAMP levels. Inhibits the activity of the calcium channel CACNA1H. Required for normal embryonic development of the adrenal gland and for normal hormonal responses to stress. Plays a role in the response to anxiogenic stimuli.
Uniprot:
P35347
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant mouse Corticotropin-releasing factor receptor 1
Sub Unit:
Heterodimer; heterodimerizes with GPER1 (By similarity). Interacts (via N-terminal extracellular domain) with CRH and UCN. Interacts with DLG1; this inhibits endocytosis of CRHR1 after agonist binding.
Subcellular Location:
Cell membrane Multi-pass membrane protein Endosome Agonist-binding promotes endocytosis.
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
CRF-R1: a G-protein coupled receptor that specifically binds corticotropin-releasing hormone, a potent mediator of endocrine, autonomic, behavioral, and immune responses to stress. Four splice-variant isoforms have been described.Protein type: Receptor, GPCR; GPCR, family 2; Membrane protein, multi-pass; Membrane protein, integralCellular Component: cell; dendrite; integral to membrane; trans-Golgi network; cytosol; intrinsic to plasma membrane; multivesicular body; cell soma; membrane; apical part of cell; plasma membrane; vesicle; endosomeMolecular Function: G-protein coupled receptor activity; signal transducer activity; corticotropin-releasing hormone binding; transmembrane receptor activity; corticotropin-releasing hormone receptor activity; protein complex binding; G-protein alpha-subunit binding; corticotrophin-releasing factor receptor activity; peptide bindingBiological Process: fear response; hormone-mediated signaling; regulation of synaptic plasticity; behavioral response to ethanol; signal transduction; G-protein signaling, adenylate cyclase activating pathway; elevation of cytosolic calcium ion concentration; behavioral response to pain; general adaptation syndrome, behavioral process; cell surface receptor linked signal transduction; epithelial cell differentiation; behavioral response to cocaine; neuropeptide signaling pathway; positive regulation of cAMP biosynthetic process; visual learning; feeding behavior; response to electrical stimulus; adrenal gland development; positive regulation of mast cell degranulation; memory; G-protein signaling, coupled to cAMP nucleotide second messenger; G-protein coupled receptor protein signaling pathway; adrenocorticotropin hormone secretion; response to hypoxia; negative regulation of epinephrine secretion; G-protein signaling, coupled to IP3 second messenger (phospholipase C activating)
UniProt Protein Details:
NCBI Summary:
The protein encoded by this gene is a G-protein coupled receptor that binds corticotropin-releasing factor (CRH) and urocortin (UCN) with high affinity. The encoded protein upregulates adenylate cyclase activity and intracellular cAMP levels. This protein is essential for the activation of signal transduction pathways that regulate diverse physiological processes including stress, reproduction, immune response, and obesity. Several transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Sep 2015]
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.
