The Rat Beta-2 Adrenergic Receptor (ADRB2) ELISA Kit is specifically designed for the accurate measurement of ADRB2 levels in rat serum, plasma, and cell culture supernatants. This kit offers high sensitivity and specificity, ensuring reliable and reproducible results for a variety of research applications.The ADRB2 receptor is a key protein involved in mediating responses to adrenaline and other stress hormones in the body. Dysregulation of ADRB2 signaling has been implicated in various diseases, including asthma, heart disease, and obesity.
Therefore, the measurement of ADRB2 levels using this ELISA kit can provide valuable insights into the role of ADRB2 in these conditions and aid in the development of novel therapeutic strategies.Overall, the Rat Beta-2 Adrenergic Receptor ELISA Kit is a powerful tool for researchers studying ADRB2 signaling pathways and their implications in various disease states.
Beta-adrenergic receptors mediate the catecholamine-induced activation of adenylate cyclase through the action of G proteins. The beta-2-adrenergic receptor binds epinephrine with an approximately 30-fold greater affinity than it does norepinephrine.
Uniprot:
P10608
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant rat Beta-2 adrenergic receptor
Sub Unit:
Binds SLC9A3R1 and GPRASP1. Interacts with ARRB1 and ARRB2. Interacts with SRC, USP20 and USP33. Interacts with VHL; the interaction, which is increased on hydroxylation of ADRB2, ubiquitinates ADRB2 leading to its degradation. Interacts with EGLN3; the interaction hydroxylates ADRB2 facilitating VHL-E3 ligase-mediated ubiquitination. Interacts (via PDZ-binding motif) with SNX27 (via PDZ domain); the interaction is required when endocytosed to prevent degradation in lysosomes and promote recycling to the plasma membrane. Interacts with CNIH4. Interacts with ARRDC3.
Research Area:
Signal Transduction
Subcellular Location:
Cell membrane Multi-pass membrane protein Early endosome Colocalizes with VHL on cell membranes. Activated receptors are internalized into endosomes prior to their degradation in lysosomes.
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
ADRB2: Beta-adrenergic receptors mediate the catecholamine- induced activation of adenylate cyclase through the action of G proteins. The beta-2-adrenergic receptor binds epinephrine with an approximately 30-fold greater affinity than it does norepinephrine. Belongs to the G-protein coupled receptor 1 family. Adrenergic receptor subfamily. ADRB2 sub-subfamily.Protein type: GPCR, family 1; Membrane protein, integral; Receptor, GPCR; Membrane protein, multi-passCellular Component: intracellular membrane-bound organelle; integral to plasma membrane; dendrite; integral to membrane; dendritic spine; caveola; membrane; axon; apical plasma membrane; cytoplasm; plasma membrane; nucleus; endosome; sarcolemma; receptor complexMolecular Function: protein homodimerization activity; G-protein alpha-subunit binding; norepinephrine binding; drug binding; epinephrine binding; PDZ domain binding; ionotropic glutamate receptor binding; protein binding; potassium channel regulator activity; enzyme binding; protein complex binding; dopamine binding; beta2-adrenergic receptor activity; B2 bradykinin receptor binding; adenylate cyclase bindingBiological Process: diet induced thermogenesis; wound healing; positive regulation of apoptosis; regulation of vasodilation; positive regulation of potassium ion transport; regulation of systemic arterial blood pressure by norepinephrine-epinephrine; female pregnancy; positive regulation of vasodilation; G-protein signaling, adenylate cyclase activating pathway; positive regulation of MAPKKK cascade; diaphragm contraction; negative regulation of smooth muscle contraction; positive regulation of cell proliferation; negative regulation of ossification; associative learning; aging; bone resorption; arrestin mediated desensitization of G-protein coupled receptor protein signaling pathway; regulation of smooth muscle contraction; synaptic transmission, glutamatergic; receptor-mediated endocytosis; negative regulation of multicellular organism growth; adenylate cyclase activation; regulation of calcium ion transport; response to testosterone stimulus; regulation of sodium ion transport; liver development; positive regulation of bone mineralization; G-protein coupled receptor protein signaling pathway; negative regulation of angiogenesis; positive regulation of the force of heart contraction by epinephrine; positive regulation of ATPase activity; response to estrogen stimulus; positive regulation of protein ubiquitination; negative regulation of inflammatory response; heat generation; brown fat cell differentiation; response to hypoxia; positive regulation of transcription from RNA polymerase II promoter; response to cold; regulation of sensory perception of pain; response to progesterone stimulus; positive regulation of skeletal muscle growth; regulation of excitatory postsynaptic membrane potential; positive regulation of heart contraction; norepinephrine-epinephrine vasodilation involved in regulation of systemic arterial blood pressure
UniProt Protein Details:
NCBI Summary:
binds adrenergic receptor agonist zinterol; plays a role in G-protein coupled receptor regulation of calcium channel current [RGD, Feb 2006]
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.
