The Mouse Mineralocorticoid Receptor (NR3C2) ELISA Kit is a powerful tool for accurately measuring levels of mineralocorticoid receptor in mouse serum, plasma, and cell culture supernatants. This kit offers high sensitivity and specificity, ensuring precise and consistent results for a variety of research purposes.The mineralocorticoid receptor, also known as NR3C2, plays a critical role in regulating water and electrolyte balance in the body. Dysregulation of the mineralocorticoid receptor has been implicated in various conditions such as hypertension, heart failure, and kidney disease, making it a valuable biomarker for studying these diseases and developing potential therapeutic interventions.
With its reliable performance and ease of use, the Mouse Mineralocorticoid Receptor (NR3C2) ELISA Kit is an essential tool for researchers studying the physiological and pathological roles of the mineralocorticoid receptor in mouse models.
Nuclear receptor subfamily 3 group C member 2, MR, Mlr
Assay Type:
Sandwich
Detection Method:
ELISA
Reactivity:
Mouse
Detection Range:
0.156-10ng/mL
Sensitivity:
0.082ng/mL
Intra CV:
6.9%
Inter CV:
8.8%
Linearity:
Sample
1:2
1:4
1:8
1:16
Serum(N=5)
99-109%
92-102%
104-114%
98-108%
EDTA Plasma(N=5)
86-95%
99-108%
101-111%
106-115%
Heparin Plasma(N=5)
105-117%
108-117%
102-112%
100-109%
Recovery:
Sample Type
Average(%)
Recovery Range(%)
Serum
85
80-91
Plasma
87
81-93
Function:
Receptor for both mineralocorticoids (MC) such as aldosterone and glucocorticoids (GC) such as corticosterone or cortisol. Binds to mineralocorticoid response elements (MRE) and transactivates target genes. The effect of MC is to increase ion and water transport and thus raise extracellular fluid volume and blood pressure and lower potassium levels.
Uniprot:
Q8VII8
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant mouse Mineralocorticoid receptor
Sub Unit:
Heteromultimeric cytoplasmic complex with HSP90, HSP70, and FKBP4, in the absence of ligand. After ligand binding, it translocates to the nucleus and binds to DNA as a homodimer and as a heterodimer with NR3C1. Binds the coactivator NCOA2. May interact with HSD11B2 in the absence of ligand. Binds the coactivators NCOA1, TIF1 and NRIP1.
Research Area:
Epigenetics
Subcellular Location:
Cytoplasm Nucleus Endoplasmic reticulum membrane Peripheral membrane protein Cytoplasmic and nuclear in the absence of ligand; nuclear after ligand-binding. When bound to HSD11B2, it is found associated with the endoplasmic reticulum membrane (By similarity).
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
MCR: Receptor for both mineralocorticoids (MC) such as aldosterone and glucocorticoids (GC) such as corticosterone or cortisol. Binds to mineralocorticoid response elements (MRE) and transactivates target genes. The effect of MC is to increase ion and water transport and thus raise extracellular fluid volume and blood pressure and lower potassium levels. Defects in NR3C2 are a cause of autosomal dominant pseudohypoaldosteronism type I (AD-PHA1). PHA1 is characterized by urinary salt wasting, resulting from target organ unresponsiveness to mineralocorticoids. There are 2 forms of PHA1: the autosomal dominant form that is mild, and the recessive form which is more severe and due to defects in any of the epithelial sodium channel subunits. In AD-PHA1 the target organ defect is confined to kidney. Clinical expression can vary from asymptomatic to moderate. It may be severe at birth, but symptoms remit with age. Familial and sporadic cases have been reported. Defects in NR3C2 are a cause of early-onset hypertension with severe exacerbation in pregnancy (EOHSEP). Inheritance is autosomal dominant. The disease is characterized by the onset of severe hypertension before the age of 20, and by suppression of aldosterone secretion. Belongs to the nuclear hormone receptor family. NR3 subfamily. 4 isoforms of the human protein are produced by alternative splicing.Protein type: DNA-binding; Nuclear receptorCellular Component: cytoplasm; endoplasmic reticulum; membrane; nucleus; receptor complexMolecular Function: DNA binding; double-stranded DNA binding; hormone binding; lipid binding; metal ion binding; mineralocorticoid receptor activity; protein binding; protein heterodimerization activity; protein homodimerization activity; sequence-specific DNA binding; steroid binding; steroid hormone receptor activity; transcription factor activity; zinc ion bindingBiological Process: cellular sodium ion homeostasis; excretion; mineralocorticoid receptor signaling pathway; regulation of cell proliferation; regulation of transcription, DNA-dependent; steroid hormone mediated signaling; transcription, DNA-dependent
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.
