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Mouse NADPH oxidase 4 (Nox4) ELISA Kit (MOEB1357)

SKU:
MOEB1357
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q9JHI8
Range:
0.312-20 ng/mL
ELISA Type:
Sandwich
Synonyms:
NOX4, KOX-1, RENOX
Reactivity:
Mouse
€649
Frequently bought together:

Description

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Mouse NADPH oxidase 4 (Nox4) ELISA Kit

The Mouse NADPH Oxidase 4 (NOX4) ELISA Kit is specifically designed for the quantitative detection of NOX4 levels in mouse serum, plasma, and cell culture supernatants. This kit offers high accuracy and precision, ensuring reliable and consistent results for various research applications.NOX4 is a key enzyme involved in the production of reactive oxygen species (ROS) and has been implicated in various physiological and pathological processes, including oxidative stress, inflammation, and cardiovascular diseases.

Monitoring NOX4 levels can provide valuable insights into the role of ROS in disease development and progression, making this kit essential for studying NOX4 biology and potential therapeutic interventions.With its high sensitivity and specificity, the Mouse NOX4 ELISA Kit is a valuable tool for researchers looking to investigate the role of NOX4 in various disease conditions and develop novel treatment strategies. Trust in this kit to deliver accurate and dependable results for your research needs.

Product Name:Mouse NADPH oxidase 4 (Nox4) ELISA Kit
SKU:MOEB1357
Size:96T
Target:Mouse NADPH oxidase 4 (Nox4)
Synonyms:Kidney oxidase-1, Kidney superoxide-producing NADPH oxidase, Renal NAD(P)H-oxidase, Superoxide-generating NADPH oxidase 4, KOX-1, Renox
Assay Type:Sandwich
Detection Method:ELISA
Reactivity:Mouse
Detection Range:0.312-20ng/mL
Sensitivity:0.156ng/mL
Intra CV:6.8%
Inter CV:8.7%
Linearity:
Sample1:21:41:81:16
Serum(N=5)102-112%109-120%104-104%93-103%
EDTA Plasma(N=5)95-105%89-99%101-111%91-101%
Heparin Plasma(N=5)95-107%111-120%91-101%99-109%
Recovery:
Sample TypeAverage(%)Recovery Range(%)
Serum9690-102
Plasma9892-104
Function:Constitutive NADPH oxidase which generates superoxide intracellularly upon formation of a complex with CYBA/p22phox. Regulates signaling cascades probably through phosphatases inhibition. May function as an oxygen sensor regulating the KCNK3/TASK-1 potassium channel and HIF1A activity. May regulate insulin signaling cascade. May play a role in apoptosis, bone resorption and lipolysaccharide-mediated activation of NFKB.
Uniprot:Q9JHI8
Sample Type:Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:Natural and recombinant mouse NADPH oxidase 4
Sub Unit:Interacts with, relocalizes and stabilizes CYBA/p22phox. Interacts with TLR4. Interacts with protein disulfide isomerase.
Research Area:Cardiovascular
Subcellular Location:Endoplasmic reticulum membrane Multi-pass membrane protein Cell junction Focal adhesion Cell membrane May localize to plasma membrane and focal adhesions.
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function:NOX4: Constitutive NADPH oxidase which generates superoxide intracellularly upon formation of a complex with CYBA/p22phox. Regulates signaling cascades probably through phosphatases inhibition. May function as an oxygen sensor regulating the KCNK3/TASK-1 potassium channel and HIF1A activity. May regulate insulin signaling cascade. May play a role in apoptosis, bone resorption and lipolysaccharide-mediated activation of NFKB. May produce superoxide in the nucleus and play a role in regulating gene expression upon cell stimulation. Isoform 3 is not functional. Isoform 4 displays an increased activity. Isoform 5 and isoform 6 display reduced activity. 7 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Oxidoreductase; Nucleolus; EC 1.6.3.-; Membrane protein, multi-pass; Membrane protein, integral; Cell cycle regulation

Cellular Component: focal adhesion; mitochondrion; membrane; endoplasmic reticulum; perinuclear region of cytoplasm; apical plasma membrane; integral to membrane; stress fiber; plasma membrane; cell junction; NADPH oxidase complex

Molecular Function:NAD(P)H oxidase activity; superoxide-generating NADPH oxidase activity; oxidoreductase activity, acting on NADH or NADPH, with oxygen as acceptor; oxidoreductase activity

Biological Process: positive regulation of apoptosis; cell morphogenesis; superoxide metabolic process; cell aging; homocysteine metabolic process; positive regulation of smooth muscle cell migration; cardiac muscle cell differentiation; positive regulation of protein kinase B signaling cascade; positive regulation of MAP kinase activity; negative regulation of cell proliferation; positive regulation of stress fiber formation; superoxide release; bone resorption

UniProt Code:Q9JHI8
NCBI GenInfo Identifier:81881891
NCBI Gene ID:50490
NCBI Accession:Q9JHI8.1
UniProt Secondary Accession:Q9JHI8,Q3TF39, Q8C3M1, Q8VCA3,
UniProt Related Accession:Q9JHI8
Molecular Weight:
NCBI Full Name:NADPH oxidase 4
NCBI Synonym Full Names:NADPH oxidase 4
NCBI Official Symbol:Nox4
NCBI Official Synonym Symbols:AI648021
NCBI Protein Information:NADPH oxidase 4; KOX-1; kidney oxidase-1; renal NAD(P)H-oxidase; superoxide-generating NADPH oxidase 4; kidney superoxide-producing NADPH oxidase
UniProt Protein Name:NADPH oxidase 4
UniProt Synonym Protein Names:Kidney oxidase-1; KOX-1; Kidney superoxide-producing NADPH oxidase; Renal NAD(P)H-oxidase; Superoxide-generating NADPH oxidase 4
Protein Family:NADPH oxidase
UniProt Gene Name:Nox4
UniProt Entry Name:NOX4_MOUSE
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.

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