Human Immunology ELISA Kits 9

Human Superoxide dismutase [Mn], mitochondrial (SOD2) ELISA Kit

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SKU:
HUEB2264
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P04179
Range:
31.2-2000 pg/mL
ELISA Type:
Sandwich
Synonyms:
SOD2, Superoxide dismutase [Mn], mitochondrial
Reactivity:
Human

Description

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Product Name:Human Superoxide dismutase [Mn], mitochondrial (SOD2) ELISA Kit
Product Code:HUEB2264
Alias:Superoxide dismutase [Mn], mitochondrial, SOD2, 1.15.1.1
Uniprot:P04179
Reactivity:Human
Range:31.2-2000 pg/mL
Detection Method:Sandwich
Size:96 Assay
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function:SOD2: Destroys superoxide anion radicals which are normally produced within the cells and which are toxic to biological systems. Genetic variation in SOD2 is associated with susceptibility to microvascular complications of diabetes type 6 (MVCD6). These are pathological conditions that develop in numerous tissues and organs as a consequence of diabetes mellitus. They include diabetic retinopathy, diabetic nephropathy leading to end-stage renal disease, and diabetic neuropathy. Diabetic retinopathy remains the major cause of new- onset blindness among diabetic adults. It is characterized by vascular permeability and increased tissue ischemia and angiogenesis. Belongs to the iron/manganese superoxide dismutase family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Mitochondrial; EC 1.15.1.1; Oxidoreductase

Chromosomal Location of Human Ortholog: 6q25.3

Cellular Component: mitochondrion; mitochondrial matrix; mitochondrial inner membrane

Molecular Function:identical protein binding; DNA binding; manganese ion binding; superoxide dismutase activity; oxygen binding

Biological Process: oxygen homeostasis; positive regulation of nitric oxide biosynthetic process; removal of superoxide radicals; heart development; locomotory behavior; response to lipopolysaccharide; response to L-ascorbic acid; post-embryonic development; protein homotetramerization; negative regulation of cell proliferation; response to selenium ion; glutathione metabolic process; regulation of mitochondrial membrane potential; acetylcholine vasodilation involved in regulation of systemic arterial blood pressure; regulation of catalytic activity; regulation of blood pressure; response to gamma radiation; hemopoiesis; negative regulation of neuron apoptosis; response to axon injury; response to electrical stimulus; response to drug; erythrophore differentiation; release of cytochrome c from mitochondria; response to superoxide; superoxide metabolic process; liver development; negative regulation of fat cell differentiation; response to manganese ion; regulation of transcription from RNA polymerase II promoter; response to silicon dioxide; response to reactive oxygen species; iron ion homeostasis; response to hyperoxia; response to cadmium ion; response to hydrogen peroxide; DNA damage response, signal transduction resulting in induction of apoptosis; age-dependent response to reactive oxygen species; detection of oxygen; response to zinc ion; negative regulation of fibroblast proliferation; response to hypoxia; neuron development; response to activity; response to cold; induction of apoptosis by oxidative stress; superoxide release; hydrogen peroxide biosynthetic process

Disease: Microvascular Complications Of Diabetes, Susceptibility To, 6

NCBI Summary:This gene is a member of the iron/manganese superoxide dismutase family. It encodes a mitochondrial protein that forms a homotetramer and binds one manganese ion per subunit. This protein binds to the superoxide byproducts of oxidative phosphorylation and converts them to hydrogen peroxide and diatomic oxygen. Mutations in this gene have been associated with idiopathic cardiomyopathy (IDC), premature aging, sporadic motor neuron disease, and cancer. Alternate transcriptional splice variants, encoding different isoforms, have been characterized. [provided by RefSeq, Jul 2008]
UniProt Code:P04179
NCBI GenInfo Identifier:134665
NCBI Gene ID:6648
NCBI Accession:P04179.2
UniProt Secondary Accession:P04179,P78434, Q16792, Q5TCM1, Q96EE6, Q9P2Z3, B2R7R1 B3KUK2, B4DL20, B4E3K9, E1P5A9,
UniProt Related Accession:P04179
Molecular Weight:222
NCBI Full Name:Superoxide dismutase
NCBI Synonym Full Names:superoxide dismutase 2, mitochondrial
NCBI Official Symbol:SOD2  
NCBI Official Synonym Symbols:IPOB; MNSOD; MVCD6  
NCBI Protein Information:superoxide dismutase [Mn], mitochondrial; superoxide dismutase [Mn], mitochondrial; indophenoloxidase B; Mn superoxide dismutase; mangano-superoxide dismutase; manganese-containing superoxide dismutase
UniProt Protein Name:Superoxide dismutase [Mn], mitochondrial
Protein Family:Superoxide dismutase
UniProt Gene Name:SOD2  
UniProt Entry Name:SODM_HUMAN
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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