Product Name:	Human Superoxide dismutase [Cu-Zn] (SOD1) ELISA Kit
SKU:	HUEB0338
Size:	96T
Target:	Human Superoxide dismutase [Cu-Zn] (SOD1)
Synonyms:	Superoxide dismutase 1, hSod1
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.156-10ng/mL
Sensitivity:	0.078ng/mL

Intra CV:

5.1%

Inter CV:

8.6%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	82-82%	98-109%	87-97%	104-113%
EDTA Plasma(N=5)	80-90%	94-107%	101-110%	93-103%
Heparin Plasma(N=5)	107-119%	104-116%	105-116%	104-114%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	88	82-94
Plasma	90	84-96

Function:

Destroys radicals which are normally produced within the cells and which are toxic to biological systems.

Uniprot:	P00441
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Superoxide dismutase [Cu-Zn]
Sub Unit:	Homodimer; non-disulfide linked. Homodimerization may take place via the ditryptophan cross-link at Trp-33. The pathogenic variants ALS1 Arg-38, Arg-47, Arg-86 and Ala-94 interact with RNF19A, whereas wild-type protein does not. The pathogenic variants ALS1 Arg-86 and Ala-94 interact with MARCH5, whereas wild-type protein does not.
Research Area:	Neurosciences
Subcellular Location:	Cytoplasm Mitochondrion Nucleus Predominantly cytoplasmic; the pathogenic variants ALS1 Arg-86 and Ala-94 gradually aggregates and accumulates in mitochondria.
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	SOD1: Destroys radicals which are normally produced within the cells and which are toxic to biological systems. Homodimer; non-disulfide linked. Homodimerization may take place via the ditryptophan cross-link at Trp-33. The pathogenic variants ALS1 Arg-38, Arg-47, Arg-86 and Ala-94 interact with RNF19A, whereas wild-type protein does not. The pathogenic variants ALS1 Arg-86 and Ala-94 interact with MARCH5, whereas wild-type protein does not. Belongs to the Cu-Zn superoxide dismutase family.
UniProt Protein Details:	Protein type:Oxidoreductase; Mitochondrial; EC 1.15.1.1; Nuclear receptor co-regulator; Apoptosis Chromosomal Location of Human Ortholog: 21q22.11 Cellular Component: dendrite cytoplasm; extracellular space; protein complex; mitochondrion; extracellular region; mitochondrial intermembrane space; cytosol; nucleoplasm; extracellular matrix; cell soma; mitochondrial matrix; cytoplasm; plasma membrane; peroxisome; cytoplasmic vesicle; nucleus Molecular Function:identical protein binding; protein binding; protein homodimerization activity; copper ion binding; zinc ion binding; chaperone binding; superoxide dismutase activity; Rac GTPase binding; protein phosphatase 2B binding Biological Process: positive regulation of catalytic activity; activation of MAPK activity; positive regulation of apoptosis; cellular iron ion homeostasis; myeloid cell homeostasis; retrograde axon cargo transport; response to antibiotic; muscle maintenance; retinal homeostasis; glutathione metabolic process; regulation of mitochondrial membrane potential; neurofilament cytoskeleton organization and biogenesis; positive regulation of superoxide release; negative regulation of neuron apoptosis; placenta development; positive regulation of cytokine production; response to drug; platelet activation; cell aging; regulation of organ growth; transmission of nerve impulse; response to reactive oxygen species; response to ethanol; heart contraction; response to heat; superoxide release; relaxation of vascular smooth muscle; removal of superoxide radicals; locomotory behavior; response to organic substance; sensory perception of sound; platelet degranulation; ovarian follicle development; regulation of blood pressure; response to axon injury; auditory receptor cell stereocilium organization and biogenesis; anterograde axon cargo transport; negative regulation of cholesterol biosynthetic process; response to nutrient levels; response to superoxide; thymus development; regulation of T cell differentiation in the thymus; response to amphetamine; superoxide metabolic process; myelin maintenance in the peripheral nervous system; regulation of multicellular organism growth; response to hydrogen peroxide; response to copper ion; spermatogenesis; regulation of protein kinase activity; blood coagulation; embryo implantation; hydrogen peroxide biosynthetic process Disease: Amyotrophic Lateral Sclerosis 1
NCBI Summary:	The protein encoded by this gene binds copper and zinc ions and is one of two isozymes responsible for destroying free superoxide radicals in the body. The encoded isozyme is a soluble cytoplasmic protein, acting as a homodimer to convert naturally-occuring but harmful superoxide radicals to molecular oxygen and hydrogen peroxide. The other isozyme is a mitochondrial protein. Mutations in this gene have been implicated as causes of familial amyotrophic lateral sclerosis. Rare transcript variants have been reported for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	P00441
NCBI GenInfo Identifier:	134611
NCBI Gene ID:	6647
NCBI Accession:	P00441.2
UniProt Related Accession:	P00441
Molecular Weight:
NCBI Full Name:	Superoxide dismutase
NCBI Synonym Full Names:	superoxide dismutase 1
NCBI Official Symbol:	SOD1
NCBI Official Synonym Symbols:	ALS; SOD; ALS1; IPOA; hSod1; HEL-S-44; homodimer
NCBI Protein Information:	superoxide dismutase [Cu-Zn]
UniProt Protein Name:	Superoxide dismutase [Cu-Zn]
UniProt Synonym Protein Names:	Superoxide dismutase 1; hSod1
Protein Family:	Sodium channel
UniProt Gene Name:	SOD1
UniProt Entry Name:	SODC_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.

ELISA	Antibodies
Human SOD1 ELISA Kit	Anti-SOD1 Antibody (CAB0274)
Human SOD1 (Superoxide Dismutase 1, Soluble) ELISA Kit (HUES02204)	Anti-SOD1 Antibody (CAB12537)
	SOD1 Antibody (PACO01525)
	CACNG5 Antibody (PACO07025)
	SOCS6 Antibody (PACO12391)