Product Name:	Human OPTN (Optineurin) ELISA Kit
Product Code:	HUFI07420
Size:	96 Assays
Alias:	Optineurin ELISA Kit, E3-14.7K-interacting protein ELISA Kit, FIP-2 ELISA Kit, Huntingtin yeast partner L ELISA Kit, Huntingtin-interacting protein 7 ELISA Kit, HIP-7 ELISA Kit, Huntingtin-interacting protein L ELISA Kit, NEMO-related protein ELISA Kit, Optic neuropathy-inducing protein ELISA Kit, Transcription factor IIIA-interacting protein ELISA Kit, TFIIIA-IntP ELISA Kit, OPTN ELISA Kit, FIP2 ELISA Kit, GLC1E ELISA Kit, HIP7 ELISA Kit, HYPL ELISA Kit, NRP ELISA Kit
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human OPTN (Optineurin) ELISA Kit concentrations in serum plasma and other biological fluids.
Sensitivity:	< 0.094ng/ml
Range:	0.156-10ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human OPTN (Optineurin) ELISA Kit and the recovery rates were calculated by comparing the measured value to the expected amount of Human OPTN (Optineurin) ELISA Kit in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	87-102	93
EDTA plasma(n=5)	85-97	93
heparin plasma(n=5)	90-105	97

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human OPTN (Optineurin) ELISA Kit and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	87-101%	89-98%	87-100%
EDTA plasma(n=5)	91-101%	83-99%	84-101%
UFH plasma(n=5)	81-93%	86-97%	83-94%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	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

UniProt Protein Function:	optineurin: Plays an important role in the maintenance of the Golgi complex, in membrane trafficking, in exocytosis, through its interaction with myosin VI and Rab8. Links myosin VI to the Golgi complex and plays an important role in Golgi ribbon formation. Negatively regulates the induction of IFNB in response to RNA virus infection. Plays a neuroprotective role in the eye and optic nerve. Probably part of the TNF-alpha signaling pathway that can shift the equilibrium toward induction of cell death. May act by regulating membrane trafficking and cellular morphogenesis via a complex that contains Rab8 and hungtingtin (HD). May constitute a cellular target for adenovirus E3 14.7, an inhibitor of TNF-alpha functions, thereby affecting cell death. Defects in OPTN are the cause of primary open angle glaucoma type 1E (GLC1E). Primary open angle glaucoma (POAG) is characterized by a specific pattern of optic nerve and visual field defects. The angle of the anterior chamber of the eye is open, and usually the intraocular pressure is increased. The disease is asymptomatic until the late stages, by which time significant and irreversible optic nerve damage has already taken place. Defects in OPTN are a cause of susceptibility to normal pressure glaucoma (NPG). Defects in OPTN are the cause of amyotrophic lateral sclerosis type 12 (ALS12). It is a neurodegenerative disorder affecting upper motor neurons in the brain and lower motor neurons in the brain stem and spinal cord, resulting in fatal paralysis. Sensory abnormalities are absent. Death usually occurs within 2 to 5 years. The etiology of amyotrophic lateral sclerosis is likely to be multifactorial, involving both genetic and environmental factors. The disease is inherited in 5-10% of the cases. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Inhibitor Chromosomal Location of Human Ortholog: 10p13 Cellular Component: nucleoplasm; Golgi membrane; Golgi apparatus; recycling endosome; perinuclear region of cytoplasm; cytoplasmic membrane-bound vesicle; cytoplasm; autophagic vacuole; trans-Golgi network; cytosol; nucleus Molecular Function:protein C-terminus binding; protein binding; polyubiquitin binding; Rab GTPase binding Biological Process: cell death; macroautophagy; Golgi to plasma membrane protein transport; negative regulation of receptor recycling; protein targeting to Golgi; mitotic cell cycle; negative regulation of I-kappaB kinase/NF-kappaB cascade; signal transduction; defense response to Gram-negative bacterium; G2/M transition of mitotic cell cycle; regulation of I-kappaB kinase/NF-kappaB cascade; Golgi organization and biogenesis Disease: Glaucoma, Normal Tension, Susceptibility To; Glaucoma, Primary Open Angle; Amyotrophic Lateral Sclerosis 12
NCBI Summary:	This gene encodes the coiled-coil containing protein optineurin. Optineurin may play a role in normal-tension glaucoma and adult-onset primary open angle glaucoma. Optineurin interacts with adenovirus E3-14.7K protein and may utilize tumor necrosis factor-alpha or Fas-ligand pathways to mediate apoptosis, inflammation or vasoconstriction. Optineurin may also function in cellular morphogenesis and membrane trafficking, vesicle trafficking, and transcription activation through its interactions with the RAB8, huntingtin, and transcription factor IIIA proteins. Alternative splicing results in multiple transcript variants encoding the same protein. [provided by RefSeq, Jul 2008]
UniProt Code:	Q96CV9
NCBI GenInfo Identifier:	317373403
NCBI Gene ID:	10133
NCBI Accession:	Q96CV9.2
UniProt Secondary Accession:	Q96CV9,Q5T672, Q5T673, Q5T674, Q5T675, Q7LDL9, Q8N562 Q9UET9, Q9UEV4, B3KP00, D3DRS4, D3DRS8,
UniProt Related Accession:	Q96CV9
Molecular Weight:	577
NCBI Full Name:	Optineurin
NCBI Synonym Full Names:	optineurin
NCBI Official Symbol:	OPTN
NCBI Official Synonym Symbols:	NRP; FIP2; HIP7; HYPL; ALS12; GLC1E; TFIIIA-INTP
NCBI Protein Information:	optineurin; FIP-2; HIP-7; nemo-related protein; huntingtin yeast partner L; E3-14.7K-interacting protein; Huntingtin interacting protein L; huntingtin-interacting protein 7; huntingtin-interacting protein L; optic neuropathy-inducing protein; transcrption
UniProt Protein Name:	Optineurin
UniProt Synonym Protein Names:	E3-14.7K-interacting protein; FIP-2; Huntingtin yeast partner L; Huntingtin-interacting protein 7; HIP-7; Huntingtin-interacting protein L; NEMO-related protein; Optic neuropathy-inducing protein; Transcription factor IIIA-interacting protein; TFIIIA-IntP
Protein Family:	Optineurin
UniProt Gene Name:	OPTN
UniProt Entry Name:	OPTN_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37 °C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37 °C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37 °C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µL of TMB substrate into each well, cover the plate and incubate at 37 °C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µL of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

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.

Antibodies

Anti-OPTN Antibody (CAB1845)

OPTN Antibody (PACO11010)

OPTN Antibody (PACO16902)

OPTN Antibody (PACO16903)