Product Name:	Mouse Cyb5r3 ELISA Kit
Product Code:	MOFI00217
Size:	96 Assays
Alias:	Cyb5r3, Diaphorase-1
Detection Method:	Sandwich ELISA
Application:	This immunoassay kit allows for the in vitro quantitative determination of Mouse Cyb5r3 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 Mouse Cyb5r3 and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse Cyb5r3 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	92-103	97
EDTA plasma(n=5)	89-104	98
UFH plasma(n=5)	88-105	99

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse Cyb5r3 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)	86-104%	87-105%	85-100%
EDTA plasma(n=5)	82-97%	82-94%	88-100%
UFH plasma(n=5)	87-100%	81-94%	83-95%

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

Q9DCN2

UniProt Protein Function:	CYB5R3: Desaturation and elongation of fatty acids, cholesterol biosynthesis, drug metabolism, and, in erythrocyte, methemoglobin reduction. Defects in CYB5R3 are the cause of methemoglobinemia CYB5R3-related (METHB-CYB5R3). A form of methemoglobinemia, a hematologic disease characterized by the presence of excessive amounts of methemoglobin in blood cells, resulting in decreased oxygen carrying capacity of the blood, cyanosis and hypoxia. There are two types of methemoglobinemia CYB5R3-related. In type 1, the defect affects the soluble form of the enzyme, is restricted to red blood cells, and causes well- tolerated methemoglobinemia. In type 2, the defect affects both the soluble and microsomal forms of the enzyme and is thus generalized, affecting red cells, leukocytes and all body tissues. Type 2 methemoglobinemia is associated with mental deficiency and other neurologic symptoms. Belongs to the flavoprotein pyridine nucleotide cytochrome reductase family. 3 isoforms of the human protein are produced by alternative promoter.
UniProt Protein Details:	Protein type:EC 1.6.2.2; Mitochondrial; Carbohydrate Metabolism - amino sugar and nucleotide sugar; Oxidoreductase Cellular Component: cytoplasm; endoplasmic reticulum; endoplasmic reticulum membrane; membrane; mitochondrial inner membrane; mitochondrial outer membrane; mitochondrion Molecular Function:ADP binding; AMP binding; cytochrome-b5 reductase activity; FAD binding; NAD binding; oxidoreductase activity; protein binding Biological Process: cholesterol biosynthetic process; cholesterol metabolic process; lipid metabolic process; steroid biosynthetic process; steroid metabolic process; sterol biosynthetic process
UniProt Code:	Q9DCN2
NCBI GenInfo Identifier:	19745150
NCBI Gene ID:	109754
NCBI Accession:	NP_084063.1
UniProt Related Accession:	Q9DCN2
Molecular Weight:	31,550 Da
NCBI Full Name:	NADH-cytochrome b5 reductase 3
NCBI Synonym Full Names:	cytochrome b5 reductase 3
NCBI Official Symbol:	Cyb5r3
NCBI Official Synonym Symbols:	Dia1; Dia-1; C85115; WU:Cyb5r3; 0610016L08Rik; 2500002N19Rik; WU:AL591952.1-001; WU:AL591952.1-002; WU:AL591952.1-003
NCBI Protein Information:	NADH-cytochrome b5 reductase 3
UniProt Protein Name:	NADH-cytochrome b5 reductase 3
UniProt Synonym Protein Names:	Diaphorase-1
Protein Family:	NADH-cytochrome b5 reductase
UniProt Gene Name:	Cyb5r3
UniProt Entry Name:	NB5R3_MOUSE

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

Step	Procedure
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.