Mouse FIX ELISA Kit (MOFI00810)- High Sensitivity

Product Name:	Mouse FIX ELISA Kit
Product Code:	MOFI00810
Size:	96 Assays
Alias:	FIX
Detection Method:	Sandwich ELISA
Application:	This immunoassay kit allows for the in vitro quantitative determination of Mouse FIX 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 FIX and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse FIX in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	88-103	96
EDTA plasma(n=5)	85-99	94
UFH plasma(n=5)	87-100	95

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse FIX 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)	90-101%	85-103%	87-100%
EDTA plasma(n=5)	87-98%	88-101%	82-94%
UFH plasma(n=5)	87-99%	86-94%	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

P16294

UniProt Protein Function:	F9: Factor IX is a vitamin K-dependent plasma protein that participates in the intrinsic pathway of blood coagulation by converting factor X to its active form in the presence of Ca(2+) ions, phospholipids, and factor VIIIa. Defects in F9 are the cause of recessive X-linked hemophilia B (HEMB); also known as Christmas disease. Mutations in position 43 (Oxford-3, San Dimas) and 46 (Cambridge) prevents cleavage of the propeptide, mutation in position 93 (Alabama) probably fails to bind to cell membranes, mutation in position 191 (Chapel-Hill) or in position 226 (Nagoya OR Hilo) prevent cleavage of the activation peptide. Defects in F9 are the cause of thrombophilia due to factor IX defect (THPH8). A hemostatic disorder characterized by a tendency to thrombosis. Belongs to the peptidase S1 family.
UniProt Protein Details:	Protein type:Secreted, signal peptide; Secreted; Protease; EC 3.4.21.22 Cellular Component: extracellular region Molecular Function:peptidase activity; hydrolase activity; serine-type peptidase activity; serine-type endopeptidase activity; calcium ion binding; catalytic activity Biological Process: hemostasis; blood coagulation; proteolysis
NCBI Summary:	This gene encodes a vitamin K-dependent serine protease that plays a critical role in the intrinsic pathway of blood coagulation. The encoded protein is an inactive zymogen that is activated by coagulation factor XIa to generate factor IXa, a heterodimer containing heavy and light chains. In association with factor VIII, membrane phospholipids and calcium ions, factor IXa cleaves the inactive zymogen factor X to generate active factor Xa. Genetic deletion of this gene in mice results in a severe bleeding phenotype. Alternative splicing of this gene results in multiple transcript variants. [provided by RefSeq, Apr 2015]
UniProt Code:	P16294
NCBI GenInfo Identifier:	73661202
NCBI Gene ID:	14071
NCBI Accession:	NP_032005.1
UniProt Secondary Accession:	P16294,Q3UES1,
UniProt Related Accession:	P16294
Molecular Weight:	160,000
NCBI Full Name:	coagulation factor IX
NCBI Synonym Full Names:	coagulation factor IX
NCBI Official Symbol:	F9
NCBI Official Synonym Symbols:	Cf9; Cf-9; AW111646
NCBI Protein Information:	coagulation factor IX; christmas factor
UniProt Protein Name:	Coagulation factor IX
UniProt Synonym Protein Names:	Christmas factor
Protein Family:	Coagulation factor
UniProt Gene Name:	F9
UniProt Entry Name:	FA9_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.

Mouse FIX ELISA Kit (MOFI00810)

Description