Product Name:	Human F5 / Factor V ELISA Kit
Product Code:	HUFI02438
Size:	96 Assays
Alias:	F5, Coagulation Factor V, Plasma Factor V, Activated protein C cofactor, PCCF, Proaccelerin, labile factor
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human F5 concentrations in serum plasma and other biological fluids.
Sensitivity:	9.375pg/ml
Range:	15.625-1000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human F5 and the recovery rates were calculated by comparing the measured value to the expected amount of Human F5 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	88-95	91
EDTA plasma(n=5)	85-102	94
UFH plasma(n=5)	90-104	99

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human F5 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)	92-105%	91-103%	91-105%
EDTA plasma(n=5)	85-100%	87-101%	86-101%
UFH plasma(n=5)	80-95%	81-92%	80-97%

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

P12259

UniProt Protein Function:	factor V: Central regulator of hemostasis. It serves as a critical cofactor for the prothrombinase activity of factor Xa that results in the activation of prothrombin to thrombin. Defects in F5 are the cause of factor V deficiency (FA5D); also known as Owren parahemophilia. It is an hemorrhagic diastesis. Defects in F5 are the cause of thrombophilia due to activated protein C resistance (THPH2). THPH2 is a hemostatic disorder due to defective degradation of factor Va by activated protein C. It is characterized by a poor anticoagulant response to activated protein C resulting in tendency to thrombosis. Defects in F5 are a cause of susceptibility to Budd- Chiari syndrome (BDCHS). A syndrome caused by obstruction of hepatic venous outflow involving either the hepatic veins or the terminal segment of the inferior vena cava. Obstructions are generally caused by thrombosis and lead to hepatic congestion and ischemic necrosis. Clinical manifestations observed in the majority of patients include hepatomegaly, right upper quadrant pain and abdominal ascites. Budd-Chiari syndrome is associated with a combination of disease states including primary myeloproliferative syndromes and thrombophilia due to factor V Leiden, protein C deficiency and antithrombin III deficiency. Budd-Chiari syndrome is a rare but typical complication in patients with polycythemia vera. Defects in F5 may be a cause of susceptibility to ischemic stroke (ISCHSTR); also known as cerebrovascular accident or cerebral infarction. A stroke is an acute neurologic event leading to death of neural tissue of the brain and resulting in loss of motor, sensory and/or cognitive function. Ischemic strokes, resulting from vascular occlusion, is considered to be a highly complex disease consisting of a group of heterogeneous disorders with multiple genetic and environmental risk factors. Defects in F5 are associated with susceptibility to pregnancy loss, recurrent, type 1 (RPRGL1). RPRGL1 is a common complication of pregnancy, resulting in spontaneous abortion before the fetus has reached viability. The term includes all miscarriages from the time of conception until 24 weeks of gestation. Recurrent pregnancy loss is defined as 3 or more consecutive spontaneous abortions. Belongs to the multicopper oxidase family.
UniProt Protein Details:	Protein type:Secreted; Protease; Secreted, signal peptide Chromosomal Location of Human Ortholog: 1q23 Cellular Component: endoplasmic reticulum lumen; ER to Golgi transport vesicle; ER-Golgi intermediate compartment membrane; extracellular region; extracellular space; Golgi membrane; membrane; plasma membrane Molecular Function:copper ion binding; protein binding Biological Process: blood circulation; blood coagulation; cellular protein metabolic process; COPII coating of Golgi vesicle; ER to Golgi vesicle-mediated transport; platelet activation; platelet degranulation; post-translational protein modification; protein amino acid N-linked glycosylation via asparagine Disease: Budd-chiari Syndrome; Factor V Deficiency; Pregnancy Loss, Recurrent, Susceptibility To, 1; Stroke, Ischemic; Thrombophilia Due To Activated Protein C Resistance
NCBI Summary:	This gene encodes an essential cofactor of the blood coagulation cascade. This factor circulates in plasma, and is converted to the active form by the release of the activation peptide by thrombin during coagulation. This generates a heavy chain and a light chain which are held together by calcium ions. The activated protein is a cofactor that participates with activated coagulation factor X to activate prothrombin to thrombin. Defects in this gene result in either an autosomal recessive hemorrhagic diathesis or an autosomal dominant form of thrombophilia, which is known as activated protein C resistance. [provided by RefSeq, Oct 2008]
UniProt Code:	P12259
NCBI GenInfo Identifier:	105990535
NCBI Gene ID:	2153
NCBI Accession:	NP_000121.2
UniProt Secondary Accession:	P12259,Q14285, Q2EHR5, Q5R346, Q5R347, Q6UPU6, Q8WWQ6 A8K6E8,
UniProt Related Accession:	P12259
Molecular Weight:	30.4 kDa
NCBI Full Name:	coagulation factor V preproprotein
NCBI Synonym Full Names:	coagulation factor V
NCBI Official Symbol:	F5
NCBI Official Synonym Symbols:	FVL; PCCF; THPH2; RPRGL1
NCBI Protein Information:	coagulation factor V
UniProt Protein Name:	Coagulation factor V
UniProt Synonym Protein Names:	Activated protein C cofactor; Proaccelerin, labile factor
Protein Family:	Coagulation factor
UniProt Gene Name:	F5
UniProt Entry Name:	FA5_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.