Mouse Coagulation Factor V ELISA Kit

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SKU:
MOFI00821
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
O88783
Sensitivity:
1.875ng/ml
Range:
3.125-200ng/ml
ELISA Type:
Sandwich ELISA, Double Antibody
Synonyms:
FV, F5, Plasma Factor V, Activated protein C cofactor, PCCF, Proaccelerin, labile factor
Reactivity:
Mouse
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Description

system_update_altデータセットsystem_update_alt安全データデータ
商品名:Mouse Coagulation Factor V ELISA Kit
製品コード:MOFI00821
サイズ:96 Assays
エイリアス:FV, F5, Plasma Factor V, Activated protein C cofactor, PCCF, Proaccelerin, labile factor
検出方法:Sandwich ELISA
申し込み:This immunoassay kit allows for the in vitro quantitative determination of Mouse FV concentrations in serum plasma and other biological fluids.
感度:1.875ng/ml
範囲:3.125-200ng/ml
保管所:4°C for 6 months
ノート:For Research Use Only
回復:Matrices listed below were spiked with certain level of Mouse FV and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse FV in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)92-10598
EDTA plasma(n=5)88-10498
UFH plasma(n=5)86-9892
直線性:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse FV and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)85-102%89-95%86-104%
EDTA plasma(n=5)82-95%83-100%87-97%
UFH plasma(n=5)87-96%82-100%82-98%
Intra Assay:CV <8%
Inter Assay:CV <10%
成分量保管所
ELISA Microplate (Dismountable)8-12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5-

必要なその他の材料と設備：

  • 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
UniprotO88783
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:Protease; Secreted; Secreted, signal peptide

Chromosomal Location of Human Ortholog: 1 H2.2|1 71.46 cM

Cellular Component: endoplasmic reticulum; extracellular region; extracellular space; Golgi apparatus; membrane

Molecular Function:copper ion binding; metal ion binding

Biological Process: blood circulation; blood coagulation; hemostasis

NCBI Summary:This gene encodes a glycoprotein coagulation factor that plays a critical role in the process of blood coagulation and hemostasis. The encoded protein is activated by thrombin, to generate a heterodimer containing heavy and light chains held together by calcium ions. About half of the mice lacking the encoded protein die at an embryonic stage possible due to abnormal yolk-sac vasculature while the remaining animals succumbed to massive hemorrhage immediately after birth. A point mutation in this gene has been shown to cause disseminated intravascular thrombosis in the perinatal period, resulting in frequent deaths of newborn mice. [provided by RefSeq, Apr 2015]
UniProt Code:O88783
NCBI GenInfo Identifier:6679731
NCBI Gene ID:14067
NCBI Accession:NP_032002.1
UniProt Secondary Accession:O88783,Q3UTQ2, Q3UV80,
UniProt Related Accession:O88783
Molecular Weight:
NCBI Full Name:coagulation factor V preproprotein
NCBI Synonym Full Names:coagulation factor V
NCBI Official Symbol:F5  
NCBI Official Synonym Symbols:Cf5; Cf-5; AI173222  
NCBI Protein Information:coagulation factor V
UniProt Protein Name:Coagulation factor V
UniProt Synonym Protein Names:Activated protein C cofactor
Protein Family:Coagulation factor
UniProt Gene Name:F5  

*ノート: プロトコルは、各バッチ/ロットに固有です。正しい手順については、キットに含まれているプロトコルに従ってください。

ステップ手順
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.

ELISAアッセイを実施する場合、可能な限り最良の結果を達成するためにサンプルを準備することが重要です。以下に、さまざまなサンプルタイプのサンプルを準備するための手順のリストを示します。

サンプルタイププロトコル
血清:

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.

プラズマ:

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.

尿および脳脊髄液:

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.

細胞培養上清:

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.

細胞溶解物:

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.

組織ホモジネート:

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.

組織溶解物:

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.

母乳:

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.

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