| Formulation | 50% glycerol/water (v/v) |
| Storage | -20°C |
| Purity | >95% by SDS-PAGE |
| Activity Determination | Clotting assay |
| Shelf Life (properly stored) | 6 months |
The activation of single chain factor V (330,000) by alpha-thrombin is represented. Arrows indicate the site at which factor V is cleaved by thrombin to yield two activation peptides (71,000 and 120,000), in addition to the heavy (94,000) and light (74,000) chains which form Va. The heavy and light chains of factor Va remain non-covalently associated in the presence of calcium ions.
Sample Gel Information:
| Gel | Novex 4-12% Bis-Tris |
|---|---|
| Load | Human Factor V, 1 µg per lane |
| Buffer | MOPS |
| Standard | SeeBluePlus 2; Myosin (191 kDa), Phosphorylase B (97 kDa), BSA (64 kDa), Glutamic Dehydrogenase (51 kDa), Alcohol Dehydrogenase (39 kDa), Carbonic Anhydrase (28 kDa), Myoglobin Red (19 kDa), Lysozyme (14 kDa) |
| Special Notes | Factor V is a very labile protein. Some degraded fragments are usually noticeable in preparations. |
Overview:
Factor V is a large, single chain, plasma glycoprotein which is an essential component in the blood coagulation cascade (1). During coagulation, the procofactor, factor V, is converted to the active cofactor, factor Va, via limited proteolysis by the serine protease alpha-thrombin (illustrated above), and less efficiently by factor Xa. The active cofactor is composed of an NH2-terminal derived heavy chain (Mr=94,000) and a COOH-terminal derived light chain (Mr=74,000) which remain non-covalently associated in the presence of calcium ions. Factor Va serves as a cofactor for the serine protease factor Xa. Factor Va and Xa assemble on a phospholipid surface in a non-covalent and calcium ion-dependent manner, to form the prothrombinase complex. The prothrombinase complex is responsible for the rapid conversion of the zymogen prothrombin to the active serine protease, alpha-thrombin. Assembly of the prothrombinase complex increases the rate at which prothrombin is converted to thrombin by nearly 300,000-fold relative to the rate with factor Xa alone. Down regulation of the prothrombinase complex is accomplished partly through the inactivation of factor Va by activated protein C.
Human factor V is prepared from fresh frozen human plasma using immunoaffinity chromatography as described by Katzmann and coworkers (2). Bovine factor V is prepared from fresh bovine plasma using conventional chromatographic techniques as described by Nesheim and coworkers (3). Purified factor V is supplied in 50% glycerol (vol/vol), and should be stored at -20°C. Purity is determined by SDS-PAGE analysis and activity is measured in a factor V clotting assay.
Properties:
| Localization | Plasma and platelets | |||||
|---|---|---|---|---|---|---|
| Plasma concentration | 7.0 µg/ml (human) (4,5) 35 µg/ml (bovine) (5) |
|||||
| Mode of action | Procofactor; activated by thrombin to form the active cofactor, factor Va | |||||
| Molecular weight | 330,000 (3) - (determined for bovine factor V) | |||||
| Extinction coefficient |
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| Structure | one subunit, 2196 amino acids (1,6) - (determined for human factor V) | |||||
| Percent carbohydrate | approximately 25% (based upon the calculated molecular weight of human factor V) |
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1，IgG抗体是抗体中分子量最小的一种，可通过胎盘输给胎儿，保护了婴儿最初六个月内免受感染。该抗体产生晚，维持时间长，消失慢，浓度高。血中检测到可作为远期感染指标。
2，IgM抗体是抗体中分子量最大的一种，不能通过胎盘输给胎儿。抗体产生最早，一经感染，快速产生，在感染初期抗感染起作用。但维持时间短，消失快。
3，灵长类动物主要有四种免疫球蛋白IgG、IgM、IgA、和IgE。
所有用于检测抗原的免疫学方法经适当改良后，均可用于抗体的检测，如IFA、ELISA、RIA、LA......由于抗原方法的敏感性提高和PCR技术的应用，使得HSV抗体在HSV感染个体中的不均一性和不稳定性影响了这类指标在临床诊断中的意义，但作为一种感染有关指标，在一定的范围和情况下，仍有必要进行检测和深入研究.目前HSV特异性抗体的检测，主要有IgG、IgM和IgA三种。
本试验根据《中国药典》2015年版紫外分光光度法吸收系数法进行制定，本试验方法采用的方法学验证内容如下：
1.线性及范围
取IgG标准品配制5个不同浓度被测样品，并进行测定，绘制标准曲线，得到回归方程及范围。
2.准确度
已回收率对准确度进行验证。以已知含量的IgG标准品配制供试品，配制三个浓度，每个浓度平均测定三次。计算回收率。
3.精密度
IgG标准品配制供试品，平行测定六次。
4.溶液稳定性试验
至少持续2h的溶液稳定性考察。
5.干扰试验
空白溶液中加入辅料，在280nm处测定吸收，确定辅料是否对样品的吸收造成干扰。
以上，请大神指正。
补充问题，在专属性验证上，仅做辅料干扰是不是太少了？是否再进行强降解实验呢？因为其中包含了蛋白质A、外源性DNA及宿主细胞蛋白残留。我是否应该针对这三种杂质进行专属性试验？该如何进行呢？
2-IgM也是一种特异性感染指标，但在人体内持续时间不长（有时是30天更短），往往是传染病的近期感染指标。呈阴性是指：你近期没有感染过结核杆菌。
