
High mobility group protein B1 (HMGB1) belongs to a family of highly conserved proteins that contain HMG box domains. Human HMGB1 is expressed as a 215 amino acid (aa) single chain polypeptide containing three domains: two N-terminal globular, 70 aa positively charged DNA binding domains (HMG boxes A and B), and a negatively charged 30 aa C-terminal region that contains only Asp and Glu. Human HMGB1 is 100% aa identical to canine HMGB1 and 99% aa identical to mouse, rat, bovine and porcine HMGB1.
HMGB1 is a widely expressed and highly abundant protein. It was originally discovered as a nuclear protein that could bend DNA. Such bending stabilizes nucleosome formation and regulates the expression of select genes upon recruitment by DNA binding proteins. It is now known that HMGB1 also plays a significant role in extracellular signaling associated with inflammation. HMGB1 is massively released into the extracellular environment during cell necrosis. It acts as an inflammatory mediator that promotes monocyte migration and cytokine secretion, and as a mediator of T cell-dendritic cell interaction. In addition, activated monocytes, macrophages, and dendritic cells also secrete HMGB1, forming a positive feedback loop that results in the release of additional cytokines and neutrophils. The cytokine activity of HMGB1 is restricted to the HMG B box, while the A box is associated with the helix-loop-helix domain of transcription factors. Although HMGB1 does not possess a classic signal sequence, it appears to be secreted as an acetylated form via secretory endolysosome exocytosis. Once secreted, HMGB1 transduces cellular signals through its high affinity receptor, RAGE and, possibly, TLR2 and TLR4.
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(1)确定抗体的名字,注意中英文名字、别名、亚型等信息。
(2)确定您的实验类型,Elisa,WB,IHC,ICC,或者是FACS。每种抗体说明书都会列出抗体经验证过适用的实验类型,如果抗体说明书没有提及的应用类型,并不意味着该抗体不适用于此种分析应用类型,而仅是说明尚未经过此种实验验证。
(3)确定实验样本的种属,Human,Mouse,Rat等。我们的抗体说明书都列出该抗体验证过的适用物种,可根据说明书列出已验证的种属来选择适合你实验的抗体。
(4)样本抗原蛋白的结构性质。了解样本抗原蛋白的结构性质有助于选择最合适的抗体,待测样本蛋白的结构域和样本在提取和处理过程中是否会变性,蛋白空间构象的改变,会影响抗体的免疫亲和反应。
(5)单多克隆抗体的选择。一般单克隆抗体特异性强,但亲和力相对小,检测抗原灵敏度相对就低;而多克隆抗体特异性稍弱,但抗体的亲和力强,灵敏度高,但易出现非特异性染色(可以通过封闭等避免)。
2. 二抗选择
(1)种属来源。主要根据一抗种属来源而决定二抗来源,如一抗是小鼠来源,那二抗就买抗小鼠的即可(羊、兔等均可)。
(2)标记物的选择。有HRP、Biotin、荧光素等标记物。免疫组化,WB二抗主要选择HRP,Biotin标记的二抗,而免疫荧光染色可按实验需要选择不同荧光素标记的二抗,如FITC、Cy3、PE等。
2 一抗是属于哪个类或亚类
二抗需与一抗的类别或亚类相匹配。这通常是针对单克隆抗体而言。多克隆抗体主要是IgG类免疫球蛋白,因此相应的二抗就是抗IgG抗体.
单克隆抗体的类别及亚类通常会在产品列表中列出,如果你的一抗是小鼠IgM,那么相应的二抗就应当是抗小鼠IgM,或是抗小鼠IgG抗体。
如果单克隆一抗是小鼠IgG的某一亚类(IgG1,IgG2a,IgG2b,IgG3),那么几乎所有的抗小鼠IgG都可以与之结合,或者你也可以选择专门针对这一亚类的二抗,例如,如果你的一抗是小鼠IgG1,那么你可以选择抗IgG1的二抗,此种抗体在双标记实验中尤其适合。
如果你不知道一抗是哪一类别或亚类,那么抗小鼠IgG是一个不错的选择,因为此种抗体可以识别大多数类型的IgG免疫球蛋白。

