![absoluteantibody/Anti-F4/80 [Cl:A3-1 (recombinant version)]/200 μg/Ab00106-2.3](images/no_picture.gif)
UniProt Accession Number of Target Protein: Q61549
Alternative Name(s) of Target: EGF-like module; Cell surface glycoprotein F4/80; EGF-like module receptor 1; EMR1 receptor; Em Gpf4; TM7; Ly-71; f480; f-480; f4 80; f 480; f-4/80; f4-80Immunogen: Thioglycollate stimulated peritoneal macrophages of mouse origin.
Specificity: This antibody recognises the mouse F4/80 antigen, a 160kD glycoprotein, expressed by murine macrophages.
Application Notes: The anti-F4/80 antibody, clone Cl:A3-1, has been very widely used in flow cytometry, microscopy and Western blotting since its generation by Austyn and Gordon in 1980. We are the only supplier of recombinant versions of Cl:A3-1 which, whilst having the same variable domains as the original, has been shown to block hybridoma derived anti-F4/80:APC binding to in vitro generated bone marrow-derived (BMDMs) and ex vivo splenic murine macrophages. This was tested using both the recombinant version of the original isotype (rat IgG2b) as well as a murinised version (IgG2a, Ab00106-2.0). Further testing on BMDMs shows that our Fc Silent™ rat IgG2b is a valuable new reagent which shows distinct labelling of F4/80-positive cell populations, whilst the isotype control shows no binding, suggesting that only the antigen binding domains are involved in binding, promoting precision of labelling. The murinised versions will be valuable tools in the study of the role of the murine macrophage in vivo without the artefacts rat-derived antigen being co-administered may harbour. Particularly the murine IgG2a (Ab00106-2.0) should be an excellent candidate for macrophage depletion studies, with the Fc Silent™ (Ab00106-2.3) version being the ideal negative control. Using our recombinant production platform, any species and isotype are possible and our Support Team (support@absoluteantibody.com) will be glad to help you with finding the right variant to match your experimental needs.
Antibody first published in:Austyn JM, Gordon S.F4/80, a monoclonal antibody directed specifically against the mouse macrophage.Eur J Immunol. 1981 Oct;11(10):805-15.PMID:7308288Note on publication:Describes the making of the antibody and its specificity for macrophages (Flow Cytometry) and proposes its potential use as a marker.
Competitive flow-cytometry assay between Absolute Antibody anti-F4/80 (Ab00106) CI:A3-1 variants and an existing commercial anti-F4/80 antibody. Mouse (Mus musculus) splenocytes were labelled ex vivo with a commercially available APC-labelled anti-F4/80 antibody and APE labelled anti-CD11b antibody and subject to flow-cytometry analysis (A), in which a small subpopulation of F4/80-CD11b positive cells may be observed. Subsets of commerical anti-F4/80 antibody-labelled splenocytes were also subsequently incubated with unlabelled versions of either the rat ( Rattus norvegicus ) IgG2b chimeric version (Ab00106-8.1, B) or mouse IgG2A chimeric (Ab00106-2.0, C) version of CI:A3-1. Loss of the F4/80-CD11b positive subpopulation may be observed, demonstrating displacement of the commercial antibody and the specificity of CI:A3-1.
In Figure 1 murine bone marrow-derived macrophages (BMDMs) were pre-blocked with rat anti-mouse CD16 & CD32 (clone FCR-4G8) and stained with non-recombinant anti-F4/80 [Cl:A3-1] conjugated to Alexa Fluor® 647 (AF647), all commercially available from competitors. InFigure 2 BMDMs were stained with recombinant anti-F4/80 [Cl:A3-1] or isotype control (anti-fluorescein [4-4-20 (enhanced)] IgG2b (Ab00102-8.1). InFigure 3 BMDMs were stained with Fc Silent™ recombinant anti-F4/80 [Cl:A3-1] (Ab00106-8.4) or isotype control (Fc Silent™ anti-fluorescein [4-4-20 (enhanced)] IgG2b (Ab00102-8.4). These were fluorescently labelled using the secondary antibody, goat IgG anti-rat IgG (H&L-chain) polyclonal antibody directly conjugated to Alexa Fluor® 647(AF647) commercially available from a competitor. Whilst inFigure 2 the highest fluorescence signal is seen with the recombinant anti-F4/80 IgG2 (the isotype of the original hybridoma-derived antibody), the isotype control IgG2b (Ab00102-8.1) shows considerable signal overlap, indicative of binding of the antibody to Fc-receptors. This illustrates the importance of isotype controls in such experiments when using conventional antibody formats particularly when Fc-blocking reagents are incompatible with the system used due to reactivity with the secondary antibody. The Fc silent™ format however overcomes this issue as seen inFigure 3, where the Fc silent™ recombinant anti-F4/80 (Ab00102-8.4) yields a strong and distinct signal, whilst the isotype control (Ab00102-8.4) shows no discernible difference to the background staining from the secondary antibody alone. Therefore, with Fc Silent™ reagents, no Fc-blocking productsare required. [Data courtesy of Lewis Taylor.]
In Figure 1 murine bone marrow-derived macrophages (BMDMs) were pre-blocked with rat anti-mouse CD16 & CD32 (clone FCR-4G8) and stained with non-recombinant anti-F4/80 [Cl:A3-1] conjugated to Alexa Fluor® 647 (AF647), all commercially available from competitors. InFigure 2 BMDMs were stained with recombinant anti-F4/80 [Cl:A3-1] or isotype control (anti-fluorescein [4-4-20 (enhanced)] IgG2b (Ab00102-8.1). InFigure 3 BMDMs were stained with Fc Silent™ recombinant anti-F4/80 [Cl:A3-1] (Ab00106-8.4) or isotype control (Fc Silent™ anti-fluorescein [4-4-20 (enhanced)] IgG2b (Ab00102-8.4). These were fluorescently labelled using the secondary antibody, goat IgG anti-rat IgG (H&L-chain) polyclonal antibody directly conjugated to Alexa Fluor® 647(AF647) commercially available from a competitor. Whilst inFigure 2 the highest fluorescence signal is seen with the recombinant anti-F4/80 IgG2 (the isotype of the original hybridoma-derived antibody), the isotype control IgG2b (Ab00102-8.1) shows considerable signal overlap, indicative of binding of the antibody to Fc-receptors. This illustrates the importance of isotype controls in such experiments when using conventional antibody formats particularly when Fc-blocking reagents are incompatible with the system used due to reactivity with the secondary antibody. The Fc silent™ format however overcomes this issue as seen inFigure 3, where the Fc silent™ recombinant anti-F4/80 (Ab00102-8.4) yields a strong and distinct signal, whilst the isotype control (Ab00102-8.4) shows no discernible difference to the background staining from the secondary antibody alone. Therefore, with Fc Silent™ reagents, no Fc-blocking productsare required. [Data courtesy of Lewis Taylor.]
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是否可以理解为纯化水得PH范围为6.3-7.6？能否直接用pH计测量？谢谢!
因为是考察不同PH对药物的影响，样品又不好改变其PH值，这种情况怎么办？希望有经验的高手指教。
我的流动相是甲醇-水（90：10）
谢谢赐教！
请进子版按格式发贴，自行修改，谢谢。
常用流动相加酸碱后PH的总结，希望大家能够提供一点自己测过的结果，谢谢先
两个CEX方法A和B测定同一单抗，结果碱性峰比例差不多，酸性峰比例相差约7%，相应主峰也差了7%左右。
具体来说，A方法酸性峰高，主峰低，碱性峰稍微低点；B方法酸性峰低，主峰高，碱性峰稍微高点；另外也做了CIEF，结果呢和A方法更接近。
仔细比较起来，AB两个方法的峰性和数量差不多，就不知道为什么会有这么大的差异。两个方法一个用的WCX柱-磷酸缓冲液，一个用SCX柱-MES缓冲液
大家帮我分析下：
1.两个方法哪个方法更准确，是以酸性峰高的为准还是什么？为什么？
2.这显著差异是由方法造成，具体原因是什么？柱子？
3.CIEF的结果和A方法更接近，是不是可以由此证明A方法更好或者CIEF的方法更好（因为CIEF更快更方便）？
欢迎讨论~
纠正下，A方法用的是Tosoh的柱子，B方法用的是SCX柱。TOSOH的柱子是7um的填料，10cm长。SCX是10um的填料。我本人TOSOH的阳离子柱子用的很少，这次信手用用，结果发现差异很大
那我现在就考虑，在以后方法开发过程中，除了通过流动相pH和组成、梯度、柱子选择来获得样品主峰和酸碱性的最大分离，还要关注各峰比例。因为之前比较方法好坏都只看分离度，尤其是主峰和邻近峰的分离度，获得最大分离度，自然可以做到主峰尽可能纯，但从未认真比较过各峰比例。这是一个大疏忽吧！
另外，CIEF和CEX方法原理还是有点差异的，所以分的是不同的异质体，原液放行两个方法肯定是都要做的。问题就是在早期细胞株筛选和工艺开发阶段，哪个方法才是又快又准。CIEF（iCE280)一般15分钟一个样，比CEX快多了。如果CIEF测得主峰要低于CEX结果，是不是真的完全可以取代CEX呢？CEX分离出的峰远比CIEF的多！
欢迎大家继续讨论~
1、弱酸和它的盐（如：HAc---NaAc）的水溶液组成；
2、弱碱和它的盐（如：NH3·H2O---NH4Cl）的水溶液组成；
3、多元弱酸的酸式盐及其对应的次级盐（如：NaH2PO4---Na2HPO4）的水溶液组成。
酸碱缓冲溶液的选型一般应根据具体情况进行选择。缓冲酸性可选用碱性缓冲液，缓冲酸性可采用碱性缓冲液。常用作缓冲溶液的酸类由弱酸及其共轭酸盐组合成的溶液具有缓冲作用。生化实验室常用的缓冲系主要有磷酸、柠檬酸、碳酸、醋酸、巴比妥酸、Tris（三羟甲基氨基甲烷）等系统，生化实验或研究工作中要慎重地选择缓冲体系，因为有时影响实验结果的因素并不是缓冲液的pH值，而是缓冲液中的某种离子。如硼酸盐、柠檬酸盐、磷酸盐和三羟甲基甲烷等缓冲剂都可能产生不需要的化学反应。
【酸碱缓冲溶液】由弱酸及其盐、弱碱及其盐组成的混合溶液，能在一定程度上抵消、减轻外加强酸或强碱对溶液酸碱度的影响，从而保持溶液的pH值相对稳定。这种溶液称为酸碱缓冲溶液。
1.直接用固体磷酸钠配制成50mM的磷酸钠溶液，再调pH到7.4；（我们试着用这个做了下，发现挂不上柱）
2.配置磷酸钠盐缓冲液：按NaH2PO4：Na2HPO4以19：81的摩尔比配制成pH7.4的缓冲液？（附一张百度出来的配方
）
3.如果是磷酸钠盐缓冲液，可以直接将50mM的NaH2PO4的水溶液用NaOH调成pH7.4吗？
再者，2和3这两个方法配制的磷酸钠盐缓冲液有什么区别？最终效果是一样的吗？如果不一样，有什么理论的知识支撑呢？个人感觉是分析化学中酸碱理论中的缓冲液那里的知识。求帮忙解答这些疑问。
另外，我还想问一下，pH对于Ni柱对His-tagged的蛋白的分离纯化影响大吗？是怎么影响的？谢谢大家了！
由弱酸及其盐、弱碱及其盐组成的混合溶液，能在一定程度上抵消、减轻外加强酸或强碱对溶液酸碱度的影响，从而保持溶液的pH值相对稳定。这种溶液称为缓冲溶液。
