电泳迁移率实验 (EMSA) 试剂盒，含 SYBR™ Green & SYPRO™ Ruby EMSA 染料

凝胶迁移实验是一种使用非变性PAGE测定DNA结合的实验。它提供了一种简单、快速并且非常灵敏的检测序列特异性DNA结合蛋白的方法。蛋白与末端标记的DNA片段结合形成一个独立的蛋白-DNA复合物。你可以用此方法检测纯化蛋白或粗提取物中的未知因子与DNA的结合。该方法也可以实现对DNA结合蛋白的亲和性、丰度、结合速率常数、解离速率常数，以及结合特异性进行定量分析。

超迁移实验是凝胶迁移DNA结合实验的一种变形，它使用抗体检测蛋白-DNA复合体中的蛋白。向一个结合反应内加入一种特异性抗体可能会产生下面几种效应。如果被抗体识别的蛋白与复合体的形成无关，那么加入抗体应当对电泳没有任何影响。如果形成复合体的蛋白被抗体识别，那么该抗体或者将阻止复合体的形成，或者将形成一个抗体-蛋白-DNA三元复合物，从而导致蛋白-DNA复合体迁移率的进一步下降（超迁移）。在蛋白结合DNA前还是结合后加入抗体，结果可能会不同（特别是当抗原表位是在蛋白与DNA结合区域时）。

A supershift assay is a method for positively identifying a protein:DNA interaction on an EMSA. An antibody (typically 1 µg) is added to the binding reaction. During electrophoresis, the antibody:protein:DNA complex migrates slowly, causing a “supershift” compared to the “shift” caused by a protein:DNA complex. Not all antibodies will cause a supershift. Some antibodies do not bind to proteins once they are bound to DNA. Some antibodies can prevent protein:DNA interactions but can still be used to confirm the identity of a protein that causes a shift in the absence of the antibody.

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EMSAs (also called gel shifts, band shifts, gel retardation assays, or mobility assays) have been used extensively for studying protein-DNA interactions. Because protein-DNA complexes migrate more slowly through a native polyacrylamide or agarose gel than DNA alone, individual protein-DNA complexes can be visualized as discrete bands within the gel using chemiluminescence or radioisotopic detection.

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A shift assay is a DNA-binding assay using nondenaturing PAGE. It provides a simple, rapid, and extremely sensitive method for detecting sequence-specific DNA-binding proteins. Proteins bind specifically to an end-labeled DNA fragment corresponding to the individual protein-DNA complexes. You can use the assay to test binding of purified proteins or of uncharacterized factors in crude extracts. This assay also permits quantitative determination of the affinity, abundance, association rate constants, dissociation rate constants, and binding specificity of DNA-binding proteins.

A supershift assay is a variation of the mobility shift DNA-binding assay that uses antibodies to identify proteins present in the protein-DNA complex.Addition of a specific antibody to a binding reaction can have one of several effects. If the protein recognized by the antibody is not involved in complex formation, addition of the antibody should have no effect. If the protein that forms the complex is recognized by the antibody, the antibody can either block complex formation or it can form an antibody-protein-DNA ternary complex and thereby specifically result in a further reduction in the mobility of the protein-DNA complex (a supershift). Results may be different depending upon whether the antibody is added before or after the protein binds DNA (particularly if there are epitopes on the DNA binding surface of the protein).

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Here is the composition of the 5X Binding Buffer:

- 50 mM Tris HCl, pH 7.4
- 750 mM KCl
- 0.5 mM DTT
- 0.5 mM EDTA

Note: This buffer is optimized for lac repressor binding to lac operator. It may not be optimal for other protein-nucleic acid interactions.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.