改造地高辛标记DNA和检测试剂盒用于凝胶阻滞实验的新方法

凝胶阻滞实验（gel retardation）,又称为电泳迁移率变动实验（Electrophoretic mobility shift assay,EMSA）,是研究蛋白和DNA相互作用的一种技术。传统的³²P标记探针的凝胶阻滞实验,具有很高的灵敏度,然而也有接触危险性的放射性同位素且不容易定量分析的缺点。最近利用非放射性标记的凝胶阻滞实验,已有很多成功的报导,该方法快捷,安全,灵活,但非放射性标记探针的凝胶阻滞试剂盒的费用却很高。在论文中,我们提供了一种改造地高辛标记DNA和检测试剂盒用于凝胶阻滞实验的新方法。首先将双链DNA探针末端引入EcoRI 粘性末端以便进行3′末端标记,然后利用价格比较便宜的地高辛标记DNA和检测试剂盒(DIG High Prime DNA Labeling and Detection Starter Kit II, Rohe) 进行探针标记和凝胶阻滞信号检测。经过多次实验参数的摸索,最终得到了成功的结果,为利用地高辛标记DNA和检测试剂盒进行凝胶阻滞实验提供了成功的例子和方法。      

A sensitive two-color electrophoretic mobility shift assay for detecting both nucleic acids and protein in gels

DNA-binding proteins are key to the regulation and control of gene expression, replication and recombination. The electrophoretic mobility shift assay (or gel shift assay) is considered an essential tool in modern molecular biology for the study of protein-nucleic acid interactions. As typically implemented, however, the technique suffers from a number of shortcomings, including the handling of hazardous (32)P-labeled DNA probes, and difficulty in quantifying the amount of DNA and especially the amount of protein in the gel. A new detection method for mobility-shift assays is described that represents a significant improvement over existing techniques. The assay is fast, simple, does not require the use of radioisotopes and allows independent quantitative determination of: (i) free nucleic acid, (ii) bound nucleic acid, (iii) bound protein, and (iv) free protein. Nucleic acids are detected with SYBR Green EMSA dye, while proteins are subsequently detected with SYPRO Ruby EMSA dye. All fluorescence staining steps are performed after the entire gel-shift experiment is completed, so there is no need to prelabel either the DNA or the protein and no possibility of the fluorescent reagents interfering with the protein-nucleic acid interactions. The ability to independently quantify each molecular species allows more rigorous data analysis methods to be applied, especially with respect to the mass of protein bound per nucleic acid.     

积磷小月菌调控蛋白Mlp21700的异源表达及其与多聚磷酸盐(Poly-P)代谢基因启动子的结合

【背景】积磷小月菌(Microlunatus phosphovorus)是重要的聚磷微生物,在好氧条件下积累聚磷酸盐并在厌氧条件分解聚磷酸盐,这个过程可能受到精细的基因调控。【目的】利用凝胶迁移实验(EMSA)分析调控蛋白Mlp21700结合的多聚磷酸盐(Poly-P)代谢基因启动子,找到Mlp21700可能的调控靶点。【方法】以积磷小月菌JN459菌株的基因组DNA为模板,PCR扩增Mlp21700编码序列,构建重组质粒p ET28a-21700并转化到大肠杆菌Transetta(DE3)菌株,诱导表达后采用非变性方法纯化获得Mlp21700融合蛋白。利用PCR方法分别扩增各个Poly-P代谢基因的启动子,用生物素标记后作为探针。采用EMSA分析Mlp21700在试验条件下是否结合启动子以及结合强度。【结果】DNA测序和酶切验证表明p ET28a-21700携带正确的Mlp21700编码序列。SDS-PAGE分析显示试验条件下Transetta(DE3)菌株大量表达可溶性Mlp21700,纯化的重组Mlp21700蛋白纯度大于90%,含量为0.64 mg/mL。EMSA分析表明在试验条件下Mlp21700能够结合Mlp26610基因ppgk和Mlp44770基因ppx的启动子。【结论】调控蛋白Mlp21700可能参与Mlp26610和Mlp44770基因的转录调控,进而调控Poly-P的分解过程。     

Scintillation proximity assay for DNA binding by human p53

Many DNA binding proteins are known to regulate gene expression. When that binding is altered, a disease state can result. A common method for measuring DNA binding, namely electrophoretic mobility shift assay (EMSA) is often used but it is not amenable to rapid screening of many samples. As an alternative method, we have developed a DNA binding assay for the tumor suppressor protein p53 in a 96-well microtiter plate format using scintillation proximity assay (SPA) beads. We have shown this assay to be sensitive (as little as 0.5 ng p53 can be detected), quick (assay completed in as little as 15 min), and easily quantitated using a microtiter plate scintillation counter We also used the assay to analyze the kinetics of the DNA binding to p53. The specificity of this p53 DNA binding SPA was confirmed using competition by oligonucleotides either from the same gene or from mutated versions of this sequence. Thus, SPA is a good alternative to gel shift assays for DNA binding and may be useful for the analysis of multiple tumor cell samples or for high-throughput screens for compounds affecting DNA binding by proteins of interest.     

Photoaffinity electrophoretic mobility shift assay using photoreactive DNA bearing 3-trifluoromethyl-3-phenyldiazirine in its phosphate backbone

Photoaffinity cross-linking enables the analysis of interactions between DNA and proteins even under denaturing conditions. We present a photoaffinity electrophoretic mobility shift assay (EMSA) in which two heterogeneous techniques―photoaffinity cross-linking using DNA bearing 3-trifluoromethyl-3-phenyldiazirine and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) analysis—are combined. To prepare the photoreactive DNA, which is an essential tool for photoaffinity EMSA, we first determined the optimal conditions for the integration of 4-(3-trifluoromethyl-3H-diazirin-3-yl)benzyl bromide to the specific site of oligonucleotide where phosphodiester linkage was replaced with phosphorothioate linkage. The photoaffinity EMSA was developed using the POU (initial letters of three genes: Pit-l, Oct-1,2, and unc-86) domain region of Oct-1 protein, which specifically bound to octamer DNA motif (ATGCAAAT). The affinity-purified recombinant POU domain proteins conjugated with glutathione-S-transferase (GST) contained three distinct proteins with molecular weights of 34, 36, and 45 kDa. The photoaffinity EMSA could clearly distinguish the individual binding abilities of three proteins on a single lane and showed that the whole POU domain protein specifically bound to octamer DNA motif by competition experiments. Using the nuclear extract of HeLa cells, the photoaffinity EMSA revealed that at least five specific proteins could bind to the octamer DNA motif. These results show that photoaffinity EMSA using 3-trifluoromethyl-3-phenyldiazirine can provide high-performance analysis of DNA-binding proteins.     

Liquid chemiluminescent DNA pull-down assay to measure nuclear receptor-DNA binding in solution

Electrophoretic mobility shift assays (EMSAs) are commonly used to investigate protein-DNA binding in vitro. However, EMSA can generate considerable amounts of undesirable waste, particularly when toxic compounds are examined. We therefore developed a novel in vitro protein-DNA binding assay called liquid chemiluminescent DNA pull-down assay, which is based on solution hybridization between digoxigenin-labeled DNA and glutathione S-transferase (GST)-fused DNA binding protein bound to glutathione-Sepharose beads.     

Multimerization of restriction fragments by magnesium-mediated stable base pairing between overhangs: a cause of electrophoretic mobility shift

The electrophoretic mobility shift assay (EMSA) or simply the "gel shift assay" is one of the most sensitive methods for studying the ability of a protein to bind to DNA. EMSAs are also widely used to investigate protein- or sequence-dependent DNA bending. Here we report that electrophoresis using physiological concentrations of Mg(2+) can cause a mobility shift of restriction fragments in nondenaturing polyacrylamide gels as the overhangs form stable base pairs. This phenomenon was observed at even 37 degrees C. The retardation was, however, more pronounced at low temperatures, where a three-nucleotide overhang 5'-GAC also caused a mobility shift. The stability of the pairing was generally high when the overhangs of four nucleotides display high GC content, while the mobility shift caused by 5'-AATT was greater than those caused by 5'-GATC, 5'-TCGA, and 5'-CTAG. This observation should be taken into account to avoid misinterpretation of the data when the EMSA, especially the circular permutation gel mobility shift assay, is performed using a running buffer that contains Mg(2+) ions. The stable adhesion between short overhangs may present an important basis for genome stability and many genetic processes occurring in living cells.     

UV cross-linking/immunoprecipitation assay: glucocorticoid receptor-adrenergic receptor gene sequence interaction

The rat beta 1-adrenergic receptor (beta 1-AR) gene contains glucocorticoid response element (GRE) half-sites at positions -2767 and -945. In electrophoretic mobility shift assay (EMSA) experiments, neither beta 1-AR GRE half-site recognized glucocorticoid receptors (GRs) obtained from baculovirus high-level expression systems or from mammalian cells. We have developed a sensitive UV cross-linking/immunoprecipitation assay, using a 524-bp fragment containing the prototypical GRE obtained from the rat tyrosine aminotransferase promoter sequence and using antibodies recognizing mammalian GR. Using this assay, we provide evidence that rat beta 1-AR gene sequences recognize mammalian GRs expressed in mouse 3T3 cells and that the site of GR interaction does not appear to specifically contain the beta 1-AR GRE half-sites. This represents one of the first reports demonstrating the utility of a UV cross-linking/immunoprecipitation assay in the detection of mammalian GR interaction with beta 1-AR sequences, is consistent with the lack of specific DNA-GR protein complexes observed in EMSA experiments using oligonucleotide probes containing the beta 1-AR GRE half-sites, and provides evidence that mammalian GR interaction occurs at complex rate beta 1-AR gene sequences.     

植物转录因子与DNA互作研究技术

转录通过调控下游基因的时空特异性表达影响植物生长发育。在转录调控机制的解析过程中, 转录因子与DNA的相互作用是关键的一环。近年来, 研究者利用酵母单杂交(Y1H)和凝胶阻滞迁移率实验(EMSA)检测转录因子能否直接结合DNA; 而瞬时表达技术则是一种检测转录因子对下游基因调控作用的便捷方式。该文对Y1H、EMSA和瞬时表达技术的原理、实验方法和相关注意事项进行详细阐述, 以期为转录因子与DNA的互作研究提供参考方法。     

Southwestern blotting in investigating transcriptional regulation

Southwestern blotting is used to investigate DNA-protein interactions. The advantage of this technique over other related methods such as electrophoretic mobility shift assay (EMSA) and DNA footprinting is that it provides information regarding the molecular weight of unknown protein factor. This method combines the features of Southern and Western blotting techniques; a denaturing SDS-PAGE is first employed to separate proteins electrophoretically based on size, and after transferring the proteins to a membrane support, the membrane-bound proteins are renatured and incubated with a (32)P-labeled double-stranded oligonucleotide probe of specific DNA sequence. The interaction of the probe with the protein(s) is later visualized by autoradiography. This technique could be combined with database searching (TransFac, http://www.gene-regulation.com/pub/databases.html#transfac), prediction of potential protein factors binding onto a target motif (e.g., Patch search), in vitro supershift EMSA and in vivo chromatin immunoprecipitation (ChIP) assays for effective identification of protein factors. The whole Southwestern blotting procedure takes approximately 4 d to complete. In this article, a commonly used protocol and expected results are described and discussed.     

近红外荧光电泳迁移率变动分析的实验方法

目的:分析最新近红外荧光标记(NIRF)电泳迁移率变动分析(EMSA)技术的优缺点,在此基础上对NIRF-EMSA技术进行实验方法的改进。方法:设计了3种NIRF-EMSA实验方法,即扫胶法、染胶法和扫膜法,通过实验比较了3种方法的实验程序复杂性、优缺点及检测成本,并将3种方法与化学发光EMSA进行了比较。结果:采用3种方法都可以取得良好的EMSA检测效果,但比较而言,间接扫膜法不仅效果良好,而且实验成本最低。结论:间接扫膜法是值得推广应用的最经济便捷的基于Odyssey红外荧光成像系统的近红外荧光EMSA实验方法。     

The nuclear protein p30 specifically interacts with a nuclear matrix attachment region from the rat genome

In our previous study, a 454 bp DNA fragment was isolated from rat genomic DNA as an element which interacts with nuclear matrix proteins, i.e. a Matrix Associated Region (MAR). Computer analyses revealed that the right half of this fragment, named RME (Rat MAR Element), possesses a high matrix association potential and is likely to be responsible for the matrix association of the whole sequence. RME was used as a probe in an electrophoretic mobility shift assay (EMSA), and with the use of Southwestern blotting, a rat liver nuclear protein which binds specifically to it was identified. Its molecular mass was estimated by SDS-PAGE as 30 kDa (p30). Polyclonal antibodies raised against protein-RME complexes caused a super-shift of specific complexes in EMSA, and bound to p30 in nuclear extracts of rat liver in Western blotting. The immunofluorescence labelling of a rat embryonic fibroblast cell monolayer with anti-p30 antibody revealed a mainly intranuclear pattern of staining.     

A customized retroviral vector confers marker gene expression in osteoclast lineage cells

Osteoclasts play a seminal role in many skeletal diseases and therefore are candidates for cell-based gene delivery systems to treat disorders of bone. As an initial step toward developing osteoclast-mediated gene delivery systems, we have made and analyzed a customized Molony-Murine leukemia virus (MMLV)-based retroviral vector containing elements of the osteoclast-specific tartrate-resistant acid phosphatase (TRAP) gene. RAW 264.7 cells were transduced with the customized vector (E3) and differentiated along macrophage or osteoclast lineages. E3 contained a truncated form of the human nerve growth factor receptor (NGFR) as a reporter gene. NGFR expression increased with RANK-ligand (RANK-L) treatment but not with macrophage (gamma-IFN/LPS treatment) differentiation. Enhanced NGFR expression peaked 48 h after RANK-L treatment. Electrophoretic mobility shift assays (EMSA) analysis of the TRAP gene regulatory elements in E3 identified a single 27 bp DNA probe, which specifically bound protein from RANK-L-treated cells. DNA sequence revealed AP-1 binding sites, and analysis with mutant probes implied that the sites were functional. EMSA supershift analysis identified Fos protein interacting with the 27 bp probe. In summary, insertion of sequence -962 to -868 from the TRAP gene into the U3 region of the MMLV LTR confers RANK-L induced retroviral gene expression via Fos family protein interaction at AP-1 sites.