Simultaneous immunoblotting analysis with activity gel electrophoresis in a single polyacrylamide gel

Chen, Hung−Wen; Chang, Geen-Dong

doi:10.1002/1522-2683(200106)22:10<1894::aid-elps1894>3.0.co;2-5

Cited by 22 publications

(9 citation statements)

References 23 publications

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“…The classical approach to this problem has been the electrophoretic supershift assay, in which an antibody against a candidate protein reduces the gel-mobility of the protein-nucleic acid complex, producing a secondary mobility shift 84 . More recently, 2-dimensional strategies in which EMSA is paired with SDS-PAGE with Western blot detection 85,86 , or paired with mass spectrometry 87,88 have started to bring the power of proteomics to bear on this problem. A detailed protocol describing EMSA paired with SDS-PAGE and mass spectrometry for protein identification has recently been published 89 .…”

Section: Anticipated Resultsmentioning

confidence: 99%

Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions

2007

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The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32 P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this article, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided.

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Section: Anticipated Resultsmentioning

confidence: 99%

Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions

2007

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“…Otherwise, the tissue extracts were mixed with an equal volume of 2-fold concentrated SDS sample buffer, boiled for 5 min, and stored at Ϫ70°C. Immunoblotting was carried out using anti-nephrosin inhibitor or anti-nephrosin antiserum (1:2000 dilution) and horseradish peroxidase-conjugated second antibody (1:10,000 dilution) after diffusion blotting onto polyvinylidene difluoride (PVDF) membrane (34). Immunoreactive bands were detected by the NiCl 2 enhancement method (35).…”

Section: Methodsmentioning

confidence: 99%

Purification and Cloning of an Endogenous Protein Inhibitor of Carp Nephrosin, an Astacin Metalloproteinase

Tsai

Chen

Huang

et al. 2004

Journal of Biological Chemistry

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Nephrosin is a newly discovered member of the astacin family. It is a secreted proteinase and is present in carp head kidney, kidney, and spleen, all of which are responsible for immune and hematopoietic functions in fish. A complex formed by nephrosin and its inhibitor was purified from carp kidney extract by heparin affinity column chromatography. The presence of the nephrosin-inhibitor complex in different tissues was examined by immunoblotting with polyclonal antisera against the purified nephrosin inhibitor and nephrosin. Both nephrosin and the nephrosin inhibitor were present mainly in gill, head kidney, kidney, and spleen. In addition, we have cloned the cDNA encoding the nephrosin inhibitor. There are two different cDNA clones possibly resulting from two different genes, and the long form contains unique tandem repeat sequences in the 3 -end. The deduced primary structure of nephrosin inhibitor is similar to that of fetuin-A, a mammalian protein present in blood, liver, cerebrospinal fluid, and cerebral cortex during fetal development. Treatment with both N-glycosidase F and O-glycosidase removed the carbohydrate moiety of the nephrosin inhibitor and decreased the apparent molecular mass from 40 to 30 kDa. The nephrosin inhibitor seems to be synthesized in liver and then secreted to the blood as a precursor. When it was distributed into hematopoietic tissues, it was processed from 67 to 40 kDa and acquired inhibitory activity. This processing phenomenon of fetuin has not been reported elsewhere. Importantly, the presence of an endogenous inhibitor of nephrosin is the first report of this kind for astacin enzymes. It is very likely that endogenous tissue inhibitors may also be present for the regulation of other astacin enzymes.

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“…Several methods are available for the measurement of stoichiometries of complexes resolved in native gels. These include the labeling of proteins and RNA with complementary radioisotopes [52], noncovalent staining with dyes or fluorophores [8,36,37], and Northern-and Western-blotting [53] with appropriate controls (see above). Where sample quantities are not limiting, a rigorous free-solution method such as analytical ultracentrifugation [54,55] should also be considered.…”

Section: Binding Stoichiometrymentioning

confidence: 99%

Detection of RNA–Protein Complexes by Electrophoretic Mobility Shift Assay

Fried

2012

Alternative Pre‐mRNA Splicing

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The electrophoretic mobility shift assay (EMSA) is commonly used to study proteinnucleic acid interactions. The technique is based on the observation that proteinbound nucleic acid molecules migrate more slowly than free nucleic acid molecules when subjected to native polyacrylamide or agarose gel electrophoresis. After electrophoresis, the distribution of species containing nucleic acid is determined by autoradiography or other sensitive imaging technique. Under appropriate conditions, the method can be used for quantitative binding analysis, but it is most widely used as a qualitative means of detecting protein-RNA complexes. In this chapter, features relevant to the design of EMSA experiments are identified, and the technical factors found to be important for the success of the assay are outlined. Theoretical BackgroundThe electrophoretic mobility shift assay (EMSA) is a simple and powerful method for detecting interactions between nucleic acids and proteins [1][2][3][4][5][6]. Although the simplicity of the method accounts for its popularity, it also conceals technical subtleties. In this chapter, the aim is to help select the most convenient EMSA variants for experimental purposes, and to avoid the most important pitfalls associated with the technique.The EMSA is based on the observation that the gel-electrophoretic mobility of a protein-nucleic acid complex is often less than that of the corresponding free nucleic acid. Current versions of the assay differ little from those originally described by Garner and Revzin [7] and Fried and Crothers [8], although precursors can be found in earlier reports [9][10][11]. Although originally developed to analyze DNA-protein complexes, the method is also useful for the characterization of RNA-protein interactions [12][13][14].The electrophoretic mobilities of RNA molecules in polyacrylamide or agarose gels depend on: (i) the sizes, shapes, and charges of the molecules; (ii) the conductivities of the gel and sample buffers; and (iii) the concentrations of the gel polymer and its degree of crosslinking [15]. Protein binding can result in complexes that differ from the parent RNA in terms of charge, size or shape; changes in any or all of these factors may produce a difference in gel-mobility that allows the detection of binding. Changes in the type of gel matrix (e.g., agarose versus polyacrylamide), its concentration, and changes in the composition of the gel buffer, can each affect electrophoretic resolution and, just as importantly, the stabilities of the protein-nucleic acid complexes within the gel [16][17][18][19]. Optimization of these factors is straightforward and can significantly improve the results of an EMSA.Alternative pre-mRNA Splicing: Theory and Protocols, First Edition. EditedThe selection of a binding substrate requires assumptions to be made about the RNA structure(s) needed for appropriate protein binding. If the structure of the RNA probe differs significantly from the native cellular binding target, the behavior of the assay system may not be repre...

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Simultaneous immunoblotting analysis with activity gel electrophoresis in a single polyacrylamide gel

Cited by 22 publications

References 23 publications

Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions

Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions

Purification and Cloning of an Endogenous Protein Inhibitor of Carp Nephrosin, an Astacin Metalloproteinase

Detection of RNA–Protein Complexes by Electrophoretic Mobility Shift Assay

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