Counterion‐dye staining for DNA in electrophoresed gels using indoine blue and methyl orange

Hwang, Sunyoung; Jin, Litai; Yoo, Gyurng-Soo; Choi, Jung-Kap

doi:10.1002/elps.200500640

Cited by 9 publications

(4 citation statements)

References 13 publications

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“…This was confirmed by our own result from methylene blue staining (data not shown). More recently, Choi et al developed the counterion−dye staining method, , which showed low background staining and hence improved sensitivity. We tried this method, but the sensitivity was poor in our hands, possibly because this method is not compatible with SDS−PAGE.…”

Section: Resultsmentioning

confidence: 99%

Use of DNA Ladders for Reproducible Protein Fractionation by Sodium Dodecyl Sulfate−Polyacrylamide Gel Electrophoresis (SDS−PAGE) for Quantitative Proteomics

2008

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In proteomics, one-dimensional (1D) SDS-PAGE is widely used for protein fractionation prior to mass spectrometric analysis to enhance dynamic range of analysis and to improve identification of low-abundance proteins. Such protein pre-fractionation works well for quantitation strategies if the proteins are labeled prior to separation. However, due to the poor reproducibility of cutting gel slices, especially when small amounts of samples are analyzed, its application in label-free and peptide labeling quantitative proteomics methods has been greatly limited. To overcome this limitation, we developed a new strategy in which a DNA ladder is mixed with the protein sample before PAGE separation. After PAGE separation, the DNA ladder is stained to allow for easy, precise and reproducible gel cutting. To this end, a novel visible DNA staining method was developed. This staining method is fast, sensitive and compatible with mass spectrometry. To evaluate the reproducibility of DNA ladder assisted gel cutting for quantitative protein fractionation, we used SILAC (stable isotope labeling with amino acids in cell culture). Our results show that the quantitative error associated with fractionation can be minimized by using the DNA assisted fractionation and multiple replicates of gel cutting. In conclusion, 1D PAGE fractionation in combination with DNA ladders can be used for label-free comparative proteomics without compromising quantitation.

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Section: Resultsmentioning

confidence: 99%

Use of DNA Ladders for Reproducible Protein Fractionation by Sodium Dodecyl Sulfate−Polyacrylamide Gel Electrophoresis (SDS−PAGE) for Quantitative Proteomics

2008

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“…For example, interactions with human serum albumin have been reported (Ito & Yamamoto, 2015). To our knowledge, methyl orange alone does not interact with DNA; however, when used in combination with other dyes like crystal violet or indoine blue, the resulting complexes are able to interact with DNA (Hwang, Jin, Yoo, & Choi, 2006;Yang, Jung, Bai, Yoo, & Choi, 2001). In such delicate conditions, another method for CMC determination should be considered.…”

Section: Troubleshootingmentioning

confidence: 99%

“…Current Protocols in Protein Science combination with other dyes like crystal violet or indoine blue, the resulting complexes are able to interact with DNA (Hwang, Jin, Yoo, & Choi, 2006;Yang, Jung, Bai, Yoo, & Choi, 2001). In such delicate conditions, another method for CMC determination should be considered.…”

Section: Of 27mentioning

confidence: 99%

Screening of Detergents for Stabilization of Functional Membrane Proteins

et al. 2018

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Membrane protein studies usually require use of detergents to extract and isolate proteins from membranes and manipulate them in a soluble context for their functional or structural characterization. However, solubilization with detergent may interfere with MP stability and may directly affect MP function or structure. Moreover, detergent properties can be affected such as critical micellar concentration (CMC) can be affected by the experimental conditions. Consequently, the experimenter must pay attention to both the protein and the behavior of the detergent. This article provides a convenient protocol for estimating the CMC of detergents in given experimental conditions. Then, it presents two protocols aimed at monitoring the function of a membrane protein in the presence of detergent. Such experiments may help to test various detergents for their inactivating or stabilizing effects on long incubation times, ranging from few hours to some days. © 2018 by John Wiley & Sons, Inc.

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“…In addition, mercury can affect human body organs through binding to the thiol groups of amino acids and proteins, thereby causing serious damage to the central nervous system, brain, kidney, and respiratory system. [14][15][16][17][18][19] The US Environmental Protection Agency (EPA) and the World Health Organization (WHO) have set the maximum allowed concentration of Hg 2+ in drinking water at 2 ppb (10 nM). 20 The determination of Hg 2+ pollution in environment is usually carried out by conventional advanced methods such as inductively coupled plasma mass spectroscopy (ICP-MS), 21 atomic absorption spectroscopy (AAS), 22 high performance liquid chromatography (HPLC), 23 and inductively coupled plasma-atomic emission spectrometry (ICP-AES).…”

Section: Introductionmentioning

confidence: 99%