An improved procedure for protein staining in polyacrylamide gels with a new type of Coomassie Brilliant Blue

Diezel, W; Kopperschläger, Gèrhard; Hofmann, E

doi:10.1016/0003-2697(72)90117-0

Cited by 479 publications

(105 citation statements)

References 10 publications

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“…Following electrophoresis, gels were fixed in 12.5% trichloroacetic acid for 5 min and stained with Coomassie Brilliant Blue (14). Prestained molecular weight protein standards were obtained from Pharmacia Biotech Inc.…”

Section: Methodsmentioning

confidence: 99%

Disulfide Bond Assignment in Human Interleukin-7 by Matrix-assisted Laser Desorption/Ionization Mass Spectroscopy and Site-directed Cysteine to Serine Mutational Analysis

Cosenza¹,

Sweeney²,

Murphy³

1997

Journal of Biological Chemistry

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Interleukin-7 (IL-7) is a proteinaceous biological response modifier that has a bioactive tertiary structure dependent on disulfide bond formation. Disulfide bond assignments in human (h)IL-7 are based upon the results of matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy and Cys to Ser mutational analyses. A gene encoding the hIL-7 was synthesized incorporating Escherichia coli codon usage bias and was used to express biologically active protein as determined by stimulation of precursor B-cell proliferation. MALDI mass spectroscopic analysis of trypsin-digested hIL-7 was performed and compared with the anticipated results of a simulated tryptic digestion. Many of the anticipated hIL-7 tryptic fragments were detected including one with a molecular mass equivalent to the sum of two polypeptides linked through a disulfide bond respectively. In single disulfide bond-forming mutants of hIL-7, the ability to stimulate cell proliferation was abolished in the presence of 2 mM dithiothreitol. The results presented strongly suggest that only a single disulfide bond is required for hIL-7 to form a tertiary structure capable of stimulating precursor B-cell proliferation.

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Section: Methodsmentioning

confidence: 99%

Disulfide Bond Assignment in Human Interleukin-7 by Matrix-assisted Laser Desorption/Ionization Mass Spectroscopy and Site-directed Cysteine to Serine Mutational Analysis

Cosenza¹,

Sweeney²,

Murphy³

1997

Journal of Biological Chemistry

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“…The pellets were suspended in water, treated with ribonuclease A (1.25 jig/A260 unit), dissolved in sample buffer and subjected to electrophoresis (2 A260 units/gel) on 15 % polyacrylamide gels (0.6 cm x 22cm, with a 1 cm 3 % polyacrylamide stacking gel) at 2 mA/gel for 15 h as described by Laemmli (1970). After electrophoresis, the gels were stained with Coomassie Brillant Blue G-250, and destained with 7% (v/v) acetic acid as described by Diezel et al (1972). Molecular-weight calibration of the gels was obtained by using ribonuclease (apparent mol.wt.…”

Section: Methodsmentioning

confidence: 99%

Differences in size, structure and function of free and membrane-bound polyribosomes of rat liver. Evidence for a single class of membrane-bound polyribosomes

Ramsey¹,

Steele²

1977

Biochemical Journal

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Free loosely bound and tightly bound polyribosomes were separated from rat liver homogenate by salt extraction followed by differential centrifugation, and several of their structural and functional properties were compared to resolve the existence of loosely bound polyribosomes and verify the specificity of the separation. The free and loosely bound polyribosomes have similar sedimentation profiles and polyribosome contents, their subunit proteins have similar electrophoretic patterns and their products of protein synthesis in vitro show a close correspondence in size and amounts synthesized. In contrast, the tightly bound polyribosomes have different properties from those of the free and loosely bound polyribosomes; their average size is significantly smaller; their polyribosome content is higher; their 60S-subunit proteins lack two components and contain four or more components not found elsewhere; their products of protein synthesis in vitro differ in size and amounts synthesized. These observations show that rat liver membranes entrap a large fraction of the free polyribosomes at low salt concentrations and that these polyribosomes are similar to those ofthe free-polyribosome fraction and are different from those of the tightly bound polyribosome fraction in size, structure and function.

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“…Over the years, there have been a remarkably large number of studies dedicated to improving solvent composition, changing dye concentration/type and developing strategies for staining protein/destaining gel matrix in 1D PAGE or isoelectric focusing (IEF) gels to potentially achieve higher levels of sensitivity [30][31][32][33][34][35][36][37][38][39][40][41][42]. This continuous drive for improvement, however, was not able to enhance the detection sensitivity of CBB below ∼30 ng of bovine serum albumin (BSA) or actin [33,[43][44][45].…”

Section: Coomassie Brilliant Bluementioning

confidence: 99%

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

2010

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Proteomics research relies heavily on visualization methods for detection of proteins separated by polyacrylamide gel electrophoresis. Commonly used staining approaches involve colorimetric dyes such as Coomassie Brilliant Blue, fluorescent dyes including Sypro Ruby, newly developed reactive fluorophores, as well as a plethora of others. The most desired characteristic in selecting one stain over another is sensitivity, but this is far from the only important parameter. This review evaluates protein detection methods in terms of their quantitative attributes, including limit of detection (i.e., sensitivity), linear dynamic range, inter-protein variability, capacity for spot detection after 2D gel electrophoresis, and compatibility with subsequent mass spectrometric analyses. Unfortunately, many of these quantitative criteria are not routinely or consistently addressed by most of the studies published to date. We would urge more rigorous routine characterization of stains and detection methodologies as a critical approach to systematically improving these critically important tools for quantitative proteomics. In addition, substantial improvements in detection technology, particularly over the last decade or so, emphasize the need to consider renewed characterization of existing stains; the quantitative stains we need, or at least the chemistries required for their future development, may well already exist.

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An improved procedure for protein staining in polyacrylamide gels with a new type of Coomassie Brilliant Blue

Cited by 479 publications

References 10 publications

Disulfide Bond Assignment in Human Interleukin-7 by Matrix-assisted Laser Desorption/Ionization Mass Spectroscopy and Site-directed Cysteine to Serine Mutational Analysis

Disulfide Bond Assignment in Human Interleukin-7 by Matrix-assisted Laser Desorption/Ionization Mass Spectroscopy and Site-directed Cysteine to Serine Mutational Analysis

Differences in size, structure and function of free and membrane-bound polyribosomes of rat liver. Evidence for a single class of membrane-bound polyribosomes

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

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