Quantitation of protein on gels and blots by infrared fluorescence of Coomassie blue and Fast Green

Luo, Shen; Wehr, Nancy B.; Levine, Rodney L.

doi:10.1016/j.ab.2005.10.048

Cited by 109 publications

(108 citation statements)

References 10 publications

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“…This method is considered generally to be less sensitive than silver or fluorescent staining techniques, but is less susceptible to between-gel variability than silver stain (25). Moreover, enhanced sensitivity has been reported when near-infrared fluorescence imaging has been used for detection (19) in which case Coomassie brilliant blue is comparable with Sypro ruby stain (9).…”

Section: Discussionmentioning

confidence: 99%

Proteomic analysis of epicardial and subcutaneous adipose tissue reveals differences in proteins involved in oxidative stress

Salgado-Somoza¹,

Teijeira-Fernández

Fernández

et al. 2010

American Journal of Physiology-Heart and Circulatory Physiology

144

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Salgado-Somoza A, Teijeira-Fernández E, Fernández AL, González-Juanatey JR, Eiras S. Proteomic analysis of epicardial and subcutaneous adipose tissue reveals differences in proteins involved in oxidative stress. Am J Physiol Heart Circ Physiol 299: H202-H209, 2010. First published April 30, 2010; doi:10.1152/ajpheart.00120.2010.-Epicardial adipose tissue (EAT) is an endocrine organ adjacent to coronary arteries and myocardium without anatomy barriers. Locally produced adipokines may reflect or affect to cardiovascular physiology and pathology. Our aim was to study the protein expression profiles of EAT and subcutaneous adipose tissue (SAT) to identify local candidate molecules characterizing EAT in patients with cardiovascular disease. EAT and SAT samples were collected from 55 patients undergoing heart surgery. Proteins from these tissues were separated by two-dimensional (2D) gel electrophoresis, and differences between them were identified by MALDI-TOF/TOF spectra. Differences in protein levels were further investigated by real-time RT-PCR and Western blots, and production of reactive oxygen species (ROS) in EAT and SAT was evaluated by nitroblue tetrazolium chloride assays. ROS production was higher in EAT than SAT. We have found mRNA differences for catalase, glutathione S-transferase P, and protein disulfide isomerase, and 2D Western blots additionally showed post-translational differences for phosphoglycerate mutase 1; all four are related to oxidative stress pathways. EAT suffers greater oxidative stress than SAT in patients with cardiovascular diseases and exhibits associated proteomic differences that suggest the possibility of its association with myocardial stress in these patients. epicardial adipose tisssue OBESITY IS ONE OF THE MAIN causes of metabolic diseases. It is associated with a chronic inflammatory response and cardiovascular diseases (34). However, risk depends significantly on the distribution of adipose tissue in the body; for example, metabolic risk factors were more associated with omental adipose tissue (OAT) than with subcutaneous adipose tissue (SAT) among 3,001 participants in the Framingham Heart Study (6). These differences are plausibly due to different adipose tissues differing in adipokine expression and the production of inflammatory reactive oxygen species (ROS), which are known to be associated with cardiovascular disease (8); OAT is associated with more circulating substances related to inflammation and oxidative stress than SAT is (24).Epicardial adipose tissue (EAT) is a visceral fat pad adjacent to coronary arteries and the myocardium (11). The amount of EAT is related to left ventricle mass (13) and to the amount of intra-abdominal visceral fat, and visceral adiposity can in fact be evaluated by means of its echocardiographic measurement (10). Like other adipose tissues, EAT is now recognized as an endocrine organ, and its proximity to myocardium and coronary arteries suggests the possibility of paracrine action on these structures. In fact, in patients with coronary ...

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Section: Discussionmentioning

confidence: 99%

Proteomic analysis of epicardial and subcutaneous adipose tissue reveals differences in proteins involved in oxidative stress

Salgado-Somoza¹,

Teijeira-Fernández

Fernández

et al. 2010

American Journal of Physiology-Heart and Circulatory Physiology

144

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“…As noted above, although several other published improvements to CBB staining protocols reportedly also deliver increased sensitivity, the assessments carried out in those studies unfortunately do not allow for their evaluation in this review [21,[51][52][53][54][55]. Similarly, alternate staining strategies have also been developed to improve protein detection sensitivity by combining CBB with other stains.…”

Section: Coomassie Brilliant Bluementioning

confidence: 99%

“…Most commercial CBB stains, usually the G form, are marketed for densitometric detection. However, CBB may be re-visited as a sensitive stain once again since recent literature has indicated that near-infrared fluorescence detection of proteins by CBB offers improved sensitivity [21,54]. Even though the densitometric use of CBB is relatively insensitive, use of this stain may be reborn in proteomics for fluorescent protein detection.…”

Section: Coomassie Brilliant Bluementioning

confidence: 99%

“…Alternate densitometric stains have not gained popularity within the proteomic research community because these stains have been unable to compete with the performance of fluorescent staining and imaging. Also, most of these alternate densitometric stains have, at best, had limited characterization or have similar detection sensitivity to cCBB and hence have not been pursued for further application in proteomics [54,[117][118][119][120][121][122][123][124][125][126].…”

Section: Additional Densitometric Stainsmentioning

confidence: 99%

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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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“…To determine protein concentration, gels were stained with Coomassie stain as described (21) and rescanned. Protein detection was in the 700 nm channel.…”

Section: Methodsmentioning

confidence: 99%

Roles for Trafficking and O-Linked Glycosylation in the Turnover of Model Cell Surface Proteins

Karabasheva

Cole

Donaldson

2014

Journal of Biological Chemistry

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Background: Plasma membrane proteins can be degraded by a number of mechanisms. Results: Turnover rates are determined by endocytosis signals and trafficking to lysosomes, ubiquitination, and ectodomain cleavage. Conclusion: Membrane trafficking to lysosomes and proteolysis at the cell surface determine protein half-life. Significance: Learning how plasma membrane proteins are turned over is critical for understanding protein homeostasis.

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Quantitation of protein on gels and blots by infrared fluorescence of Coomassie blue and Fast Green

Cited by 109 publications

References 10 publications

Proteomic analysis of epicardial and subcutaneous adipose tissue reveals differences in proteins involved in oxidative stress

Proteomic analysis of epicardial and subcutaneous adipose tissue reveals differences in proteins involved in oxidative stress

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

Roles for Trafficking and O-Linked Glycosylation in the Turnover of Model Cell Surface Proteins

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