Two-Dimensional Polyacrylamide Gel Electrophoresis of Proteins Labeled with the Fluorophore Monobromobimane Prior to First-Dimensional Isoelectric Focusing: Imaging of the Fluorescent Protein Spot Patterns Using a Cooled Charge-Coupled Device

Urwin, Valerie E.; Jackson, Peter

doi:10.1006/abio.1993.1082

Cited by 58 publications

(25 citation statements)

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“…Although mBB can be used to prelabel protein prior to IEF for 2DE, like other reactive dyes, it only detects a specific reactive group, so those proteins lacking thiols go undetected. Pre-labeling may also affect the mobility of some low MW proteins [150].…”

Section: Reactive Fluorescent Dyesmentioning

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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Section: Reactive Fluorescent Dyesmentioning

confidence: 99%

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

2010

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“…The compound monobromobimane (TMB) has been used to fluorescently label sulfhydryl groups in protein solutions prior to electrophoresis and to study lymphoid cell line patterns in comparison to silver stain with equal protein loads [106]. Small changes in pI and M r have been found for TMBlabeled proteins as well as variations in relative spot intensities.…”

Section: Modified Cysteine Residuesmentioning

confidence: 99%

Protein stains for proteomic applications:Which, when, why?

Crawford²,

2006

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This review recollects literature data on sensitivity and dynamic range for the most commonly used colorimetric and fluorescent dyes for general protein staining, and summarizes procedures for the most common PTM-specific detection methods. It also compiles some important points to be considered in imaging and evaluation. In addition to theoretical considerations, examples are provided to illustrate differential staining of specific proteins with different detection methods. This includes a large body of original data on the comparative evaluation of several pre-and post-electrophoresis stains used in parallel on a single specimen, horse serum run in 2-DE (IPG-DALT). A number of proteins/protein spots are found to be over-or under-revealed with some of the staining procedures.

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“…We and others [11][12][13] have previously examined conditions for labeling proteins with thiolreactive dyes, and defined the basic properties these dyes must exhibit in order to function as viable candidates; these include high extinction coefficients, good quantum yields, pH insensitivity of fluorescence, low photo-bleaching, minimal effects on protein pIs, a high degree of chemical specificity, and the ability to achieve reproducible saturation of protein residues. In our study [11], we noted that thiourea, an important component of many protein solubilization cocktails, severely inhibited labeling of protein thiols by iodoacetamide dyes, but not by a maleimide dye, in agreement with others who observed similar reactivities [14].…”

Section: Introductionmentioning

confidence: 99%

Saturation fluorescence labeling of proteins for proteomic analyses

Pretzer

Wiktorowicz

2008

Analytical Biochemistry

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We present here an optimized and cost-effective approach to saturation fluorescence labeling of protein thiols for proteomic analysis. We investigated a number of conditions and reagent concentrations including a disulfide reducing agent (TCEP), pH, incubation time, linearity of labeling, and saturating dye: protein thiol ratio with protein standards to gauge specific and nonspecific labeling. Efficacy of labeling under these conditions was quantified using specific fluorescence estimation, defined as the ratio of fluorescence pixel intensities and Coomassie-stained pixel intensities of bands after digital imaging. Factors leading to specific vs. non-specific labeling in the presence of thiourea are also discussed.We have found that reproducible saturation of available Cys residues of the proteins used as labeling standards (human carbonic anhydrase I, enolase, α-lactalbumin) is achieved at 50-100-fold excess of the uncharged maleimide-functionalized BODIPY™ dyes over Cys. We confirm our previous findings and those of others that the maleimide dyes are not impacted by the presence of 2M thiourea. Moreover, we establish that 2 mM TCEP used as reductant is optimal. We also establish further that labeling is optimal at pH 7.5 and complete after 30 min. Low non-specific labeling was gauged by the inclusion of non-Cys containing proteins (horse myoglobin, bovine carbonic anhydrase) to the labeling mixture. We also show that the dye exhibits little to no effect on the two dimensional mobilities of labeled proteins derived from cells.

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Two-Dimensional Polyacrylamide Gel Electrophoresis of Proteins Labeled with the Fluorophore Monobromobimane Prior to First-Dimensional Isoelectric Focusing: Imaging of the Fluorescent Protein Spot Patterns Using a Cooled Charge-Coupled Device

Cited by 58 publications

References 0 publications

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

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

Protein stains for proteomic applications:Which, when, why?

Saturation fluorescence labeling of proteins for proteomic analyses

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