Detection of Five Nanograms of Protein by Two-Minute Nile Red Staining of Unfixed SDS Gels

Fj, Alba; Bermúdez, Antonio; Bartolomé, Salvador; Daban,

doi:10.2144/96214bm12

Cited by 31 publications

(30 citation statements)

References 4 publications

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“…Not only was a filter (Corning 051) required to remove excitation energy but since the Gilford was designed to read absorbance, it was necessary to calculate the antilogarithm of peaks to obtain a measurement of signal. It gradually became more common to transilluminate gels with UV light to detect fluorescently stained proteins [20,157,158,182,184,186,190,191,195,246,247]. Subsequently, photographs of transilluminated gels had to be scanned by a densitometer before quantitative analysis could be undertaken; again, all without the aid of local computers that are now considered standard lab equipment.…”

Section: Equipment Innovationsmentioning

confidence: 99%

“…The maximum signal detectable by this camera was 10 5 electrons while the read noise or minimum signal was 1 electron. The range of detection afforded by this system presented an important development in the routine quantification of proteins and the application of CCD based systems for imaging gels still remains one of the most prevalent technologies in use today [193,194,196,197,236,[247][248][249].…”

Section: Equipment Innovationsmentioning

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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Section: Equipment Innovationsmentioning

confidence: 99%

Section: Equipment Innovationsmentioning

confidence: 99%

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

2010

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“…The dried gels were exposed to the bio-imaging plate for 2 h at room temperature, and the plates were analyzed using an imaging plate reader and bio-imaging analyzer (BAS 2000, Fujifilm, Tokyo, Japan). Protein bands in SDS-PAGE gels under the reducing condition were visualized by the fluorescent dye Nile Red (14).…”

Section: Materials-[mentioning

confidence: 99%

120- and 160-kDa Receptors for Endogenous Mitogenic Peptide, Phytosulfokine-α, in Rice Plasma Membranes

Matsubayashi¹,

Sakagami

2000

Journal of Biological Chemistry

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“…7 Since then, this procedure has been in common use for the identification of electrophoretically separated proteins by peptide mass and mass spectrometric microsequencing. 8,9 In recent years, alternative sensitive and non-fixative staining of proteins in gels using fluorescent dyes such as Nile Red, 10 SYPRO Red and SYPRO Orange 11,12 have also been described. Although the non-covalent Nile Red dye permits rapid dye protein detection in sodium dodecyl sulfate (SDS) gel in a few minutes and is useful for protein microsequencing or immunodetection, 10,12,13 it is less sensitive than imidazole-zinc or silver stains.…”

Section: Introductionmentioning

confidence: 99%

“…8,9 In recent years, alternative sensitive and non-fixative staining of proteins in gels using fluorescent dyes such as Nile Red, 10 SYPRO Red and SYPRO Orange 11,12 have also been described. Although the non-covalent Nile Red dye permits rapid dye protein detection in sodium dodecyl sulfate (SDS) gel in a few minutes and is useful for protein microsequencing or immunodetection, 10,12,13 it is less sensitive than imidazole-zinc or silver stains. Contrariwise, the two new fluorescent protein gel stains SYPRO Orange and SYPRO Red dyes have a sensitivity of 1-10 ng of protein, 11,12,14 equalling that of silver stain, 7 making it possible to detect even minimally expressed proteins.…”

Section: Introductionmentioning

confidence: 99%

Novel procedure for the identification of proteins by mass fingerprinting combining two-dimensional electrophoresis with fluorescent SYPRO Red staining

et al. 2000

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The fluorescent sensitive SYPRO Red dye was successfully employed to stain proteins in two-dimensional gels for protein identification by peptide mass fingerprinting. Proteins which are not chemically modified during the SYPRO Red staining process are well digested enzymatically in the gel and hence the resulting peptides can be efficiently eluted and analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A SYPRO Red two-dimensional gel of a complex protein extract from Candida albicans was analysed by MALDI-TOF MS. The validity of SYPRO Red staining was demonstrated by identifying, via peptide mass fingerprinting, 10 different C. albicans proteins from a total of 31 selected protein spots. The peptide mass signal intensity, the number of matched peptides and the percentage of coverage of protein sequences from SYPRO Red-stained proteins were similar to or greater than those obtained in parallel with the modified silver protein gel staining. This work demonstrates that fluorescent SYPRO Red staining is compatible with the identification of proteins separated on polyacrylamide gel and that it can be used as an alternative to silver staining. As far as we know, this is the first report in which C. albicans proteins separated using 2-D gels have been identified by peptide mass fingerprinting. The improved technique described here should be very useful for carrying out proteomic studies.

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Detection of Five Nanograms of Protein by Two-Minute Nile Red Staining of Unfixed SDS Gels

Cited by 31 publications

References 4 publications

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

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

120- and 160-kDa Receptors for Endogenous Mitogenic Peptide, Phytosulfokine-α, in Rice Plasma Membranes

Novel procedure for the identification of proteins by mass fingerprinting combining two-dimensional electrophoresis with fluorescent SYPRO Red staining

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