A comparison of silver stain and SYPRO Ruby Protein Gel Stain with respect to protein detection in two-dimensional gels and identification by peptide mass profiling

Lopez, Mary F.; Berggren, Kiera N.; Chernokalskaya, Elena; Лазарев, А. Ф.; Robinson, Myra; Patton, Wayne F.

doi:10.1002/1522-2683(200011)21:17<3673::aid-elps3673>3.3.co;2-d

Cited by 56 publications

(69 citation statements)

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“…SYPRO Ruby was selected as the staining method for the HCP studies. SYPRO Ruby was compared to silver stain for the detection of proteins in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS–PAGE) by Lopez et al () and the study demonstrated both had very similar sensitivity. In addition, SYPRO Ruby also presented advantages over silver stain including a broad linear dynamic range and enhanced recovery of peptides from in‐gel digest for mass spectrometry studies.…”

Section: The Complexity Of Host Cell Protein Populationsmentioning

confidence: 99%

Host cell protein testing by ELISAs and the use of orthogonal methods

Zhu-Shimoni¹,

Yu²,

Nishihara³

et al. 2014

Biotech & Bioengineering

139

127

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Host cell proteins (HCPs) are among the process-related impurities monitored during recombinant protein pharmaceutical process development. The challenges of HCP detection include (1) low levels of residual HCPs present in large excess of product protein, (2) the assay must measure a large number of different protein analytes, and (3) the population of HCP species may change during process development. Suitable methods for measuring process-related impurities are needed to support process development, process validation, and control system testing. A multi-analyte enzyme-linked immunosorbent assay (ELISA) is the workhorse method for HCP testing due to its high throughput, sensitivity and selectivity. However, as the anti-HCP antibodies, the critical reagents for HCP ELISA, do not comprehensively recognize all the HCP species, it is especially important to ensure that weak and non-immunoreactive HCPs are not overlooked by the ELISA. In some cases limited amount of antibodies to HCP species or antigen excess causes dilution-dependent non-linearity with multi-product HCP ELISA. In our experience, correct interpretation of assay data can lead to isolation and identification of co-purifying HCP with the product in some cases. Moreover, even if the antibodies for a particular HCP are present in the reagent, the corresponding HCP may not be readily detected in the ELISA due to antibody/antigen binding conditions and availability of HCP epitopes. This report reviews the use of the HCP ELISA, discusses its limitations, and demonstrates the importance of orthogonal methods, including mass spectrometry, to complement the platform HCP ELISA for support of process development. In addition, risk and impact assessment for low-level HCPs is also outlined, with consideration of clinical information.

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Section: The Complexity Of Host Cell Protein Populationsmentioning

confidence: 99%

Host cell protein testing by ELISAs and the use of orthogonal methods

Zhu-Shimoni¹,

Yu²,

Nishihara³

et al. 2014

Biotech & Bioengineering

139

127

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“…After electrophoresis, the protein gel can be stained for visualisation, quantification and comparison. Apart from the most commonly used Coomassie brilliant blue and silver stain for global protein detection, fluorescent dyes such as Sypro Orange, Sypro Red or Sypro Ruby, which provide additional benefits such as high sensitivity, simplicity and wide dynamic range 12,13 . Using size‐ and charge‐matched fluorescent CyDyes, two‐dimensional fluorescence difference gel electrophoresis (2‐D DIGE) technique has been developed recently, which provides a quantitative dimension to the traditional 2D PAGE technique 14,15 .…”

Section: What Are the Proteomic Technologies?mentioning

confidence: 99%

Application of proteomic technology in eye research: a mini review

Lam

Chun

et al. 2008

Clinical and Experimental Optometry

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Proteomics is a rapidly growing research area for the study of the protein cognate of genomic data. This review gives a brief overview of the modern proteomic technology. In addition to general applications of proteomics, we highlight its contribution to studying the physiology of different ocular tissues. We also summarise the published proteomic literature in the broad context of ophthalmic diseases, such as cataract, agerelated maculopathy, diabetic retinopathy, glaucoma and myopia. The proteomic technology is a useful research tool and it will continue to advance our understanding of a variety of molecular processes in ocular tissues and diseases.

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“…Hundreds to thousands of individual protein spots can be visualized depending on protein loading conditions and staining methodology [54][55][56]. Standard staining approaches include Coomassie Brilliant Blue [54] and silver [57,58], as well as a variety of fluorescent dyes [58][59][60][61]. One of the most powerful comparative ways of analyzing proteomes is fluorescence difference in-gel electrophoresis [62], which uses 2-CyDye or 3-CyDye systems to differentially label proteins belonging to dissimilar protein mixtures prior to gel electrophoretic separation [63][64][65].…”

Section: Gel Electrophoretic Protein Separa-tionmentioning

confidence: 99%

On-Membrane Digestion Technology for Muscle Proteomics

Ohlendieck¹

2013

J. Memb. Separ. Tech.

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High-resolution two-dimensional gel electrophoresis and in-gel digestion are routinely used for large-scale protein separation and peptide generation in mass spectrometry-based proteomics, respectively. However, the combination of isoelectric focusing in the first dimension and polyacrylamide slab gel electrophoresis in the second dimension is not suitable for the proper separation of integral proteins and high-molecular-mass proteins. In addition, ingel trypsination may not result in a high degree of efficient digestion levels for the production of large numbers of peptides in the case of certain protein species. The application of gradient one-dimensional gel electrophoresis and onmembrane digestion can overcome these technical problems and be extremely helpful for the comprehensive identification of proteins that are underrepresented in routine two-dimensional gel electrophoretic approaches. This review critically examines the general application of on-membrane digestion techniques in proteomics and its recent application for the identification of very large integral membrane proteins from skeletal muscle by mass spectrometry. This includes the discussion of proteomic studies that have focused on the proteomic characterization of the membrane cytoskeletal protein dystrophin from sarcolemma vesicles and the ryanodine receptor calcium release channel of the sarcoplasmic reticulum from skeletal muscle.

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A comparison of silver stain and SYPRO Ruby Protein Gel Stain with respect to protein detection in two-dimensional gels and identification by peptide mass profiling

Cited by 56 publications

References 0 publications

Host cell protein testing by ELISAs and the use of orthogonal methods

Host cell protein testing by ELISAs and the use of orthogonal methods

Application of proteomic technology in eye research: a mini review

On-Membrane Digestion Technology for Muscle Proteomics

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