Solubilization of proteins for electrophoretic analyses

Rabilloud, Thierry

doi:10.1002/elps.1150170503

Cited by 304 publications

(225 citation statements)

References 137 publications

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“…Nine individual proteins were separated with several proteins displaying multiple isoforms and a new sub class of the giardins was described, alpha giardin, which migrated at 33 kDa. The overall number of proteins and the molecular mass range was decreased when compared to the SDS-PAGE analysis of Crossley and Holberton (1983), however this is not unexpected due to the difference in solubilisation techniques used in 2D-PAGE and SDS-PAGE (Rabilloud, 1996). Sodium dodecyl sulphate is one of the most effective protein solubilisation agents; however it coats the protein molecules in a negative charge.…”

Section: Cytoskeletonmentioning

confidence: 89%

“…This charge would interfere with the iso-electric focusing used in the first dimension of 2D-PAGE, which relies on proteins having no net charge at their pI, therefore ruling out SDS for protein solubilisation for 2D-PAGE. The buffers for 2D-PAGE must use zwitterionic detergents, which are not as effective at solubilising proteins, resulting in the decreased number of proteins visible in 2D-PAGE (Rabilloud, 1996).…”

Section: Cytoskeletonmentioning

confidence: 99%

“…Neither CWP1 nor CWP2 were identified on the 2D-PAGE gel, however, their presence was confirmed using western blots analysis of ESV protein separated using SDS-PAGE. Again the solubilisation techniques needed for 2D-PAGE analysis were not enough to effectively solubilise the CWPs (Rabilloud, 1996).…”

Section: Cell Biologymentioning

confidence: 99%

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Proteomic analysis of Giardia: Studies from the pre- and post-genomic era

Steuart¹

2010

Experimental Parasitology

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The proteome of Giardia duodenalis has been under study for the last 25 years and has lead to the discovery of valuable information on the biology and variation of the parasite.Proteomic techniques, mainly SDS-PAGE and 2D-PAGE, have been used to investigate protein variation, cellular structure and host parasite interactions. This has allowed for the identification of assemblage and host specific proteins, structural proteins, proteins released by trophozoites upon exposure to host cell monolayers and immunoreactive proteins. These data are important in understanding the pathogenesis of G. duodenalis infections, as well as highlighting potential drug and vaccine targets. There is however a large amount of future work needed to fully understand the proteome of this parasite.

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Section: Cytoskeletonmentioning

confidence: 89%

Section: Cytoskeletonmentioning

confidence: 99%

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Proteomic analysis of Giardia: Studies from the pre- and post-genomic era

Steuart¹

2010

Experimental Parasitology

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“…In general, most protocols involve an initial treatment such as microdialysis, precipitation, or sonication to remove common contaminants such as nucleic acids, salt ions, and lipids, followed by the addition of denaturants, reducing and alkylating agents, detergents, and chaotropes to facilitate protein solubility. 7,8 Reductants such as dithiothreitol (DTT) and dithioerythritol (DTE) are commonly used to prevent protein oxidation and reduce disulfide bonds. Resulting sulfhydryl groups are often alkylated with iodoacetamide or iodoacetic acid to prevent reformation of intra-and interprotein disulfide bonds.…”

Section: Sample Preparationmentioning

confidence: 99%

Two-dimensional protein electrophoresis: From molecular pathway discovery to biomarker discovery in neurological disorders

Choe

Werner

Lee

2006

NeuroRX

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Summary: Two-dimensional protein electrophoresis (2-DE) has undergone many technical improvements in the past 30 years, resulting in an analytical method that is unparalleled in the resolution of complex protein mixtures and capable of quantifying changes in protein expression from a wide variety of tissues and samples. The technique has been applied in many studies of neurologic disease to identify changes in spot patterns that correlate with disease. The true power of the technique emerges when it is coupled to state-of-the-art methods in mass spectrometry, which enable identification of the protein or proteins contained within a spot of interest on a 2-DE map. Investigators have successfully applied the technique to gain improved understanding of neurologic disease mechanisms in humans and in animal models and to discover biomarkers that are useful in the clinical setting. An important extension to these efforts that has not been realized thus far is the desire to profile changes in protein expression that result from therapy to help relate disease-modifying effects at the molecular level with clinical outcomes. Here we review the major advances in 2-DE methods and discuss specific examples of its application in the study of neurologic diseases.

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“…Based on their different solubility, cereal proteins have been sequentially extracted and analyzed [7][8][9]. Prolamins and glutelins, also called storage proteins, are the main proteins in cereals and, as previously indicated, they are not soluble in water.…”

Section: Introductionmentioning

confidence: 99%

Development of a capillary electrophoresis method for the determination of soybean proteins in soybean–rice gluten‐free dietary products

2006

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Development of a capillary electrophoresis method for the determination of soybean proteins in soybean-rice gluten-free dietary products CE has been applied for the first time to the simultaneous separation of soybean and rice proteins. Treated and untreated capillaries with different effective lengths as well as separation media at different pHs were tested. For that purpose, samples and standard solutions were prepared in 25:75 ACN-water media containing 0.3% v/v acetic acid. The use of an untreated capillary of 50 cm effective length together with an 80 mM borate buffer (pH 8.5) modified with 20% v/v ACN and UV detection at 254 nm were the conditions working the best. These conditions enabled the determination of soybean proteins in gluten-free dietary commercial products elaborated with soybean protein and/or soybean flour and rice flour using the standard additions calibration method. The method was linear up to 26 mg/mL of soybean proteins, the precision (expressed as RSD) was always better than 6%, and recoveries obtained for soybean proteins when spiking commercial products were very close to 100%.

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Solubilization of proteins for electrophoretic analyses

Cited by 304 publications

References 137 publications

Proteomic analysis of Giardia: Studies from the pre- and post-genomic era

Proteomic analysis of Giardia: Studies from the pre- and post-genomic era

Two-dimensional protein electrophoresis: From molecular pathway discovery to biomarker discovery in neurological disorders

Development of a capillary electrophoresis method for the determination of soybean proteins in soybean–rice gluten‐free dietary products

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