Cyanate formation and electrophoretic behavior of proteins in gels containing urea

Cole, Elaine G.; Mecham, Dale K.

doi:10.1016/0003-2697(66)90129-1

Cited by 49 publications

(11 citation statements)

References 7 publications

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“…Extremely well-defined bands in PAMU-PAGE cannot be described as artifacts due to carbamylation in 5 M urea. Carbamylation of amino groups in the presence of aqueous urea does make protein relatively less basic (Cole and Mecham 1966). But carbamylation occurs only above pH 5.5 (Duesberg and Rueckert 1965).…”

Section: Resultsmentioning

confidence: 99%

BIOCHEMICAL STUDIES ON BLACK GRAM (PHASEOLUS MUNGO): I. Solubilization and Electrophoretic Characterization of The Proteins.

Padhye

Salunkhe

1977

J Food Biochemistry

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Protein content of 60 mesh, dehydrated, defatted black gram (Phaseolus mungo) meal was 28.5%. Sodium carbonate (0.5-1.0%), t e tmsodium pyrophosphate (0.5%), and sodium dodecyl sulfate (0.5-5%) extracted more than 76 gram Lowry's protein per 100 gram Kjeldahl protein. On the considerations of contaminating residue in the final product and disruption o f native structure of the proteins these chemical agents were unsuitable. Sodium sulfate at 10% level was judged to be the best protein solubilizer. The phenol-acetic acidmercaptoethanol-urea (PAMU) system in polyacrylamide gel electrophoresis was more efficient in resolving protein subunits than the sodium dodecyl sulfate (SDS) gel system. PAMU dissociated total black gram proteins in one major and 7-8 other sharp bands. Proteins separated on PAMU gel were run on the f l a t bed polyacrylamide gel containing SDS system. The proteins were then separated in 13 subunits, the molecular weight o f the major one was 55,000. Defatting with n-hexane improved protein extraction by 6%. Solubilized proteins contained 81% globulins, 13% albumins, 4% ethanol soluble proteins, and 2% acetic acid soluble proteins.

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Section: Resultsmentioning

confidence: 99%

BIOCHEMICAL STUDIES ON BLACK GRAM (PHASEOLUS MUNGO): I. Solubilization and Electrophoretic Characterization of The Proteins.

Padhye

Salunkhe

1977

J Food Biochemistry

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“…Second, it prevents proteins from many enzymatic digestions, resulting in incompletely digested peptides. Third, it leads to unexpected chromatographic retention time in protein/peptide separation and unpredicted masses, thus increasing the complexity of samples [7]. Fourth, it affects peptide and protein identification and accurate quantification by reducing the ionization efficiency and signal intensity of the detected peaks [8].…”

Section: Introductionmentioning

confidence: 99%

Inhibition of protein carbamylation in urea solution using ammonium-containing buffers

Sun

Zhou

Yang

et al. 2014

Analytical Biochemistry

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Urea solution is one of the most commonly employed protein denaturants for protease digestion in proteomic studies. However, it has long been recognized that urea solution can cause carbamylation at the N-termini of proteins/peptides and at the side chain amino groups of lysine and arginine residues. Protein/peptide carbamylation blocks protease digestion and affects protein identification and quantification in mass spectrometry analysis by blocking peptide amino groups from isotopic/isobaric labeling and changing peptide charge states, retention times and masses. In addition, protein carbamylation during sample preparation makes it difficult to study in vivo protein carbamylation. In this study, we compared the peptide carbamylation in urea solutions of different buffers and found that ammonium containing buffers were the most effective buffers to inhibit protein carbamylation in urea solution. The possible mechanism of carbamylation inhibition by ammonium containing buffers is discussed, and a revised procedure for the protease digestion of proteins in urea and ammonium containing buffers was developed to facilitate its application in proteomic research.

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“…Deionization is most easily carried out by passage of the solution over a mixed-bed ion exchange resin (e.g., Dowex 501-D) and most easily evaluated by measurement of specific conductance of the eluate (which should approximate that of distilled water). The most widely used nonionic solute, 8 M urea, has to be prepared freshly from "ultrapure " reagent or deionized just prior to use, in order to minimize the possibility of formation of artifact bands due to the carbamylation reaction (Cole and Mecham, 1966). Use ofTriton X-IOD has many advantages relative to urea: it does not perturb protein conformation, it does not have to be added to the gel, since it is carried by the protein sampIe into the gel, and it does not interfere with the ordinary staining procedures when used in this fashion (Hearing et al, 1976).…”

Section: Procedures 01 Polymerization Olone-dimensional Vertical Gel Slabmentioning

confidence: 99%

Analytical and Preparative Polyacrylamide Gel Electrophoresis

Chrambach

Rodbard

Jovin

et al. 1976

Methods of Protein Separation

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Cyanate formation and electrophoretic behavior of proteins in gels containing urea

Cited by 49 publications

References 7 publications

BIOCHEMICAL STUDIES ON BLACK GRAM (PHASEOLUS MUNGO): I. Solubilization and Electrophoretic Characterization of The Proteins.

BIOCHEMICAL STUDIES ON BLACK GRAM (PHASEOLUS MUNGO): I. Solubilization and Electrophoretic Characterization of The Proteins.

Inhibition of protein carbamylation in urea solution using ammonium-containing buffers

Analytical and Preparative Polyacrylamide Gel Electrophoresis

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