Models of protein modification in Tris–glycine and neutral pH Bis–Tris gels during electrophoresis: Effect of gel pH

Hachmann, John; Amshey, Joseph W.

doi:10.1016/j.ab.2005.04.015

Cited by 25 publications

(19 citation statements)

References 20 publications

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“…Alkaline conditions result in a higher reactivity [328,329], therefore it is advisable to utilize neutral or slightly acidic gel systems [332]. A comparison between Laemmli gels [28] (pH 9.5) and a Bis-Tris-MES system with pH 7.2 confirms that electrophoresis at near neutral pH reduces the risk of protein modifications [333].…”

Section: Methods Comparison and Limitations Of Preparative Electrophormentioning

confidence: 93%

Preparative isolation of protein complexes and other bioparticles by elution from polyacrylamide gels

Seelert

Krause

2008

Electrophoresis

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Due to its unmatched resolution, gel electrophoresis is an indispensable tool for the analysis of diverse biomolecules. By adaptation of the electrophoretic conditions, even fragile protein complexes as parts of intracellular networks migrate through the gel matrix under sustainment of their integrity. If the thickness of such native gels is significantly increased compared to the analytical version, also high sample loads can be processed. However, the cage-like network obstructs an in-depth analysis for deciphering structure and function of protein complexes and other species. Consequently, the biomolecules have to be removed from the gel matrix into solution. Several approaches summarized in this review tackle this problem. While passive elution relies on diffusion processes, electroelution employs an electric field to force biomolecules out of the gel. An alternative procedure requires a special electrophoresis setup, the continuous elution device. In this apparatus, molecules migrate in the electric field until they leave the gel and were collected in a buffer stream. Successful isolation of diverse protein complexes like photosystems, ATP-dependent enzymes or active respiratory supercomplexes and some other bioparticles demonstrates the versatility of preparative electrophoresis. After liberating particles out of the gel cage, numerous applications are feasible. They include elucidation of the individual components up to high resolution structures of protein complexes. Therefore, preparative electrophoresis can complement standard purification methods and is in some cases superior to them.

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Section: Methods Comparison and Limitations Of Preparative Electrophormentioning

confidence: 93%

Preparative isolation of protein complexes and other bioparticles by elution from polyacrylamide gels

Seelert

Krause

2008

Electrophoresis

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“…Although the use of high concentrations of urea in the purification of proteins poses risks for carbamylation, this generally occurs under conditions which promote cyanate production such as highly alkaline environments (.pH 10) and elevated temperatures (.377C) [57,58]. Although the use of preparative electrophoresis for the preparation of proteins poses a risk for adduct formation with acrylamide [59][60][61][62], the adducts were not identified in the preparative SDS-PAGE isolation and structural analysis of dimeric MER-45 kDa hGH [31], which employed the same buffers and pHs for electrophoresis used in the present work. Human GH is extremely stable to conformational changes in urea at concentrations below 9.1 M at pHs between 5 and 9 [63].…”

Section: Discussionmentioning

confidence: 99%

Preparative alkaline urea gradient PAGE: Application to purification of extraordinarily‐stable disulfide‐linked homodimer of human growth hormone

et al. 2007

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The 40-60 pituitary human growth hormone (hGH) isoforms are so similar in their physico-chemical properties (charge, size, hydrophobicity) that the limited resolutions of chromatographic separation methodologies have not permitted most of them to be isolated. However, application of high-resolution preparative alkaline urea gradient PAGE has facilitated isolation of a disulfide-linked mercaptoethanol-resistant (MER) 45 kDa hGH dimer. Human pituitary extracts were separated by Sephadex G-100 chromatography under alkaline conditions. Pooled fractions containing MER-45 kDa hGH, as determined by SDS-PAGE, were then separated by Sephadex G-100 chromatography under acidic conditions followed by diethylaminoethyl (DEAE) anion-exchange chromatography. Pooled DEAE fractions containing MER-45 kDa hGH and other hGH isoforms were then separated by preparative electrophoresis in an alkaline polyacrylamide gradient (5-20%) slab gel containing 8 M urea into five distinct protein zones. One electroeluted zone contained pure MER-45 kDa hGH. The dimeric hGH isoform was immunoreactive at low concentrations (effective dose to produce 50% response (ED(50)) +/- S.E. = 58 +/- 5.00 pM) in a hGH radioimmunoassay, similar to that of standard monomeric hGH (ED(50) +/- S.E. = 22.93 +/- 3.90 pM), indicating that is was conformationally intact. Alkaline urea gradient PAGE is a valuable tool for preparative separation of structurally similar proteins such as isoforms of the hGH family.

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“…The purified enzyme was concentrated, dialyzed against 20 mM sodium acetate buffer (pH 5.0) using a Vivaspin 20 concentrator (10,000 MWCO, Sartorius AG, Goettingen, Germany) and stored at 4°C until use. The purity and size of the protein was analyzed by SDS-PAGE using precast NuPAGE 4 - 12% polyacrylamide Bis-Tris gels (Life Technologies, Carlsbad, CA, USA) (Hachmann and Amshey [2005]). All proteins were identified by N-terminus sequence analysis.…”

Section: Methodsmentioning

confidence: 99%

Xylanase (GH11) from Acremonium cellulolyticus: homologous expression and characterization

Watanabe

Inoue

et al. 2014

AMB Expr

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Cellulosic materials constitute most of the biomass on earth, and can be converted into biofuel or bio-based materials if fermentable sugars can be released using cellulose-related enzymes. Acremonium cellulolyticus is a mesophilic fungus which produces a high amount of cellulose-related enzymes. In the genome sequence data of A. cellulolyticus, ORFs showing homology to GH10 and GH11 xylanases were found. The xylanases of A. cellulolyticus play an important role in cellulolytic biomass degradation. Search of a draft genome sequence of A. cellulolyticus for xylanase coding regions identified seven ORFs showing homology to GH 11 xylanase genes (xylA, xylB, xylC, xylD, xylE, xylF and xylG). These genes were cloned and their enzymes were prepared with a homologous expression system under the control of a glucoamylase promoter. Six of the seven recombinant enzymes were successfully expressed, prepared, and characterized. These enzymes exhibited optimal xylanase activity at pH 4.0 – 4.5. But this time, we found that only XylC had enormously higher relative activity (2947 U•mg −1) than the other xylanases at optimum pH. This result is surprising because XylC does not retain a carbohydrate-binding module 1 (CBM-1) that is necessary to bind tightly own substrate such as xylan. In this study, we discuss the relationship between activity, pH and sequence of seven xylanases in A. cellulolyticus.

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Models of protein modification in Tris–glycine and neutral pH Bis–Tris gels during electrophoresis: Effect of gel pH

Cited by 25 publications

References 20 publications

Preparative isolation of protein complexes and other bioparticles by elution from polyacrylamide gels

Preparative isolation of protein complexes and other bioparticles by elution from polyacrylamide gels

Preparative alkaline urea gradient PAGE: Application to purification of extraordinarily‐stable disulfide‐linked homodimer of human growth hormone

Xylanase (GH11) from Acremonium cellulolyticus: homologous expression and characterization

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