Recovery of Native Proteins from Preparative Electrophoresis Gel Slices by Reverse Polarity ElutioN

As, Abramovitz; Randolph,; Mehra, Aruna S.; Christakos, Sylvia

doi:10.1080/10826068408070629

Cited by 12 publications

(4 citation statements)

References 20 publications

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Section: Methodsmentioning

confidence: 99%

“…27 The predominant band of ~70 kDa, presumably comprised of pertactin, was excised from the gel and electroeluted as described. 1 The electroeluted material was concentrated and re-equilibrated with PBS over an ultrafiltration device. c Purified pertactin (0.5 mL, ~60 μg) was emulsified by sonication with an equal volume of adjuvent d and injected intradermally into a Bordetella-free New Zealand White rabbit at 25 sites along the back.…”

Section: Pertactin Purification and Polyclonal Rabbit Serummentioning

confidence: 99%

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Cilia-associated bacteria in fatal Bordetella bronchiseptica pneumonia of dogs and cats

et al. 2016

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Bordetella bronchiseptica frequently causes nonfatal tracheobronchitis, but its role in fatal pneumonia is less recognized. Our study evaluated histologic identification of cilia-associated bacteria as a method for diagnosis of B. bronchiseptica pneumonia. Cases of fatal bronchopneumonia were studied retrospectively, excluding neonates and cases of aspiration pneumonia, minor lung lesions, or autolysis. The study population comprised 36 canine and 31 feline cases of bronchopneumonia. B. bronchiseptica was identified in 8 of 36 canine and 14 of 31 feline cases based on immunohistochemistry (IHC) using serum from a rabbit hyperimmunized with pertactin, PCR testing (Fla2/Fla12), and/or bacterial culture data when available. Of these, IHC was positive in 4 canine and 7 feline cases, PCR was positive in 8 canine and 14 feline cases, and B. bronchiseptica was isolated in 2 of 5 canine and 3 of 9 feline cases tested. Examination of histologic sections stained with hematoxylin and eosin revealed bronchial cilia-associated bacteria in 4 of 36 canine and 5 of 31 feline cases; these were all positive by IHC and PCR. The presence of cilia-associated bacteria had been noted in the pathology report for only 2 of these 9 cases. Thus, the presence of cilia-associated bacteria seems frequently overlooked by pathologists, but is a diagnostically significant feature of B. bronchiseptica pneumonia. A specific diagnosis of B. bronchiseptica pneumonia is important because it suggests primary or opportunistic bacterial pneumonia rather than aspiration pneumonia, and because of the risk of animal-to-animal transmission of B. bronchiseptica, the availability of vaccines for disease prevention, and the potential zoonotic risk to immunocompromised pet owners.

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Section: Methodsmentioning

confidence: 99%

Section: Pertactin Purification and Polyclonal Rabbit Serummentioning

confidence: 99%

Cilia-associated bacteria in fatal Bordetella bronchiseptica pneumonia of dogs and cats

et al. 2016

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“…Purification of IDH was achieved by electroelution from a nondenaturing polyacrylamide gel by the method of Abramovitz et al (1984) as described in the Hoefer Scientific Instruments catalog (1989). Concentrated eluent (1.9 ml) was applied to a 9% (w/ v) nondenaturing polyacrylamide gel (3 mm) and electrophoresed for 3 h at 60 mA.…”

Section: Methodsmentioning

confidence: 99%

Characterization of a heat-stable NADP-dependent isocitrate dehydrogenase from the obligate thermophile Thermoleophilum minutum YS-4

Novotny

Perry

1991

Appl Microbiol Biotechnol

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A thermostable NADP-dependent isocitrate dehydrogenase (IDH; EC. 1.1.1.42) was purified from the obligately thermophilic hydrocarbonoclastic bacterium Thermoleophilum minutum YS-4 (ATCC 35265). This was accomplished by affinity chromatography and electroelution from a nondenaturing polyacrylamide gel. The enzyme has an Mr of 60000 and is composed of two identical subunits of Mr 30 500. The amino acid composition has an Arg/Lys ratio of 4:1 and very high levels of glycine. Under nondenaturing conditions, the enzyme has a distinct difference in electrophoretic mobility relative to IDHs obtained from other genera including the genus Thermus. The secondary structure consists of 16% a-helix, 20% fl-sheet, 25% fl-turn and 37% random coil as determined by circular dichroism spectroscopy. The optimum pH and temperature for activity were 7.2 and 75 ° C respectively and the apparent Km values for DL-isocitrate and N A D P + were 33 IXM, and 48 lXM, respectively. The enzyme requires divalent cations, such as Mn 2+ or Mg 2+ for activity. NAD + cannot substitute for N A D P +. Oxaloacetate plus glyoxylate exert considerable inhibition on IDH activity while other glycolytic and tricarboxylic acid cycle intermediates have a lesser effect, p-Chloromercuribenzoic acid was inhibitory to the I D H although isocitrate and Mn 2+ offered some protection from this inactivation. The enzyme is thermostable, retaining 84% and 57% of initial activity after incubation for 1 h at 60 ° and 70 ° C, respectively. Isocitrate provided protection from thermal inactivation allowing the I D H to maintain 21% activity after 1 h at 80 ° C.

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“…A small volume of buffer is filled in and the chamber is sealed by a dialysis membrane. In this case, electroelution is performed in opposite direction to the preceding electro-phoresis [153][154][155]. Instead of using a dialysis membrane, a combination of two solutions can cause retaining of the protein.…”

Section: Reversed Electrophoresis Using a Discontinuous Conductivity mentioning

confidence: 98%

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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Recovery of Native Proteins from Preparative Electrophoresis Gel Slices by Reverse Polarity ElutioN

Cited by 12 publications

References 20 publications

Cilia-associated bacteria in fatal Bordetella bronchiseptica pneumonia of dogs and cats

Cilia-associated bacteria in fatal Bordetella bronchiseptica pneumonia of dogs and cats

Characterization of a heat-stable NADP-dependent isocitrate dehydrogenase from the obligate thermophile Thermoleophilum minutum YS-4

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

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