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

2008 ◽  
Vol 29 (12) ◽  
pp. 2617-2636 ◽  
Author(s):  
Holger Seelert ◽  
Frank Krause
Keyword(s):  
Protein Complexes ◽  
Polyacrylamide Gels ◽  
Preparative Isolation

scholarly journals Stabilizing labile DNA-protein complexes in polyacrylamide gels

2010 ◽  
Vol 31 (4) ◽  
pp. 648-653 ◽  
Author(s):  
Nina Y. Sidorova ◽  
Stevephen Hung ◽  
Donald C. Rau
Keyword(s):  
Protein Complexes ◽  
Polyacrylamide Gels

scholarly journals Stabilizing Labile DNA-Protein Complexes in Polyacrylamide Gels

2012 ◽  
Vol 102 (3) ◽  
pp. 484a
Author(s):  
Nina Y. Sidorova ◽  
Stevephen Hung ◽  
Donald C. Rau
Keyword(s):  
Protein Complexes ◽  
Polyacrylamide Gels

PEPPI-MS: Strategies to Enhance the Extraction of Electrophoretically Separated Proteins from Polyacrylamide Gels and Their Application to Top-Down/native Mass Spectrometry

2019 ◽  
Author(s):  
Ayako Takemori ◽  
Lissa C Anderson ◽  
Victoria M. Harman ◽  
Philip Brownridge ◽  
David Butcher ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Complexes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Native Mass Spectrometry ◽  
Polyacrylamide Gels ◽  
Top Down ◽  
Native Page ◽  
Intact Proteins ◽  
Molecular Weights ◽  
High Yields

Polyacrylamide gel electrophoresis (PAGE) is a powerful technique for separating proteins from complex biological samples. However, the difficulty in recovering proteins with high yields from polyacrylamide matrices often precludes further analyses of intact proteins. Here, we propose a novel experimental workflow named Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS (‘PEPPI-MS’), which allows intact mass spectrometry (MS) of PAGE separated proteins. We discovered that staining proteins with certain Coomassie brilliant blue formulations immediately after PAGE improves the efficiency of extraction in a medium with pH 7–11. Post-staining, proteins spanning a broad range of molecular weights were recovered efficiently in a 10-minute procedure. High recovery yields were also obtained from dried and archived gels. This workflow is effective for top-down proteomics analysis of the target molecular region in the gel. An alternative procedure was developed for the extraction of protein complexes exceeding 400 kDa, which were separated using native PAGE, from unstained gels. Non-covalent hemoglobin tetramer, purified from cell lysate with two-dimensional native PAGE and extracted with the mild detergent octyl-β-Dglucopyranoside, was amenable for native MS analysis. We anticipate that the established workflow will facilitate the purification, storage, and transport of proteins destined for detailed characterization by MS.

PEPPI-MS: Strategies to Enhance the Extraction of Electrophoretically Separated Proteins from Polyacrylamide Gels and Their Application to Top-Down/native Mass Spectrometry

2019 ◽  
Author(s):  
Ayako Takemori ◽  
Lissa C Anderson ◽  
Victoria M. Harman ◽  
Philip Brownridge ◽  
David Butcher ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Complexes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Native Mass Spectrometry ◽  
Polyacrylamide Gels ◽  
Top Down ◽  
Native Page ◽  
Intact Proteins ◽  
Molecular Weights ◽  
High Yields

Polyacrylamide gel electrophoresis (PAGE) is a powerful technique for separating proteins from complex biological samples. However, the difficulty in recovering proteins with high yields from polyacrylamide matrices often precludes further analyses of intact proteins. Here, we propose a novel experimental workflow named Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS (‘PEPPI-MS’), which allows intact mass spectrometry (MS) of PAGE separated proteins. We discovered that staining proteins with certain Coomassie brilliant blue formulations immediately after PAGE improves the efficiency of extraction in a medium with pH 7–11. Post-staining, proteins spanning a broad range of molecular weights were recovered efficiently in a 10-minute procedure. High recovery yields were also obtained from dried and archived gels. This workflow is effective for top-down proteomics analysis of the target molecular region in the gel. An alternative procedure was developed for the extraction of protein complexes exceeding 400 kDa, which were separated using native PAGE, from unstained gels. Non-covalent hemoglobin tetramer, purified from cell lysate with two-dimensional native PAGE and extracted with the mild detergent octyl-β-Dglucopyranoside, was amenable for native MS analysis. We anticipate that the established workflow will facilitate the purification, storage, and transport of proteins destined for detailed characterization by MS.

Visualization of stable ferritin complexes with palladium, rhodium and iridium nanoparticles detected by their catalytic activity in native polyacrylamide gels

2017 ◽  
Vol 46 (40) ◽  
pp. 13690-13694 ◽  
Author(s):  
Vladimir Pekarik ◽  
Marie Peskova ◽  
Roman Guran ◽  
Jiri Novacek ◽  
Zbynek Heger ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Metal Nanoparticles ◽  
Protein Complexes ◽  
Direct Visualization ◽  
Polyacrylamide Gels ◽  
Iridium Nanoparticles

Direct visualization of proteins and the catalytic activity of metal nanoparticles from metal–protein complexes in native polyacrylamide gels.

Electron Microscopy and image analysis of nucleo-protein complexes

1994 ◽  
Vol 52 ◽  
pp. 88-89
Author(s):  
E. H. Egelman ◽  
X. Yu
Keyword(s):  
Electron Microscopy ◽  
Image Analysis ◽  
Normal Cell ◽  
Genetic Recombination ◽  
Protein Complexes ◽  
Chromosomal Rearrangements ◽  
Reca Protein ◽  
E Coli ◽  
Helical Polymer ◽  
Specific Protease

The RecA protein of E. coli has been shown to mediate genetic recombination, regulate its own synthesis, control the expression of other genes, act as a specific protease, form a helical polymer and have an ATPase activity, among other observed properties. The unusual filament formed by the RecA protein on DNA has not previously been shown to exist outside of bacteria. Within this filament, the 36 Å pitch of B-form DNA is extended to about 95 Å, the pitch of the RecA helix. We have now establishedthat similar nucleo-protein complexes are formed by bacteriophage and yeast proteins, and availableevidence suggests that this structure is universal across all of biology, including humans. Thus, understanding the function of the RecA protein will reveal basic mechanisms, in existence inall organisms, that are at the foundation of general genetic recombination and repair.Recombination at this moment is assuming an importance far greater than just pure biology. The association between chromosomal rearrangements and neoplasms has become stronger and stronger, and these rearrangements are most likely products of the recombinatory apparatus of the normal cell. Further, damage to DNA appears to be a major cause of cancer.

Structure of contact sites between the outer and inner mitochondrial membranes investigated by HVEM tomography

1996 ◽  
Vol 54 ◽  
pp. 966-967
Author(s):  
C.A. Mannella ◽  
K.F. Buttle ◽  
K.A. O‘Farrell ◽  
A. Leith ◽  
M. Marko
Keyword(s):  
Liver Mitochondrion ◽  
Adenine Nucleotide ◽  
Protein Complexes ◽  
Mitochondrial Membranes ◽  
Electron Microscopic ◽  
Contact Sites ◽  
Transmission Electron ◽  
Precursor Proteins ◽  
Membrane Contacts ◽  
And Function

Early transmission electron microscopy of plastic-embedded, thin-sectioned mitochondria indicated that there are numerous junctions between the outer and inner membranes of this organelle. More recent studies have suggested that the mitochondrial membrane contacts may be the site of protein complexes engaged in specialized functions, e.g., import of mitochondrial precursor proteins, adenine nucleotide channeling, and even intermembrane signalling. It has been suggested that the intermembrane contacts may be sites of membrane fusion involving non-bilayer lipid domains in the two membranes. However, despite growing interest in the nature and function of intramitochondrial contact sites, little is known about their structure.We are using electron microscopic tomography with the Albany HVEM to determine the internal organization of mitochondria. We have reconstructed a 0.6-μm section through an isolated, plasticembedded rat-liver mitochondrion by combining 123 projections collected by tilting (+/- 70°) around two perpendicular tilt axes. The resulting 3-D image has confirmed the basic inner-membrane organization inferred from lower-resolution reconstructions obtained from single-axis tomography.

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