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Hermann Schägger - One of the best experts on this subject based on the ideXlab platform.

  • blue native page
    Nature Protocols, 2006
    Co-Authors: Ilka Wittig, Hanspeter Braun, Hermann Schägger
    Abstract:

    Blue native PAGE (BN-PAGE) can be used for one-step isolation of protein complexes from biological membranes and total cell and tissue homogenates. It can also be used to determine native protein masses and oligomeric states and to identify physiological protein–protein interactions. Native complexes are recovered from gels by electroelution or diffusion and are used for 2D crystallization and electron microscopy or analyzed by in-gel activity assays or by native Electroblotting and immunodetection. In this protocol, we describe methodology to perform BN-PAGE followed by (i) native extraction or native Electroblotting of separated proteins, or (ii) a second dimension of tricine-SDS-PAGE or modified BN-PAGE, or (iii) a second dimension of isoelectric focusing (IEF) followed by a third dimension of tricine-SDS-PAGE for the separation of subunits of complexes. These protocols for 2D and 3D PAGE can be completed in 2 and 3 days.

  • tricine sds page
    Nature Protocols, 2006
    Co-Authors: Hermann Schägger
    Abstract:

    Tricine-SDS-PAGE is commonly used to separate proteins in the mass range 1-100 kDa. It is the preferred electrophoretic system for the resolution of proteins smaller than 30 kDa. The concentrations of acrylamide used in the gels are lower than in other electrophoretic systems. These lower concentrations facilitate Electroblotting, which is particularly crucial for hydrophobic proteins. Tricine-SDS-PAGE is also used preferentially for doubled SDS-PAGE (dSDS-PAGE), a proteomic tool used to isolate extremely hydrophobic proteins for mass spectrometric identification, and it offers advantages for resolution of the second dimension after blue-native PAGE (BN-PAGE) and clear-native PAGE (CN-PAGE). Here I describe a protocol for Tricine-SDS-PAGE, which includes efficient methods for Coomassie blue or silver staining and Electroblotting, thereby increasing the versatility of the approach. This protocol can be completed in 1-2 d.

  • Tricine-SDS-PAGE,”
    2006
    Co-Authors: Hermann Schägger
    Abstract:

    Tricine-SDS-PAGE is commonly used to separate proteins in the mass range 1-100 kDa. It is the preferred electrophoretic system for the resolution of proteins smaller than 30 kDa. The concentrations of acrylamide used in the gels are lower than in other electrophoretic systems. These lower concentrations facilitate Electroblotting, which is particularly crucial for hydrophobic proteins. Tricine-SDS-PAGE is also used preferentially for doubled SDS-PAGE (dSDS-PAGE), a proteomic tool used to isolate extremely hydrophobic proteins for mass spectrometric identification, and it offers advantages for resolution of the second dimension after blue-native PAGE (BN-PAGE) and clear-native PAGE (CN-PAGE). Here I describe a protocol for Tricine-SDS-PAGE, which includes efficient methods for Coomassie blue or silver staining and Electroblotting, thereby increasing the versatility of the approach. This protocol can be completed in 1-2 d

Akira Tsugita - One of the best experts on this subject based on the ideXlab platform.

  • amino acid sequence of trichoanguina a ribosomal inactivating protein from trichosanthes anguina seeds
    Journal of Biomedical Science, 1996
    Co-Authors: Luping Chow, Masaharu Kamo, Jungyaw Lin, Shiuanhuei Wang, Yoshio Ueno, Akira Tsugita
    Abstract:

    In this study, we sequenced a new type I ribosome-inactivating protein, trichoanguina, from the seeds ofTrichosanthes anguina (snake gourd). Trichoanguina is a basic glycoprotein having an apparent molecular mass of 35.0 kD and possessing strong ribosome-inactivating activity. Trichoanguina was cleaved with cyanogen bromide and partially digested with thermolysin, chymotrypsin, trypsin andStaphylococcus aureus V8 protease. The subsequent peptide fragments were separated by SDS-polyacrylamide gel electrophoresis, followed by Electroblotting to polyvinylidene difluoride membranes and then sequencing. The sequencing of trichoanguina was completed, consisting of 245 amino acid residues. The sequencing of trichoanguina revealed a considerable homology to trichosanthin anda-trichosanthin, which are known as abortifacient, ribosome-inactivating and antihuman immunodeficiency virus proteins, with 46.7% and 55.6% amino acid identities, respectively. The sequence conserves two active sites: Glu-158 and Arg-161.

Girault H. H. - One of the best experts on this subject based on the ideXlab platform.

  • Scanning Electrochemical Microscopy as a Readout Tool for Protein Electrophoresis
    'American Chemical Society (ACS)', 2007
    Co-Authors: Zhang M., Wittstock G., Shao Y., Girault H. H.
    Abstract:

    Scanning electrochemical microscopy (SECM) was used to image silver-stained proteins on a poly(vinylidene difluoride) membrane. The method is based on measuring the current at a scanning microelectrode in the feedback mode. The electrochemical feedback is caused by the redox-mediated etching of the isolated 5−10-nm-diameter silver nanoparticles formed during the staining process. Several parameters, such as the redox mediator and the staining protocol, were optimized to ensure a high resolution and a low detection limit, i.e., 0.5 ng of bovine serum albumin (4 × 10-14 mol) distributed on an area of 1 mm2 (4 × 10-16 mol·cm-2). Images of β-lactoglobulin A and myoglobin bands after gel electrophoretic separation and Electroblotting were obtained in order to demonstrate that SECM can be employed as a sensitive and quantitative readout method for detection of proteins after gel electrophoresis. An additional advantage is that the silver staining can be removed, allowing further downstream mass spectrometry analysis

  • Scanning electrochemical microscopy as a readout tool for protein electrophoresis
    analytical chemistry, 2007
    Co-Authors: Zhang Meiqin, Wittstock Gunther, Shao Yuanhua, Girault H. H.
    Abstract:

    Scanning electrochemical microscopy (SECM) was used to image silver-stained proteins on a poly(vinylidene difluoride) membrane. The method is based on measuring the current at a scanning microelectrode in the feedback mode. The electrochemical feedback is caused by the redox-mediated etching of the isolated 5-10-nm-diameter silver nanoparticles formed during the staining process. Several parameters, such as the redox mediator and the staining protocol, were optimized to ensure a high resolution and a low detection limit, i.e., 0.5 ng of bovine serum albumin (4 x 10(-14) mol) distributed on an area of 1 mm(2) (4 x 10(-16) mol center dot cm(-2)). Images of beta-lactoglobulin A and myoglobin bands after gel electrophoretic separation and Electroblotting were obtained in order to demonstrate that SECM can be employed as a sensitive and quantitative readout method for detection of proteins after gel electrophoresis. An additional advantage is that the silver staining can be removed, allowing further downstream mass spectrometry analysis.Chemistry, AnalyticalSCI(E)EI0ARTICLE134833-48397

Gunnar Houen - One of the best experts on this subject based on the ideXlab platform.

  • silver staining of proteins on Electroblotting membranes and intensification of silver staining of proteins separated by polyacrylamide gel electrophoresis
    Analytical Biochemistry, 2002
    Co-Authors: Birgitte Kjaer Sorensen, Peter Hojrup, Erik Ostergard, Charlotte Svaerke Jorgensen, Jan J Enghild, Lisa Rebekka Ryder, Gunnar Houen
    Abstract:

    A fast and convenient method for silver staining of proteins on Electroblotting membranes was developed based on Gallyas' histochemical intensifier and applied to human endothelial cell proteins separated by one- and two-dimensional electrophoresis and electroblotted to polyvinyl difluoride membranes. The method allowed detection of proteins on membranes with a sensitivity equal to the sensitivity of the most sensitive silver-staining protocols for electrophoresis gels. Also, the method was compatible with preceding immunostaining on the same membrane. Furthermore, an intensifying method for proteins in silver-stained SDS-PAGE gels was developed based on Gallyas' histochemical intensifier. This method was applied to proteins separated by one- and two-dimensional gel electrophoresis and visualized by one of several silver-staining methods. Maximal intensification was achieved for the less sensitive but fast acidic silver-staining protocols, but even for the very sensitive alkaline protocols a significant increase in signal to noise ratio was obtained. In particular, negatively stained or invisible proteins on the silver-stained gels were found to be visualized by the Gallyas stain. Proteins from silver-stained and Gallyas-stained gels were identified by mass spectrometry, and the intensification procedure was fully compatible with mass spectrometry.

Jd Zhao - One of the best experts on this subject based on the ideXlab platform.

  • Evidence that HetR protein is an unusual serine-type protease
    proceedings of the national academy of sciences of the united states of america, 1998
    Co-Authors: Rb Zhou, Xc Wei, Jiang N, Yq Dong, Kl Hsi, Jd Zhao
    Abstract:

    The hetR gene plays a very important role in cell differentiation of heterocystous cyanobacteria, To understand the mechanism of the hetR gene product in regulation of heterocyst differentiation, the recombinant HetR protein (rHetR) was overproduced in Escherichia coli, Purified rHetR was unstable and degraded easily in solution, Phenylmethanesulfonyl fluoride, a serine-type protease inhibitor, prevented the degradation and was shown to modify covalently rHetR, Dansyl fluoride (DnsF), another serine-type protease inhibitor, also covalently modifies rHetR as shown by electrophoresis and Electroblotting of the labeled rHetR and by MS. The labeling of rHetR with phenylmethanesulfonyl fluoride and DnsF was at the same site of rHetR and required Ca2+. S179N-rHetR, a mutant protein from strain 216 of Anabaena PCC 7120, which cannot differentiate heterocysts because of the mutation, was also overproduced and characterized. Although S170N-rHetR still can be labeled with DnsF, no proteolysis was observed, suggesting that Ser179 is involved in proteolytic activity. DnsF-labeled rHetR was digested with trypsin, and the labeled peptide was isolated and sequenced, The labeled peptide matches a sequence from HetR, These results show that HetR is a protease.Multidisciplinary SciencesSCI(E)7794959-49639