Protein Blotting and Immunoblotting

Bini, Luca; Liberatori, Sabrina; Magi, Barbara; Marzocchi, Barbara; Raggiaschi, Roberto; Pallini, Vitaliano

doi:10.1007/978-3-642-57105-3_6

Cited by 4 publications

(1 citation statement)

References 53 publications

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“…After one-dimensional (1D) and 2D electrophoresis proteins were electroblotted on to nitrocellulose membrane. Before 2D immunodetection the membranes were reversibly stained with 0.2 % (w\v) Ponceau S in 3 % (w\v) trichloroacetic acid for 3 min [18] to facilitate the further computer-aided matching of immunoreactive spots on the silver-and Coomassie Blue-stained protein maps. Immunoreactive spots were detected using an enhanced chemiluminescence (ECL) detection kit (Pierce) and peroxidase-conjugated secondary antibodies.…”

Section: Protein Identification By Western Blottingmentioning

confidence: 99%

Extensive temporally regulated reorganization of the lipid raft proteome following T-cell antigen receptor triggering

Bini

Pacini²,

Liberatori

et al. 2003

Biochemical Journal

137

117

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Signalling by immunoreceptors is orchestrated at specific plasma membrane microdomains, referred to as lipid rafts. Here we present a proteomics approach to the temporal analysis of protein association with lipid rafts following T-cell antigen receptor (TCR) triggering. We show that TCR engagement promotes the temporally regulated recruitment of proteins participating in the TCR signalling cascade to lipid rafts. Furthermore, TCR triggering results in profound modifications in the composition of lipid rafts involving a number of proteins associated either directly or indirectly with both plasma and intracellular membranes. Raft-associated proteins can be clustered according to their temporal profile of raft association. The data identify lipid rafts as highly dynamic structures and reveal a dramatic impact of surface TCR triggering not only on components of the TCR signalling machinery but also on proteins implicated in a number of diverse cellular processes.

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Section: Protein Identification By Western Blottingmentioning

confidence: 99%

Extensive temporally regulated reorganization of the lipid raft proteome following T-cell antigen receptor triggering

Bini

Pacini²,

Liberatori

et al. 2003

Biochemical Journal

137

117

View full text Add to dashboard Cite

show abstract

Expression of the small tyrosine phosphatase (Stp1) inSaccharomyces cerevisiae: A study on protein tyrosine phosphorylation

et al. 2001

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Small tyrosine phoshatase 1 (Stp1) is a Schizosaccharomyces pombe low-molecular-mass phosphotyrosine-phosphatase 50% identical to Saccharomyces cerevisiae Ltp1. In order to investigate the role of Stp1 in yeast, a mutant was generated having the characteristic of a dominant negative molecule. Changes in protein tyrosine phosphorylation in S. cerevisiae proteome in response to Stp1 or its dominant negative mutant expression were analyzed by high-resolution two-dimensional (2-D) electrophoresis. The most remarkable result is the modification by phosphorylation on tyrosine of several proteins involved in carbohydrate metabolism. Twelve proteins were identified on the basis of their positions in the anti-phosphotyrosine immunoblot of the 2-D electrophoresis. Ten of these present tyrosyl residues that are within the consensus sequence for protein kinase CK2 (casein kinase-2). These data open the possibility for the identification of Stp1 substrates in yeast and provide hints about the nature of tyrosine phosphorylating agents in yeast and in other organisms where bona fide tyrosine kinases are lacking.

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Proteomic approach to the identification of voltage‐dependent anion channel protein isoforms in guinea pig brain synaptosomes

et al. 2004

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Voltage-dependent anion channel (VDAC) proteins are small, abundant, pore-forming proteins belonging to the eukaryotic mitochondrial porins. At least three different VDAC genes have been identified in vertebrates. VDAC proteins are known to play an essential role in cellular metabolism and in the early stages of apoptosis. A proteomic approach, consisting of two-dimensional gel electrophoresis followed by two-dimensional immunoblotting with anti-VDAC and anti-phosphotyrosine antibodies and by matrix-assisted laser desorption/ionization-time of flight mass spectrometry, was exploited to define the expression pattern of VDAC isoforms in guinea pig brain synaptosomes, both in normoxic and hypoxic conditions. In this way a total of five different VDAC isoforms were identified, as both VDAC1 and VDAC2 were detected in more than one electrophoretic spot. Moreover, VDAC isoforms selectively undergo hypoxia-induced tyrosine phosphorylation, suggesting that tyrosine phosphorylation may contribute to the modulation of VDAC protein function/conformation or interaction with other proteins in hypoxic conditions.

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Protein Blotting and Immunoblotting

Cited by 4 publications

References 53 publications

Extensive temporally regulated reorganization of the lipid raft proteome following T-cell antigen receptor triggering

Extensive temporally regulated reorganization of the lipid raft proteome following T-cell antigen receptor triggering

Expression of the small tyrosine phosphatase (Stp1) inSaccharomyces cerevisiae: A study on protein tyrosine phosphorylation

Proteomic approach to the identification of voltage‐dependent anion channel protein isoforms in guinea pig brain synaptosomes

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