Electroelution of fixed and stained membrane proteins from preparative sodium dodecyl sulfate-polyacrylamide gels into a membrane trap

Jacobs, Enno; Clad, Andreas

doi:10.1016/0003-2697(86)90033-3

Cited by 122 publications

(49 citation statements)

References 15 publications

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“…Indeed, the mass difference between two isoforms or between the isoform and the expected theoretical molecular mass would allow drawing a solid hypothesis on the nature of the protein modification. Efforts have been made to extract proteins from 2D polyacrylamide gels using various methods, including electroelution (12)(13)(14)(15), electrotransfer on membranes (16 -18), and passive elution (19 -21). These methods, however, are not adapted for the further determination of the molecular mass of picomoles of protein isoforms using MS. For example, electroelution uses large elution volumes leading to significant losses of material.…”

Section: Molecular and Cellular Proteomics 2:483-493 2003mentioning

confidence: 99%

Characterization of Protein Variants and Post-Translational Modifications: ESI-MSn Analyses of Intact Proteins Eluted from Polyacrylamide Gels

Claverol

Burlet‐Schiltz

Gairin

et al. 2003

Molecular & Cellular Proteomics

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We have developed a strategy to characterize protein isoforms, resulting from single-point mutations and posttranslational modifications. This strategy is based on polyacrylamide gel electrophoresis separation of protein isoforms, mass spectrometry (MS) and MS n analyses of intact proteins, and tandem MS analyses of proteolytic peptides. We extracted protein isoforms from polyacrylamide gels by passive elution using SDS, followed by nanoscale hydrophilic phase chromatography for SDS removal. We performed electrospray ionization MS analyses of the intact proteins to determine their molecular mass, allowing us to draw hypotheses on the nature of the modification. In the case of labile post-translational modifications, like phosphorylations and glycosylations, we conducted electrospray ionization MS n analyses of the intact proteins to confirm their presence. Finally, after digestion of the proteins in solution, we performed tandem MS analyses of the modified peptides to locate the modifications. Using this strategy, we have determined the molecular mass of 5-10 pmol of a protein up to circa 50 kDa loaded on a gel with a 0.01% mass accuracy. The efficiency of this approach for the characterization of protein variants and post-translational modifications is illustrated with the study of a mixture of -casein isoforms, for which we were able to identify the two major variants and their phosphorylation site and glycosylation motif. We believe that this strategy, which combines two-dimensional gel electrophoresis and mass spectrometric analyses of geleluted intact proteins using a benchtop ion trap mass spectrometer, represents a promising approach in proteomics. Molecular & Cellular Proteomics 2:483-493, 2003.The most widely used approach in proteomics using mass spectrometry (MS) 1 is referred to as "bottom up" strategy. In this approach, protein identification is achieved after protein separation by one-or two-dimensional (1D or 2D) polyacrylamide gel electrophoresis followed by protein digestion usually with trypsin, analyses of the resulting peptide mixture using various MS approaches, and data base search. To overcome limitations of 2D gel electrophoresis, alternative techniques based on multidimensional liquid chromatography have been recently developed (1, 2). The "bottom up" strategy has allowed the identification of thousands of proteins in complex mixtures (3). The low protein sequence coverage often obtained using the "bottom up" approach hinders the characterization of post-translational modifications, singlepoint mutations, and truncated forms of a protein. Moreover, the molecular mass of the intact protein is not directly accessible. Recently, "top down" approaches have been described, which are based on mass spectrometric analyses of intact proteins (4). Protein identification is obtained after data base search using the measured protein molecular mass and protein sequence tags determined from MS and tandem MS (MS/MS) analyses of the intact protein, respectively. The critical step in this strategy is t...

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Section: Molecular and Cellular Proteomics 2:483-493 2003mentioning

confidence: 99%

Characterization of Protein Variants and Post-Translational Modifications: ESI-MSn Analyses of Intact Proteins Eluted from Polyacrylamide Gels

Claverol

Burlet‐Schiltz

Gairin

et al. 2003

Molecular & Cellular Proteomics

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“…The antibodies against CbiB2 in the serum collected after the last injection were purified by affinity binding to an antigen column. To obtain the CbiB2 column, the immunity protein was electroeluted after electrophoresis and covalently linked to Sepharose CL-6B (Pharmacia) by established methods (19,37).…”

Section: Vol 177 1995 Immunity Protein Of Carnobacteriocin B2 1145mentioning

confidence: 99%

Characterization of the protein conferring immunity to the antimicrobial peptide carnobacteriocin B2 and expression of carnobacteriocins B2 and BM1

et al. 1995

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Cloning of a 16-kb DNA fragment from the 61-kb plasmid of Carnobacterium piscicola LV17B into plasmidless C. piscicola LV17C restores the production of the plasmid-encoded carnobacteriocin B2 and the chromosomally-encoded carnobacteriocin BM1 and restores the immune phenotype. This fragment also has sufficient genetic information to allow the expression of carnobacteriocin B2 and its immunity in a heterologous host. The gene locus (cbiB2) responsible for immunity to carnobacteriocin B2 is located downstream of the structural gene for carnobacteriocin B2 and encodes a protein of 111 amino acids (CbiB2). CbiB2 was expressed in Escherichia coli as a fusion of the maltose-binding protein and CbiB2. The fusion protein was purified on an amylose column and cleaved with factor Xa, and pure CbiB2 was isolated by high-performance liquid chromatography. The N-terminal amino acid sequence and mass spectrometry (molecular weight [mean ؎ standard error], 12,662.2 ؎ 3.4) of the purified protein agree with the information deduced from the nucleotide sequence of cbiB2. Western blot (immunoblot) analysis indicates that the majority of the intracellular pool of this immunity protein is in the cytoplasm and that a smaller proportion is associated with the membrane. CbiB2 confers immunity to carnobacteriocin B2, but not to carnobacteriocin BM1, when it is expressed in homologous or heterologous hosts. No protective effect is observed for sensitive cells growing in the presence of the bacteriocin when the immunity protein is added to the medium. The purified immunity protein does not show significant binding to microtiter plates coated with carnobacteriocin B2 and is not able to inactivate the bacteriocin in solution.

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“…P26 and p29 were isolated from Raji cell lysate by means of electroelution (11) after preparative SDS-13 % PAGE . After further purification on an SDS-10-18% gradient polyacrylamide gel, the electrotransfer on PVDF for the protein sequencing was performed .…”

mentioning

confidence: 99%

Autoantibodies against triosephosphate isomerase. A possible clue to pathogenesis of hemolytic anemia in infectious mononucleosis.

Ritter

Brestrich

Nellen

et al. 1990

The Journal of Experimental Medicine

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Various clinical and pathological conditions can develop after EBV infections depending on the age ofthe individual. Whereas young children show a silent seroconversion, adolescents with acute EBV infection develop infectious mononucleosis (IM) in half of the cases . Being a lymphotropic virus, EBV can trigger an unspecific B cell stimulation besides the specific immune response. This may cause a big rise of class G and M Igs in the sera. Little is known about their specificity. Heterophil antibodies appear that agglutinate sheep, horse, and bovine erythrocytes (1, 2). Cold antibody with anti-i specificity was found in 8% to 60-70% of the IM patients (3, 4). In addition, antibodies are found that react with components of the cytoskeleton (5) or possess antinuclear features (6). None of these antibodies always appears in acute EBV infection, thus, their significance for the pathogenesis of IM is not clear. This may be different in the antibodies we recently detected in all sera of persons acutely infected with EBV These antibodies possess specificity for the glycolytic enzyme, triosephosphate isomerase (TPI). Volume 171 February 1990 565-570 Materials and Methods Brief Definitive ReportPreparing the Antigens. Frozen cells (1 ml packed volume) and disrupted tissues were allowed to thaw, and were subsequently resuspended in equal volumes of modified lysis buffer (7) containing 9.5 M urea, 2% (wt/vol) NP-40, and 5% 6-ME; further extraction was done by five cycles of freezing and thawing (-20°C/room temperature). The cell lysate was then centrifuged at 3,000 g for 15 min. The supernatant fluid was used for antigen preparation by SDS-PAGE followed by immunoblotting. The protein concentration (20 mg/ml) was measured by the method of Neuhoff et al. (8) .PAGE. Composition and electrophoresis of SDS-13 % (wt/vol) polyacrylamide slab gels were performed as reported by Laemmli (9) . The electrotransfer of proteins from SDS gels to hydrophobe polyvinylilenedifluoride (PVDF) membrane (Millipore Continental Water Systems, Bedford, MA) was performed by transversal electrophoresis (10).Immunostaining the Blots. For the detection of antibodies, sera, as well as secondary antibodies conjugated with peroxidase, were diluted in 20% FCSTBS (vol/vol) . Each antibody

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Electroelution of fixed and stained membrane proteins from preparative sodium dodecyl sulfate-polyacrylamide gels into a membrane trap

Cited by 122 publications

References 15 publications

Characterization of Protein Variants and Post-Translational Modifications: ESI-MSn Analyses of Intact Proteins Eluted from Polyacrylamide Gels

Characterization of Protein Variants and Post-Translational Modifications: ESI-MSn Analyses of Intact Proteins Eluted from Polyacrylamide Gels

Characterization of the protein conferring immunity to the antimicrobial peptide carnobacteriocin B2 and expression of carnobacteriocins B2 and BM1

Autoantibodies against triosephosphate isomerase. A possible clue to pathogenesis of hemolytic anemia in infectious mononucleosis.

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