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200 µg of tissue lysate protein extracts were separated on 2-DE and stained with Colloidal Coomassie G-250 and acquired as an image (2-DE-cCBB). In parallel 40 µg of total protein extract was separated by 2-DE and subsequently transferred onto PVDF membrane for incubation with antibodies directed against phosphorylated proteins. With a marker pen, landmarks points (some are highlighted by arrows in the Figure) were set around the membrane to be used for computer assisted merging and matching of images of phosphorylated proteins as acquired with the Abs WB-Yset, WB-pSset, WB-4G10, WB-MPM-2. Subsequently, protein spots on the same membrane were revealed by cCCB-post staining and the image was recorded (not shown). Using the marker added landmarks, the images were superimposed and combined by the image analysis software to create a virtual image showing the phosphoproteins and the total proteins together (WB-virtual).
Source publication
Reversible protein phosphorylation is an essential mechanism in the regulation of diverse biological processes, nonetheless is frequently altered in disease. As most phosphoproteome studies are based on optimized in-vitro cell culture studies new methods are in need to improve de novo identification and characterization of phosphoproteins in extrac...
Contexts in source publication
Context 1
... demonstrate the utility of the developed method lung tissue of mice was analyzed and protein extracts were separated by 2-D PAGE. Representative 2-DE maps of tissue lysates and immunoblots are shown in Figure 2. On average 450630 protein spots per gel were visualized by colloidal CCB staining (cCCB) at a pI range of 3-11. ...Context 2
... the detection of serine phosphorylated proteins, a set of 5 different anti-phosphoserines (pSset) and the MPM-2 MAbs were used (Supplementary Table S2). After development on PVDF membranes (Figure 2) with the pSset of monoclonal antibodies, the corresponding spots were excised from the 2-DE cCBB stained gel, in-gel digested with trypsin to overall yield 149 unique proteins (Table S1). In Figure 3 an example of the specificity of the pSset of MAbs is given. ...Context 3
... the detection of tyrosine phosphorylated proteins a set of 4 different monoclonal anti phosphotyrosine (pYset) and the 4G10 MAb were used (Table S2). With the pYmix MAbs (Figure 2) 54 unique proteins were detected and the corresponding spots on 2- DE-cCBB stained gels were excised. Thereafter trypic digestes were prepared and analysed by MALDI-MS. ...Similar publications
Nanocomposites are given preference over the individual materials due to the combined properties of the components involved. Ceria has a high efficiency in phosphopeptide enrichment as well as in dephosphorylation. Iron oxide and tin oxide are chosen as counter metal oxides to synthesize the ceria nanocomposites using a co-precipitation method. The...
Citations
... However, the restricted number of spots generated and the manual stage of matching limited the correction efficiency of this approach. Several authors have reported post-staining of the membrane, using colloidal gold particles [34,37,53], silver [54,60] or CCB [55,61] after immunodetection. This approach presented the advantage of staining the proteomic map from each immunoblot, but it implied the treatment of different images generated from 2 distinct steps. ...
... But the superimposition of these 2 maps remained a manual procedure even if they used image analysis software (Adobe Photoshop) [63,64]. Some authors have also suggested adding a 'pen' landmark to facilitate the superimposition of the antigenic map on its related stained transferred protein map image [61]. The defect common to all these approaches was the generation of multiple images that had to be aligned manually. ...
Serological proteome analysis (SERPA) combines classical proteomic technology with effective separation of cellular protein extracts on two-dimensional gel electrophoresis, western blotting, and identification of the antigenic spot of interest by mass spectrometry. A critical point is related to the antigenic target characterization by mass spectrometry, which depends on the accuracy of the matching of antigenic reactivities on the protein spots during the 2D immunoproteomic procedures. The superimposition, based essentially on visual criteria of antigenic and protein spots, remains the major limitation of SERPA. The introduction of fluorescent dyes in proteomic strategies, commonly known as 2D-DIGE (differential in-gel electrophoresis), has boosted the qualitative capabilities of 2D electrophoresis. Based on this 2D-DIGE strategy, we have improved the conventional SERPA by developing a new and entirely fluorescence-based bi-dimensional immunoproteomic (FBIP) analysis, performed with three fluorescent dyes. To optimize the alignment of the different antigenic maps, we introduced a landmark map composed of a combination of specific antibodies. This methodological development allows simultaneous revelation of the antigenic, landmark and proteomic maps on each immunoblot. A computer-assisted process using commercially available software automatically leads to the superimposition of the different maps, ensuring accurate localization of antigenic spots of interest.
