A general method for the rapid characterization of tyrosine-phosphorylated proteins by mini two-dimensional gel electrophoresis

Abstract

Our preliminary results are reported in the investigation of the tyrosine phosphorylation cascade triggered by the stimulation of the insulin receptor in the adipocyte cell line 3T3-L1 using a mini two-dimensional gel electrophoresis approach. The minigel format, 8 x 10 cm, was found sufficiently resolving and reproducible to study complex biological samples while considerably increasing throughput and lowering costs compared to larger gel formats. Consequently, we used the minigel format to rapidly screen a large number of samples, of which only the most relevant were then analyzed by optimized, preparative two-dimensional gels. The accurate localization and relative quantification of tyrosine-phosphorylated proteins was performed using a nonradioactive triple labeling method. After transfer onto polyvinylidene difluoride (PVDF) membranes, proteins were stained with Sypro Ruby to verify the separation quality and to localize the general region of interest for immunostaining. The membranes were subsequently blocked with polyvinylpyrrolidone-40 and probed with the relevant antibodies for visualization of the phosphorylated proteins by chemiluminescence. Finally, membranes were stained with colloidal gold to obtain a pattern reminiscent of the silver staining of a polyacrylamide gel. We believe that the presented strategy can be generalized for any gel application in which a protein has to be detected and identified based on its immunoreactivity.

New approaches to proteomic analysis of breast cancer

“…The low molecular weight ionized proteins and peptides are recorded as mass signature peaks as they strike a detector plate based on their differential time of flight, and the data are displayed as a standard chromatograph (Figs. [2][3][4][5]. SELDI analysis software allows data to be viewed as a standard mass chromatograph or as a gel-like density graph, as shown in Figs.…”

“…In addition to providing quantitative data, proteomics can provide additional qualitative information that transcriptional analyses cannot. This includes post-translational modifications such as acetylation, ubiquitination, phosphorylation [3,4], or glycosylation [5,6]. Proteomics of subcellular organs have been reported, including the mitochondrion and the cell membrane [7][8][9].…”

Characterization of Phosphorylated Proteins Using Mass Spectrometry

“…As an alternative to radioactive analysis, immunoaffinity approaches have been employed either in an immunoblotting or protein array format [20,21]. The challenge with affinity based methods is that they do not directly identify the modified residue unless a site specific antibody is available.…”

Simultaneous red/green dual fluorescence detection on electroblots using BODIPY TR-X succinimidyl ester and ELF 39 phosphate

“…More recently, a serial staining procedure that employs colloidal silver stain instead of an organic dye has been introduced to improve detection sensitivity [4]. The fluorescent SYPRO Ruby protein blot stain (Molecular Probes, Eugene, OR, USA) has been successfully incorporated into immunodetection procedures employing colorimetric, fluorescent or chemiluminescent detection reagents [5][6][7]. Successful immunodetection after SYPRO Ruby dye staining may be aided by the reversibility of the stain, which is washed away during the blocking step, prior to incubation with antibodies.…”

“…Conventional, colorimetric or chemiluminescent immunoblotting procedures have been performed after visualizing total protein profiles on blots by colloidal gold staining [9,10]. Membranes stained with colloidal gold generate heavy background signal after applying chemiluminescence based immunodetection methods, however, and acceptable results are obtained only when membranes are partially stained so that proteins are barely visible [7]. A method for the detection of proteins electroblotted to PVDF membranes was developed exploiting direct fluorescence detection of 2-methoxy-2,4-diphenyl-2(2H)furanone (MDPF) labeled proteins on PVDF membranes using a UV transilluminator followed by chemiluminescent immunodetection [11,12].…”