to expand a large - scale culture of bg01 cells for this assay, a collagenase - and trypsin - based passaging method was used [ 23 ]. while these conditions have been associated with the accumulation of trisomies of chromosomes 12, 17 and x [ 24 ], the ease of use of these cultures and similarity in gene expression and differentiation potential to karyotypically normal bg01 hescs [ 11, 24, 25 ] make them suitable for such large scale applications. for the powerblot screen, whole cell lysate from bg01 hescs was separated on five 4 – 15 % gra dien t gels. each blot contained size markers and 39 lanes. each lane was screened with 1 – 8 antibod ies in combinations that had been predetermine d to enable ac curate identification of well - separated proteins ( fig. 1a – e ). the gels and blots were performed in duplicate and expressed proteins were identified by their predicted size and verified by visual inspection. figure 1powerblot analysis of undifferentiated bg01 hescs. this large - scale western blot consisted of five gels run in duplicate and probed with 934 antibod ies. ( a - e ) one set of blots is shown at a contrast that highlights most bands. ( f ) a representative lane ( gel c, lane 24 ) aligned with protein markers used for band identification. ( g ) scatter - plot of the normalized average intensity ( i. u. ) values for each protein indicating a linear relationship between duplicate blots. datasets for this analysis are in additional tables 1 and 2. a total of 545 antibod ies detected bands of appropriate size, which could be compressed to 529 proteins with unique swissprot identification numbers ( fig. 1a – e and additional file 1 ). an enlargement of a representative lane ( lane 24 of blot c ) alongside protein markers is shown in fig. 1f. thirteen proteins including akt, cav eol in1 and erk1 were detected in multiple lanes using the same or different antibod ies. information on the antibod y catalogue number and dilution, band intensity for each repeat and the averaged value, description of protein function, and entrez gene and swissprot database identification numbers is shown in additional file 1. three hundred and eighty three antibod ies did not detect bands in this screen, indicating lack of expression, or possibly technical issues with detection under standard conditions ( additional file 1 ). the size of the detected proteins ranged from 15 kd ( gs15 ) to 280 kd ( abp - 280 ). the average intensity of the detected proteins ranged from 195 to 117926 normalized intensity units ( i. u. ), with an average of 5367 i. u. the proteins with the highest band intensity were the b2 bradykinin receptor ( 117926 i. u. ), whilst the proteins with the lowest intensity that could be verified by visual inspection were inhibitor 2 ( 247 i. u. ), finally, the consistency of this assay was demonstrated by plotting the normalized average intensity values for each protein, which revealed a linear relationship between the duplicate samples ( fig. 1g ). kinexus analysis of hescsa more focused screen was used to profile expression of protein kinases, phosphatases and phosphorylated sites in cell signaling molecules in hescs. the kinexus assays contained 140 antibod ies to these related classes of proteins and phospho - sites. karyotypically normal bg03 hescs grown on a fibronectin matrix in mef - cm [ 26 ] were used for this analysis, and whole cell lysate was separated on four 12. 5 % gels for western blotting. eighty five immunoreactive bands were identified, representing 38 protein kinases and 16 phosphatases, their isoforms, and 22 phosphorylated sites in signaling molecules ( fig. 2a – d, additional file 1 ). sixty - four antibod ies did not detect their corresponding antigen ( additional file 1 ). figure 2kinexus blots of undifferentiated bg03 cells. four blots were used to probe bg03 lysate with ( a, b ) 76 antibod ies for protein kinases, ( c ) 27 antibod ies for phosphatases and ( d ) 37 antibod ies for phosphoylated sites in cell signaling molecules. identified bands are indicated ( * ). datasets for this analysis are in additional tables 1 and 2. functional classification of proteins expressed in hescsthe powerblot and kinexus assays identified a diverse range of proteins expressed in hescs. to further annotate these data, the detected proteins were ordered into 18 subgroups based on protein function ( additional file 2 ). for example, 16 factors with known or implied roles in the regulation of self - renewal or pluripotency of mescs or hescs, such as oct4 [ 27 ], stat3 [ 28 ], members of the fgf [ 29 ], pi3 kinase [ 30 ], src [ 31 ] or mapk pathways [ 32 ], and phosphorylated isoforms of gsk3, stat3 and p38 mapk, were grouped under " pluripotency " ( fig. 3a and additional file 2 ). another functional group ( cell surface ) consisted of 20 transmembrane or cell surface proteins ( additional file 2 ). this included several receptors for peptides and growth factors, such as neurotensin receptor 3, the b2 bradykinin, endothelin 1, and thrombin receptors, and the glial derived neurotrophic factor receptor α ( fig. 3b ). these molecules may be useful as targets for cell sorting experiments, and expression of these receptors could identify bioactive peptides or growth factors that may influence hesc self - renewal or differentiation. figure 3functional classification and mobility variants of proteins detected in hescs. ( a ) proteins with known or suggested roles in self - renewal are shown, including oct4, stat3, smad2 / 3 and fgf2 ( additional table 2, " pluripotency " ). isoforms of fgf2, and phospho - gsk3 are indicated ( * ). ( b ) cell surface proteins are shown, including connexin 43, e - cad and gdnfrα ( additional table 2, " cell surface " ). other functional classes of proteins are indicated in additional table 2. ( c ) a total of 42 proteins, including fgf2, hsp70 and erk1, were found to have multiple bands in either the powerblot or kinexus blots. these bands migrated closely but were sufficiently separated from other detected proteins. bands predicted to be isoforms of the indicated protein are highlighted in some panels ( * ). other functional classification of the proteins detected by the powerblot screen included: transcription factors ( 71 proteins ), nucleus and nuclear transport ( 144 ), cytoskeleton ( 75 ), cell adhesion ( 45 ), map kinase pathway ( 24 ), protein kinase a ( 13 ), protein kinase c ( 20 ), tyrosine kinases ( 15 ), adaptors and tyrosine kinase substrates ( 51 ), protein phosphatases ( 17 ), gtpases and regulators ( 42 ), calcium signaling ( 23 ), cell cycle ( 87 ), apoptosis ( 61 ), membrane research ( 62 ), and other functions ( 51 ) ( additional file 1 ). some proteins were included in multiple functional categories due to overlapping properties, such as aim - 1, which was included in the cell cycle as well as in the nucleus / nuclear transport categories. the kinexus expression data was organized separately into cell signaling - related functional groups ( additional file 1 ).