statistical analysisoutcome measurement for each experiment was reported as mean±sem. all data were analyzed using spss 11 for mac os x ( spss inc. ). significance of inter - group differences was performed by applying student ' s t - test where appropriate. the one - way anova analysis was used to compare group differences for the forelimb use as the dependant variable and groups as the single independent factor variable. differences between the groups were determined using bonferroni ' s post hoc test. a p - value of less than 0. 05 was considered to be statistically significant. backgroundmuch of our current knowledge of the molecular expression profile of human embryonic stem cells ( hescs ) is based on transcriptional approaches. these analyses are only partly predictive of protein expression however, and do not shed light on post - translational regulation, leaving a large gap in our knowledge of the biology of pluripotent stem cells. resultshere we describe the use of two large - scale western blot assays to identify over 600 proteins expressed in undifferentiated hescs, and highlight over 40 examples of multiple gel mobility variants, which are suspected protein isoforms and / or post - translational modifications. twenty - two phosphorylation events in cell signaling molecules, as well as potential new markers of undifferentiated hescs were also identified. we confirmed the expression of a subset of the identified proteins by immunoflu orescence and correlated the expression of transcript and protein for key molecules in active signaling pathways in hescs. these analyses also indicated that hescs exhibit several features of polarized epithelia, including expression of tight junction proteins. conclusionour approach complements proteomic and transcriptional analysis to provide unique information on human pluripotent stem cells, and is a framework for the continued analyses of self - renewal. human embryonic stem cells ( hescs ) are pluripotent cells isolated from the inner cell mass of the blastocyst [ 1 ]. they can be maintained for prolonged periods in culture and differentiate to representatives of the three germ layers as well as trophoblasts and germ cells. this differentiation potential may be used to model certain aspects of human embryogenesis, including the development and differentiation of pluripotent and other stem cell types during the processes of gastrulation, neurogenesis and organogenesis. thus, hescs provide a unique and powerful system to study otherwise intractable aspects of human development. furthermore, these approaches have the potential to provide differentiated cell types for cell replacement therapies of degenerative disor ders such as parkinson ' s disease and type i diabetes [ 2, 3 ]. before these cell therapy applications are developed, an understanding of the molecular and cellular mechanisms that drive self - renewal and differentiation is required. fundamental to this understanding is the elucidation of the transcriptome and proteome of hescs, using approaches that lay a framework for functional analyses of the unique properties of these cells. large - scale gene expression analyses such as microarray, massive parallel signature sequencing ( mpss ), expressed sequenced tag ( est ) enumeration, and serial analysis of gene expression ( sage ) have been used to compare multiple hesc lines [ 4 - 7 ]; hescs to germ cell tumors [ 8 ]; or to differentiated derivatives in embryoid bodies [ 9 - 11 ] or neural populations [ 12 ]. these approaches have highlighted an expanded set of transcripts that mark the pluripotent state [ 4, 13, 14 ], cross - species commonalities in the molecular profile of escs [ 6, 12, 15 ], prominent receptors expressed by hescs [ 8 ] and pathways that may play a role in the regulation of pluripotency [ 16, 17 ]. nevertheless, cataloguing the cellular transcriptome is only predictive of protein expression and typically does not shed light on post - transcriptional regulation. for example, while tens of thousands of transcripts can be followed simultaneously with sage, microarrays and mpss, these methods do not routinely detect differences in transcript splice variants, or polyadenylation status. these differences may have profound effects on translation, as well as the isoform and function of the protein produced. finally, numerous post - translational modifications are known to regulate protein function, including enzymatic cleavage, covalent coupling to other molecules, glyco sylation, phosphorylation and ubiquitination. these issues all highlight potential shortfalls in our understanding of the hesc proteome. several practical approaches for proteomic analyses are currently available, the most established of which is the 2 - dimensional ( 2d ) separation of proteins by polyacr ylamide gel electrophoresis ( page ). hplc - tandem mass spectrometry ( hplc - ms / ms ) based technology is rapidly evolving and has recently been used to detect protein expression in multiple cell types. an alternate approach is the recent large - scale adaptation of standard western blotting [ 18 ]. in this procedure, a large well is used to separate the sample by page and lanes are created on the membrane containing immobilized protein with the use of a manifold. compatible combinations of primary antibod ies are predetermine d, with the criterion of being able to identify proteins that do not co - migrate. different combinations of primary antibod ies are added to each well, with appropriate dilutions of each primary antibod y so that expressed proteins are detected in a single condition. the scalability of the system depends on defining suitable combinations of primary antibod ies, with up to 1000 antibod ies in 200 lanes being used in the largest screens thus far. detection software is used to identify proteins based on their expected and observed gel mobility. unlike 2d page and hplc - ms / ms, large - scale western blotting only identifies proteins for which antibod ies are already available. while this is not an appropriate screen for identifying uncharacterized proteins, it greatly simplifies the verification and functional analyses of proteins that are detected. in addition, this approach is highly flexible, and if desired can be focused to particular sets of proteins or protein function, such as cell signaling molecules. importantly, the foundation of this approach is the large amount of data on individual antibod ies, which are already available and characterized in the literature. more recently, two research groups have conducted proteomic analyses of hescs using ms [ 19 - 22 ]. in the present study, we used two large - scale western blot systems to examine the expression of > 1000 proteins in hescs and detected > 600 proteins that were grouped into 18 functional classes. in addition, we identified 42 examples of multiple bands for a single protein, likely to be protein isoforms and / or post - translational modifications, and 22 phosphorylation events in cell signaling molecules. we correlated the expression of members of key active pathways in our transcriptional and proteomic databases and confirmed the validity of this approach. using these approaches we identified new markers for undifferentiated hescs and highlighted unrecognized epithelial characteristics of hescs. our data confirm the importance of proteomic analyses in complementing transcriptional profiling and provide a framework for continued analyses of the molecular and cellular biology of pluirpotent hescs. powerblot analysis of hescswe first employed a large - scale western blot screen, the powerblot system, to profile protein expression in undifferentiated hescs. this system used 934 antibod ies toward proteins representing 22 diverse classes of function, such as transcription factors, the map kinase ( mapk ) pathway, and apoptosis, among others.