title: Evidence of multimeric forms of HSP70 with phosphorylation on serine and tyrosine residues--implications for roles of HSP70 in detection of GI cancers.
abstract: BACKGROUND
  Heat-shock protein70 (HSP70) are intracellular protein chaperones, with emerging evidence of their association with various diseases. We have previously reported significantly elevated plasma-HSP70 (pHSP70) in pancreatic cancer. Current methods of pHSP70 isolation are ELISA-based which lack specificity due to cross-reactivity by similarities in the amino-acid sequence in regions of the protein backbone resulting in overestimated HSP70 value.
  
  
  METHODS
  This study was undertaken to develop a methodology to capture all isoforms of pHSP70, while further defining their tyrosine and serine phosphorylation status.
  
  
  RESULTS
  The methodology included gel electrophoresis on centrifuged supernatant obtained from plasma incubated with HSP70 antibody-coupled beads. After blocking non-specific binding sites, blots were immunostained with monoclonal-antibody specific for human-HSP70, phosphoserine and phosphotyrosine.
  
  
  CONCLUSIONS
  Our novel immunocapture approach has distinct advantages over the commercially available methods of pHSP70 quantification by allowing isolation of molecular aggregates of HSP70 with additional ability to precisely distinguish phosphorylation state of HSP70 molecules at serine and tyrosine residues.
authors.affiliation: Division of Gastroenterology, Department of Medicine, Johns Hopkins University/Sinai Hospital Program in Internal Medicine, USA E-mail : sdutta@lifebridgehealth.org, dutts31@yahoo.com.
authors.forename: Anand
authors.initials: A
authors.lastname: Dutta
authors.forename: Mohit
authors.initials: M
authors.lastname: Girotra
authors.forename: Nipun
authors.initials: N
authors.lastname: Merchant
authors.forename: Padmanabhan
authors.initials: P
authors.lastname: Nair
authors.forename: Sudhir Kumar
authors.initials: SK
authors.lastname: Dutta
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D003429:Cross Reactions / Q000276:immunology; D005770:Gastrointestinal Neoplasms / Q000276:immunology / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation / Q000276:immunology / Q000502:physiology*; D012694:Serine / Q000276:immunology / Q000378:metabolism*; D014443:Tyrosine / Q000276:immunology / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D018840:HSP70 Heat-Shock Proteins; D014443:Tyrosine; D012694:Serine