Evidence of Multimeric Forms of HSP70 with Phosphorylation on Serine and Tyrosine Residues – Implications for Roles of HSP70 in Detection of GI Cancers

Background: Heat-shock protein70 (HSP70) are intracellular protein chaperones, with emerging evidenceof 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 specificitydue to cross-reactivity by similarities in the amino-acid sequence in regions of the protein backbone resulting inoverestimated HSP70 value. Materials and
Methods: This study was undertaken to develop a methodology tocapture 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 incubatedwith HSP70 antibody-coupled beads. After blocking non-specific binding sites, blots were immunostainedwith monoclonal-antibody specific for human-HSP70, phosphoserine and phosphotyrosine.
Conclusions: Ournovel immunocapture approach has distinct advantages over the commercially available methods of pHSP70quantification by allowing isolation of molecular aggregates of HSP70 with additional ability to preciselydistinguish phosphorylation state of HSP70 molecules at serine and tyrosine residues.