title: Antibody cross-linking and target elution protocols used for immunoprecipitation significantly modulate signal-to noise ratio in downstream 2D-PAGE analysis.
abstract: BACKGROUND
  Immunoprecipitation and subsequent 2D-PAGE/mass spectrometry are powerful tools to study post-translational protein modifications. Often disregarded in this workflow is the impact of the chemical cross-linker upon antibody affinity, as well as incomplete elution of primary target protein in buffers commonly used in 2D-PAGE. This may impede detection of non-abundant protein isoforms.
  
  
  RESULTS
  Here we have compared cross-linking of antibodies to Dynabeads® Protein A by using DMP or BS3, as well as the efficiency of various target elution buffers prior to 2D-PAGE separation. BS3 cross-linking generally resulted in less non-specific binding than DMP, whereas DMP cross-linking gave overall higher yield of target protein. Regardless of the cross-linker used, incomplete elution of target protein was observed with conventional glycine- or urea-based buffers. Conversely, complete elution was obtained with 2% hot SDS and subsequent dilution in urea buffer containing 4% CHAPS, to 0.2% final SDS yielded perfectly focused gels suitable for mass spectrometry analysis.
  
  
  CONCLUSIONS
  Careful choice of Ig cross-linker as well as efficient elution of target protein in SDS prior to downstream 2D-PAGE may be key factors to analyze low-abundance proteins enriched by magnetic bead immunoprecipitation.
authors.affiliation: Department of Cancer Research and Molecular Medicine and The FUGE proteomics laboratory, Norwegian University of Science and Technology, N-7006 Trondheim, Norway.
authors.forename: Mirta Ml
authors.initials: MM
authors.lastname: Sousa
authors.affiliation: Department of Cancer Research and Molecular Medicine and The FUGE proteomics laboratory, Norwegian University of Science and Technology, N-7006 Trondheim, Norway.
authors.forename: Kristian W
authors.initials: KW
authors.lastname: Steen
authors.affiliation: Department of Cancer Research and Molecular Medicine and The FUGE proteomics laboratory, Norwegian University of Science and Technology, N-7006 Trondheim, Norway.
authors.forename: Lars
authors.initials: L
authors.lastname: Hagen
authors.affiliation: Department of Cancer Research and Molecular Medicine and The FUGE proteomics laboratory, Norwegian University of Science and Technology, N-7006 Trondheim, Norway.
authors.forename: Geir
authors.initials: G
authors.lastname: Slupphaug