Antibody cross-linking and target elution protocols used for immunoprecipitation significantly modulate signal-to noise ratio in downstream 2D-PAGE analysis

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. bs3cross-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.conclusion: 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. © 2011 sousa et al; licensee biomed central ltd.