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

作者：Sousa Mirta M L; Steen Kristian W; Hagen Lars; Slupphaug Geir^*

来源：Proteome Science, 2011, 9: 45.

DOI：10.1186/1477-5956-9-45

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 (R) Protein A by using DMP or BS(3), as well as the efficiency of various target elution buffers prior to 2D-PAGE separation. BS(3) 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.
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.

• 出版日期2011-8-4