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.
CITATION STYLE
Sousa, M. M. L., Steen, K. W., Hagen, L., & Slupphaug, G. (2011). Antibody cross-linking and target elution protocols used for immunoprecipitation significantly modulate signal-to noise ratio in downstream 2D-PAGE analysis. Proteome Science, 9. https://doi.org/10.1186/1477-5956-9-45
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