Abstract
BackgroundImmunoprecipitation 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.ResultsHere 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.ConclusionCareful 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.
Highlights
Immunoprecipitation and subsequent 2D-PAGE/mass spectrometry are powerful tools to study posttranslational protein modifications
Immunoprecipitation (IP), in the magnetic bead format, is a highly efficient method to enrich endogenous proteins from complex mixtures [1]. It is especially convenient for analysis of low abundance proteins and protein isoforms, and is by far faster than conventional column chromatography methods that include the additional risk of introducing artificial protein modifications due to the often lengthy purification schemes
We find that a main advantage of BS3 is general lower non-specific binding of proteins compared to dimethyl pimelimidate (DMP)
Summary
Immunoprecipitation and subsequent 2D-PAGE/mass spectrometry are powerful tools to study posttranslational 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 2DPAGE. This may impede detection of non-abundant protein isoforms. Potential isoforms resulting from e.g. A general problem with IP, notwithstanding the downstream electrophoretic method, is that a large number of proteins other than the antigen are generally observed in the resultant gels and of which most are non- bound to the affinity matrix. BS3 is an N-hydroxysulfosuccinimide (NHS) ester that targets the primary amine groups, but have additional cross-reactivity towards other nucleophilic groups in proteins, including tyrosines, serines and threonines [12,13]
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