Improved elution conditions for native co-immunoprecipitation

Abstract

Background: Native immunoprecipitation followed by protein A-mediated recovery of the immuno-complex is a powerful tool to study protein-protein interactions. A limitation of this technique is the concomitant recovery of large amounts of immunoglobulin, which interferes with down-stream applications such as mass spectrometric analysis and Western blotting. Here we report a detergent-based "soft" elution protocol that allows effective recovery of immunoprecipitated antigen and binding partners, yet avoids elution of the bulk of the immunoglobulin.

Methodology/principal findings: We assessed the performance of the soft elution protocol using immunoprecipitation of Adaptor protein complex 1 (AP-1) and associated proteins as a test case. Relative to conventional elution conditions, the novel protocol substantially improved the sensitivity of mass spectrometric identification of immunoprecipitated proteins from unfractionated solution digests. Averaging over three independent experiments, Mascot scores of identified AP-1 binding partners were increased by 39%. Conversely, the estimated amount of recovered immunoglobulin was reduced by 44%. We tested the protocol with five further antibodies derived from rabbit, mouse and goat. In each case we observed a significant reduction of co-eluting immunoglobulin.

Conclusions/significance: The soft elution protocol presented here shows superior performance compared to standard elution conditions for subsequent protein identification by mass spectrometry from solution digests. The method was developed for rabbit polyclonal antibodies, but also performed well with the tested goat and mouse antibodies. Hence we expect the soft elution protocol to be widely applicable.

Figure 1. Graphical overview: Immunoprecipitation, standard vs improved elution protocol, and down-stream analysis.

The diagram highlights key steps of the soft elution protocol, and shows how the comparison with standard elution conditions was performed. A complete description of the process can be found in File S1.

Figure 2. SDS-PAGE analysis of standard and improved elution protocols.

Native immunoprecipitation of the AP-1γ subunit from HeLa cell lysates was performed as described in File S1. Immuno-complexes were recovered by addition of protein A sepharose beads. Prior to elution, beads were split into two equal aliquots. One was subjected to typical high SDS/high heat elution conditions (“standard elution”, lane 1), the other to our “soft” elution protocol (lane 2). Following soft elution, beads were re-eluted using the standard protocol (lane 3), to recover protein still bound to the beads. Thus, lane 3 shows the proportion of immunoglobulin (IgG) that is avoided through soft elution. Proteins in selected bands were identified by mass spectrometry (small arrows). For each band only the top scoring hit is shown. AP-1β, AP-1γ and AP-1µ are components of the AP-1 complex; p200 and γ-Synergin are known AP-1 associated proteins. IgG: rabbit Ig gamma chain C region. Approximate molecular weights are indicated (MW, in kD). Gels were stained with Coomassie G-250.

Figure 3. Relative abundance of immunoglobulin (Ig) in IP eluates.

The amount of rabbit Ig present in IP eluates was estimated by summation of emPAI values. Average total Ig in standard eluates was set to 1, and average total Ig present in soft elution eluates was expressed as a fraction of 1. The figure shows the results of three independent experiments (error bars  =  SEM).

Figure 4. Performance of the improved elution protocol with various rabbit antibodies.

Immunoprecipitations and SDS-PAGE were performed as in Figure 2, with the indicated antibodies (all rabbit polyclonal). Immunoprecipitated proteins were eluted from the protein A sepharose beads using standard conditions (lane 1), or soft-elution (lane 2). Soft-eluted beads were then subjected to standard elution conditions, to recover any remaining material (lane 3). Hence, lane 3 shows the proportion of immunoglobulin (Ig) avoided through soft elution. Lane 4 shows molecular weight markers (MW, in kD). Gels were stained with Coomassie G-250. Small arrows indicate the precipitated primary antigens (identified by molecular weight and through comparison with Western blots). Arrowheads indicate IgG heavy chain bands (identified by molecular weight and abundance). Asterisks indicate examples of proteins that co-precipitate with the primary antigen. The figure shows that soft elution substantially reduces the amount of co-eluting Ig for all tested antibodies, whilst allowing efficient recovery of co-precipitating proteins.

Figure 5. Performance of the improved elution protocol with mouse and goat antibodies.

Immunoprecipitations and SDS-PAGE were performed as in Figures 2 and 4, with an IgG_2a mouse monoclonal antibody (mAb) against AP-1γ (left panel), or a goat polyclonal antibody (pAb) against CALM (right panel). Gels were stained with Coomassie G-250. Small arrows indicate the precipitated primary antigens (identified by molecular weight and through comparison with Western blots). Please note that CALM occurs in two isoforms (62 kD and 72 kD). Arrowheads indicate IgG heavy chain bands (identified by molecular weight and abundance). The figure shows how the soft elution protocol performs with non-rabbit antibodies. In case of the goat polyclonal antibody, co-elution of Ig is almost completely avoided through soft elution.