Journal: The Journal of Biological Chemistry

Article Title: Efficient Qualitative and Quantitative Determination of Antigen-induced Immune Responses *

doi: 10.1074/jbc.M116.736660

Figure Lengend Snippet: Evaluation of a small scale mouse serum IgG purification using the PhyNexus automated MEA system. A , SDS-PAGE analysis of samples obtained after various steps of the purification process. Samples maintained under non-reducing ( left ) and reducing ( right ) conditions were run in parallel on individual pre-cast polyacrylamide gels. Markers are in lane 1 ; flow-through in lane 2 ; washes in lanes 3–5 (DPBS; DPBS + 1 m NaCl; DPBS, respectively), and sequential elutions in lanes 6–9. H and L denote IgG heavy and light chain bands in the reducing gel. B , continuous sedimentation coefficient ( s ) and distribution of purified mouse IgGs in 60 m m sodium acetate, pH 5.0, buffer after AUC. s 20, w 0 denotes the apparent corrected sedimentation coefficient calculated using the buffer's density and viscosity. The 95% peak represents IgG monomers.

Article Snippet: After the pre-packed PhyTip columns and reagent plates were loaded onto the MEA instrument platform, the mouse antibodies were isolated using a method programmed with PhyNexus MEA software (version 1.0.17) consisting of the following steps: 1) pre-wash and equilibration of the resin with PBS; 2) capture of IgGs from the diluted 500-μl mouse serum samples with the ProPlus affinity resins; 3) washes with PBS and PBS plus 1 m NaCl to remove other serum proteins; 4) elution of bound IgGs from the resins with 30 m m sodium acetate, pH 3.0, three times, followed by one elution with 30 m m sodium acetate, pH 2.5, to ensure the full removal of any remaining bound IgGs from the resins; and 5) neutralization using 10% (by volume) of 300 m m sodium acetate, pH 9.0, to bring the final formulation to 60 m m sodium acetate, pH 5.0.

Techniques: Purification, SDS Page, Sedimentation