This protocol describes two options for the native (nondenaturing) elution of protein complexes obtained by affinity capture. The first approach involves the elution of complexes purified through a tag that includes a human rhinovirus 3C protease (PreScission protease) cleavage site sequence between the protein of interest and the tag. Incubation with the protease cleaves immobilized complexes from the affinity medium. The second approach involves the release of protein A-tagged protein

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... s using a competitive elution reagent called PEGylOx. The degree of purity of the native assemblies eluted is sample dependent and strongly influenced by the affinity capture. It should be noted that the efficiency of native elution is commonly lower than that of elution by a denaturing agent (e.g., SDS) and the release of the complex will be limited by the activity of the protease or the inhibition constant (K i ) of the competitive release agent. However, an advantage of native release is that some nonspecifically bound materials tend to stay adsorbed to the affinity medium, providing an eluted fraction of higher purity. Finally, keep in mind that the presence of the protease or elution peptide could potentially affect downstream applications; thus, their removal should be considered. MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous material used in this protocol. Reagents Desalting spin columns (40 kDa molecular mass cutoff) (optional; see Step 14) Depending on the volume of the eluted fraction, use either Micro Bio-Spin Columns with Bio-Gel P-30 (Bio-Rad 732-6223) or Zeba Micro Spin Desalting Columns, 75 µL, with 40 kDa molecular mass cutoff (Thermo Scientific 87764). These columns give equivalent results, depleting the peptide >100-fold. Digestion buffer (for Steps 1-10 only) Determine the optimal composition of this buffer for each protein complex of interest. A suggested formulation to work from is 20 mM K-HEPES (pH 7.4), 150 mM sodium chloride, 110 mM potassium acetate, 2 mM magnesium chloride, 0.1% Tween 20, and 1 mM DTT. PEGylOx native elution solution (saturated solution; >2 mM) (for Steps 11-15 only) PEGylOx is an amino-terminally PEGylated peptide of primary sequence DCAWHLGELVWCT, cyclized by oxidation of the cysteines to cystine. It can be synthesized by standard Fmoc solid-phase synthesis methods. The PEGylOx solution can be prepared using a solvent of your choosing. As long as the saturation concentration of 1 These two authors contributed equally.