Figure 3
One-step purification of BC2-tagged proteins using the BC2 nanotrap.
(a) For immunoprecipitation soluble protein fractions of bacterial lysates either expressing GFP with a C-terminal BC2 tag (GFPBC2T) or solely GFP were incubated with the BC2-Nb immobilized on agarose (BC2 nanotrap). Input (I), non-bound (NB) and bound fractions (B) were separated by SDS-PAGE and visualized either by Coomassie Blue (top) or by immunoblot analysis (bottom). (b) The BC2 nanotrap efficiently binds its epitope under harsh conditions. GFPBC2T derived from bacterial extracts was incubated at increasing levels of SDS (0–2%), Urea (0–4 M) or guanidinium hydrochloride (0–3 M) and precipitated as described. Shown are the Input (I) and bound fractions at indicated conditions. (c) BC2-tagged proteins are efficiently released using alkaline pH or peptide elution. GFPBC2T bound to the BC2 nanotrap was subjected either to elution with sodium thiocyanate (NaSCN, 1–3 M), acidic elution (0.2 M glycine pH 1–2.5), alkaline elution (pH 10–12) or peptide elution (0–1 mM). Aliquots of released (R) and bound (B) fractions were analyzed by immunoblotting with an anti-GFP antibody. (d) The BC2 nanotrap bind BC2-tagged proteins from human cell lysates irrespectively whether the BC2 tag is located on the N- or the C-terminus. For immunoprecipiation of BC2-tagged proteins soluble protein fractions of HEK293T cells expressing BC2T eGFP, eGFPBC2T or solely eGFP were incubated with the BC2 nanotrap as described. Input (I), non-bound (NB) and bound fractions (B) were separated by SDS-PAGE and visualized either by Coomassie Blue staining (top) or immunoblot analysis (bottom).
