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. 2009 Sep 15;284(47):32906–32913. doi: 10.1074/jbc.M109.034322

FIGURE 4.

FIGURE 4.

Electrophilic affibody binds its target specifically. A, nucleophiles free in solution inhibit covalent cross-linking weakly. We incubated affibody-EBA with MBP-ZSPA N6C in the presence of varying concentrations of reduced glutathione at pH 8.0 for 5 h at 37 °C and analyzed complex formation by SDS-PAGE and Coomassie staining. B, electrophilic affibody binds its target specifically in the presence of serum. Affibody-EBA was incubated with MBP-ZSPA N6K alone or in the presence of fetal calf serum. We imaged total proteins present by SDS-PAGE and Coomassie staining (left panel), whereas we detected complexes containing MBP-ZSPA N6K or that had reacted with affibody-EBA by immunoblotting with anti-T7 tag antibody (right panel). The band at ∼30 kDa is a minor impurity of the affibody-EBA sample not visible by Coomassie staining. C, electrophilic affibody binds its target specifically in the presence of cell lysate. Affibody-EBA was incubated with MBP-ZSPA N6K alone or in the presence of HeLa cell lysate and analyzed as in B.