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. Author manuscript; available in PMC: 2014 Mar 26.

Published in final edited form as: Biochemistry. 2011 Jul 8;50(30):6567–6578. doi: 10.1021/bi200553e

Recombinant GST-Pin1 was cleaved by thrombin, and the Pin1 protein concentrated, incubated with recombinant HI-2 and applied to a Superose 12 HR 10/300 column, as described under Materials & Methods. Aliquots of the eluted fractions were immunoblotted with anti-Pin1 (dashed lines) and anti-human I-2 (dotted lines) antibodies using dot blot technique with a 96 well vacuum manifold. (A) Elution profile for mixture of Pin1 and wild type HI-2. (B) Elution profile for mixture of Pin1 and I68A substituted form of HI-2. (C) Elution profile for mixture of Pin1 and truncated form of HI-2(1–152). (D) Superimposition of separate elution profiles for Pin1 and wild type HI-2. Inset is image of Coomassie stained SDS-PAGE of lane 1, molecular size standards; lane 2, the purified Pin1; and lane 3 purified HI-2 protein used in these experiments. (E) Superimposition of separate elution profiles for purified proteins used as molecular size standards: catalase (260 kDa, 10.8 ml), immunoglobulin (150 kDa, 12.0 ml), and ovalbumin (43 kDa, 13.8 ml).

Recombinant GST-Pin1 was cleaved by thrombin, and the Pin1 protein concentrated, incubated with recombinant HI-2 and applied to a Superose 12 HR 10/300 column, as described under Materials & Methods. Aliquots of the eluted fractions were immunoblotted with anti-Pin1 (dashed lines) and anti-human I-2 (dotted lines) antibodies using dot blot technique with a 96 well vacuum manifold. (A) Elution profile for mixture of Pin1 and wild type HI-2. (B) Elution profile for mixture of Pin1 and I68A substituted form of HI-2. (C) Elution profile for mixture of Pin1 and truncated form of HI-2(1–152). (D) Superimposition of separate elution profiles for Pin1 and wild type HI-2. Inset is image of Coomassie stained SDS-PAGE of lane 1, molecular size standards; lane 2, the purified Pin1; and lane 3 purified HI-2 protein used in these experiments. (E) Superimposition of separate elution profiles for purified proteins used as molecular size standards: catalase (260 kDa, 10.8 ml), immunoglobulin (150 kDa, 12.0 ml), and ovalbumin (43 kDa, 13.8 ml).