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. Author manuscript; available in PMC: 2010 Mar 20.
Published in final edited form as: J Mol Biol. 2009 Jan 23;387(1):175–191. doi: 10.1016/j.jmb.2009.01.030

Figure 8. Binding of KCTD5 and GRASP55 in solution.

Figure 8

a. KCTD5 (K5) co-purifies with GRASP55 (G55) when co-expressed in HEK293 cells. Starting material (SM) was detergent-soluble lysate that was incubated with antibody to flag to yield immunoprecipitate (IP); SM and IP were resolved by SDS-PAGE and visualized by Western blot (IB) with antibody to myc or flag. Upper panels show that GRASP55 is isolated specifically with KCTD5 and not seen either in the absence of KCTD5 or GRASP55. Lower panels show KCTD5 is isolated in complexes with GRASP55. Numbers indicate apparent mass in kDa.

b. KCTD5 and GRASP55 studied by SPR. Upper panel depicts binding of full length GRASP55 (blue line) or truncated GRASP55 (1–263, red line) to KCTD5 (residues 34–234) immobilized on the surface. GRASP proteins injected at 5 μM. Measured biophysical parameters listed in Table 2. Lower panel shows that GRASP55 does not bind to the KCTD5 N-terminal domain.

c. Equilibrium sedimentation shows association of KCTD5 and GRASP55. Here, KCTD5 residues 34–234 (9.2 μM) and full length GRASP55 (blue line, 2.6 μM) or truncated GRASP55 (1–263, red line, 2.5 μM) are shown at 24,000 rpm in the lower panel. The upper panel shows residual difference between the calculated fit and experimental data. Nine data sets (three speeds, six concentrations) were analyzed globally using SEDPHAT 4.3 software (Methods) yields an equilibrium binding affinity of 1.41 ± 0.26 μM and 8.26 ± 1.6 μM for full-length and truncated GRASP55, respectively. Estimated partial specific volumes and solvent densities of KCTD5 with full length and truncated GRASP55 were 0.733 cm3 g−1 and 0.99823 g ml−1, respectively.

d. Kv4.2 and KChIP2 form complexes but not KCTD5 on co-expression in COS cells. Lysates were incubated with antibody to 1D4, resolved by SDS-PAGE and the interaction visualized by Western blot analysis with antibodies to KChIP2 and HA. Upper panels show that KChIP2 is purified with Kv4.2. Lower panels show that KCTD5 does not co-immunoprecipitate with Kv4.2 in the presence or absence of KChIP2 despite synthesis of the protein. Numbers indicate apparent mass in kDa.