Figure 1. CLASP2 is a monomer in solution, but it can self-associate through its C-term. (A) Size exclusion chromatograms of recombinant human CLASP2 FL and CLASP2 C-term. The elution positions of the protein standards used to calibrate the column are shown. (B) Coomassie blue–stained SDS-PAGE showing the sedimentation behavior of recombinant human CLASP2 FL upon sucrose gradient centrifugation. The positions of catalase, IgGs, BSA, and STI added to the CLASP2 FL sample before centrifugation are indicated. Input, 4 µg of the recombinant CLASP2 FL protein analyzed in the gradient; IgGs H.c. and IgGs L.c., heavy and light chains of IgG, respectively. (C and D) Extracts from mitotic-enriched HeLa cells were subjected to SEC (C) and sucrose gradient centrifugation (D). Collected fractions were precipitated with TCA and analyzed by SDS-PAGE/Western blot with anti-CLASP2 and anti-tubulin antibodies (upper and lower panels, respectively). Input, 100 µg of mitotic-enriched HeLa cell extract. * indicates an unknown protein occasionally recognized by the anti-CLASP2 antibody. (C) Fraction 9 represents a void volume of the column. (E and F) Recombinant CLASP2 C-term was subjected to density gradient centrifugation in the presence of either 400 mM (E) or 150 mM (F) NaCl, as described above for CLASP2 FL. Input, mixture of recombinant CLASP2 C-term plus protein standards that were loaded on the sucrose gradients; IgGs H.c. and IgGs L.c., as in B. Numbers to the left in B–F indicate the molecular masses of the protein standards in kD. (G) Summary of Rs (nm), S, and estimated MWs (kD) of the different CLASP2 proteins. (H) Analysis of recombinant CLASP2 C-term (26.5 µM) monodispersity by DLS. (I) Determination of recombinant CLASP2 C-term Dm by DLS. The linear regression extrapolated from the graphical representation of CLASP2 C-term Dm (μm2/s) at different concentrations (μM) allows the determination of the interaction parameter (−0.0012 µM−1), demonstrating the existence of attractive intermolecular interactions. (J) Dilution ITC of CLASP2 C-term. The raw calorimetric dilution titration profile (top) and the integrated heat of dissociation (bottom) are shown for CLASP2 C-term, BSA, and ITC buffer, as indicated.
Figure 1.

CLASP2 is a monomer in solution, but it can self-associate through its C-term. (A) Size exclusion chromatograms of recombinant human CLASP2 FL and CLASP2 C-term. The elution positions of the protein standards used to calibrate the column are shown. (B) Coomassie blue–stained SDS-PAGE showing the sedimentation behavior of recombinant human CLASP2 FL upon sucrose gradient centrifugation. The positions of catalase, IgGs, BSA, and STI added to the CLASP2 FL sample before centrifugation are indicated. Input, 4 µg of the recombinant CLASP2 FL protein analyzed in the gradient; IgGs H.c. and IgGs L.c., heavy and light chains of IgG, respectively. (C and D) Extracts from mitotic-enriched HeLa cells were subjected to SEC (C) and sucrose gradient centrifugation (D). Collected fractions were precipitated with TCA and analyzed by SDS-PAGE/Western blot with anti-CLASP2 and anti-tubulin antibodies (upper and lower panels, respectively). Input, 100 µg of mitotic-enriched HeLa cell extract. * indicates an unknown protein occasionally recognized by the anti-CLASP2 antibody. (C) Fraction 9 represents a void volume of the column. (E and F) Recombinant CLASP2 C-term was subjected to density gradient centrifugation in the presence of either 400 mM (E) or 150 mM (F) NaCl, as described above for CLASP2 FL. Input, mixture of recombinant CLASP2 C-term plus protein standards that were loaded on the sucrose gradients; IgGs H.c. and IgGs L.c., as in B. Numbers to the left in B–F indicate the molecular masses of the protein standards in kD. (G) Summary of Rs (nm), S, and estimated MWs (kD) of the different CLASP2 proteins. (H) Analysis of recombinant CLASP2 C-term (26.5 µM) monodispersity by DLS. (I) Determination of recombinant CLASP2 C-term Dm by DLS. The linear regression extrapolated from the graphical representation of CLASP2 C-term Dm (μm2/s) at different concentrations (μM) allows the determination of the interaction parameter (−0.0012 µM−1), demonstrating the existence of attractive intermolecular interactions. (J) Dilution ITC of CLASP2 C-term. The raw calorimetric dilution titration profile (top) and the integrated heat of dissociation (bottom) are shown for CLASP2 C-term, BSA, and ITC buffer, as indicated.

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