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Fig. 5. Binding of EhCaBP1 to actin. (A) Immunoprecipitation of amoebic proteins with anti-EhCaBP1 antibody and western analysis with anti-actin antibody. E. histolytica cell lysate was used for immunoprecipitation using anti-EhCaBP1 antibody and protein A beads. The immunoprecipitate was eluted from beads in SDS-PAGE buffer and separated on a 10% SDS-PAGE gel. The proteins were immobilized on nitrocellulose by electrophoretic transfer for immunostaining with anti-actin antibody and anti-mouse IgG linked to peroxidase. The bound antibody was detected using the ECL system. Lanes 1: immunoprecipitated amoebic proteins; lane 2: control precipitation with only protein A beads; lane 3: total lysate (10 µg protein). Molecular mass is indicated. (B) Co-sedimentation of EhCaBP1 with F-actin. Purified recombinant EhCaBP1 (5 µM), 
-actinin (5 µM) or bovine serum albumin (5 µM) were incubated with F-actin (5 µM), in polymerization buffer containing salts, followed by ultracentrifugation to separate the soluble and pellet fractions. Pellet (p) and supernatant (s) were resolved in 15% SDS-PAGE followed by Coomassie Blue staining. Total pellet fraction and one fourth of total supernatant was loaded for gel analysis. (C) Immunoblotting of EhCaBP1 co-sedimented with F-actin. Different amount of EhCaBP1 (1.6 µM, 6 µM and 17 µM) was incubated with F-actin in polymerization buffer containing salt as described in B. The pellet and supernatant fraction after ultracentrifugation were resolved in 15% SDS-PAGE followed by western analysis with anti-EhCaBP1 antibody as described in Materials and Methods. The amount of EhCaBP1 co-sedimented was determined by densitometry. The binding affinity was computed using a Scatchard plot. (D) Scatchard plot for EhCaBP1-actin interaction. Different wells of a microtiter plate were coated with 50 µl of 5 µM actin overnight at 4°C. After blocking with BSA, EhCaBP1 was added at the indicated concentrations ranging from 10 µM to 0.1 µM, followed by incubation with anti-EhCaBP1 antibody. The amount of bound EhCaBP1 was determined using anti-rabbit alkaline phosphatase-linked IgG. The amount of product formed was detected at 405 nm. (E) Co-sedimentation of EhCaBP1, delcenEhCaBP1 and EhCaBP2 with actin. Purified EhCaBP1, delcenEhCaBP1 or EhCaBP2 (5 µM), were incubated with F-actin (5 µM) in sedimentation buffer, followed by ultracentrifugation to separate the soluble and pellet fractions as described in Materials and Methods. Pellet and supernatant fractions were resolved in 15% SDS-PAGE followed by Coomassie Blue staining. Arrow indicates the position of soluble EhCaBP1 (14 kDa). (F) Calcium requirement for binding of EhCaBP1 to actin. Binding was carried out by solid phase assay in the presence of excess calcium (5 mM) or EGTA (2 mM). The plot shows the relative mean intensity (as the percentage ±s.d.) obtained from three independent experiments.
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