Micro capillary tube

On silica gel plates, the main components of the crude nhhE were qualitatively identified (Merck, silica gel 60F-254 TLC aluminum sheet 20 × 20 cm). Using the Drummond micro capillary tube on the plate, 20 µL of 5% n-hexane fraction diluted with methanol was manually spotted.
The screening program used a variety of mobile phases with varying concentrations (Table 1). After complete solvent evaporation, the separated compounds were visualized with a UV lamp (365 and 254 nm) and developed with a vanillin/H₃PO₄ spray reagent (the composing materials were attained from Sigma-Aldrich, St. Louis, MO, USA) before being heated to 110 °C [25 (link)]. Different spots with R_f values were measured and calculated.

Spectrin–actin–4.1R ternary complex formation was measured by the falling ball viscometry assay, as described (59 (link), 60 (link)). In brief, spectrin, F-actin, and GST-h4.1R mutants at appropriate concentrations were mixed in 10 mM Tris–Cl (pH 7.6), 20 mM KCl, 2 mM MgCl2, and 1 mM ATP. A 100 μl microcapillary tube (Drummond Scientific) was filled with this solution, sealed at one end, and incubated at the indicated temperature. After incubation, the tube was placed at a 70° angle from the horizontal; a steel ball (diameter 0.6 mm, Tsubaki-Nakashima Co) was placed under the meniscus of the solution using a magnet; and the time required by the ball to fall a specific distance (6 cm) at 25 °C was measured. Sedimentation velocity was calculated from the falling time (F_t, s/cm) and converted to apparent viscosity (mPa·s) using glycerol standards of known viscosity. The apparent viscosity of reactions containing only spectrin and F-actin was used as the control. If the ball did not move from its initial position for 120 s, the protein mixture was thought to form a gel. In most measurements, spectrin and F-actin concentrations were 10 and 250 μg/ml, respectively; to this, GST-h4.1R recombinants were added at the concentrations of 0 to 20 μg/ml, yielding a spectrin:actin:4.1R M ratio of 1:125:0–5.