Western Blot Analysis of Cassava Proteins

The extraction and quantification of total protein were carried out from 1 g of frozen cassava roots according to Maass et al. [88 (link)] with modifications of Cuellar et al. [89 (link)]. Proteins were resolved on a 12% SDS-polyacrylamide gel and blotted to 0.45 μM PVDF membranes (ThermoFischer). The blotting was conducted in a Mini Trans-Blot^® Cell (Bio-Rad) apparatus. Membranes were blocked with 5% skim milk powder in Tris-buffered saline, washed, and incubated in Tris-buffered saline plus 0.1% Tween 20 with either the polyclonal antibodies anti-PSY [56 (link)] or anti-OR [51 (link)]. After washing, the membranes were incubated for 1 h with a horseradish peroxidase- conjugated goat anti-Rabbit IgG antibody (Invitrogen, Cat.# G-21234) in a 1% skim milk solution in Tris-buffered saline plus 0.1% Tween 20. The membranes were washed and immunoblots were developed with Pierce^™ ECL Western Blotting Substrate (ThermoFischer). After striping the membrane with peroxidase [90 (link)], an anti-actin antibody (Sigma-Aldrich, Cat.#A0480) and a horseradish peroxidase-conjugated goat anti-Mouse IgG antibody (Invitrogen, Cat.# G-21040) were used to reprobe the immunoblot. Protein signals were quantified with ImageJ [91 (link)].