Encyclopedia of Experiments: Biology
A subscription to JoVE is required to view this content.
Transcript
To quantify soluble protein content in a plant tissue, in a tube, begin with an appropriate amount of ground, lyophilized plant tissue.
Add sodium hydroxide, an alkali, to the tube and sonicate. The alkaline environment with the mechanical forces disrupt the cells, releasing intracellular contents, including macromolecules - soluble proteins and carbohydrates. Incubate at higher temperatures.
In the presence of heat, alkali cause protein hydrolysis, forming smaller peptides and increasing protein solubility. Additionally, carbohydrates get degraded.
Centrifuge the mixture. Collect the solubilized protein-containing supernatant. Add hydrochloric acid to neutralize the pH.
Treat the sample with trichloroacetic acid, TCA. TCA disrupts the hydration shell surrounding the proteins, leading to protein aggregation and precipitation.
Centrifuge and remove the TCA-containing supernatant. Wash the protein pellet with ice-cold acetone to remove any residual TCA, which may interfere with soluble protein estimation.
Air-dry the acetone. Resuspend the protein pellet in sodium hydroxide. Dilute with deionized water. Transfer to the wells of a multi-well plate.
Add an acidic solution of Coomassie Brilliant Blue G-250 dye. Under acidic conditions, the dye binds to the basic amino acid residues in the solubilized proteins, resulting in a blue protein-dye complex.
Use a spectrophotometer to measure the absorbance of the protein-dye complex, proportional to the soluble protein content in the plant tissue.
