My internship in laboratory on 2026.1.23

Experiment for Determining Protein Content in Pine Needles
Principle: Coomassie Brilliant Blue Method

1. Grind the pre-cooled centrifuge tube containing pine needle extract and small steel beads in a tissue homogenizer to fully disrupt cells and release intracellular proteins. (Pre-cooling prevents protein degradation by proteases.)
2. Add PBS buffer to the centrifuge tube and mix well.
3. Centrifuge the tube at high speed and low temperature for 20 minutes.
4. After centrifugation, transfer the supernatant to a new centrifuge tube.
5. Prepare a series of protein standard solutions with known concentrations (e.g., using Bovine Serum Albumin, BSA) diluted in PBS buffer to form a concentration gradient (see table for ratios). Typically, 6–7 different concentrations are prepared.
6. Aliquot a specific volume of each standard solution and the sample supernatant into the wells of a microplate. Each standard concentration and each sample should be loaded in triplicate to minimize random error.
7. Add Coomassie Brilliant Blue G-250 dye solution to each well, mix immediately, and incubate at room temperature in the dark for 10 minutes.
8. Place the microplate in a microplate reader and measure the absorbance (OD) of each well at 594 nm. A higher absorbance value indicates a deeper blue color, more bound dye, and thus a higher protein concentration in the well.

Compare the measured sample absorbance values with a protein standard curve to determine the protein concentration in the samples. The standard curve is plotted with protein concentration on the x-axis and absorbance values on the y-axis.
Then we can use the concentration to calculate the protein content.