Quantifying Lignin in Cell Walls

Protein-free cell wall sample (20 mg) was placed into a screw-cap centrifuge tube containing 0.5 ml of 25% acetyl bromide (v/v in glacial acetic acid) and incubated at 70°C for 30 min. After complete digestion, the sample was quickly cooled in an ice bath, and then mixed with 0.9 ml of 2 M NaOH, 0.1 ml of 5 M hydroxylamine-HCl, and a volume of glacial acetic acid sufficient for complete solubilization of the lignin extract (4 ml for soybean tissues or 6 ml for sugarcane bagasse). After centrifugation (1,400×g, 5 min), the absorbance of the supernatant was measured at 280 nm [21] (link). A standard curve was generated with alkali lignin (Aldrich 37, 096-7) and the absorptivity (ε) value obtained was 22.9 g⁻¹ L cm⁻¹. The results were expressed as mg lignin g⁻¹ cell wall.