Comparative Analyses of Universal Extraction Buffers for Assay of Stress Related Biochemical and Physiological Parameters

Abstract

Comparative efficiency of three extraction solutions, including the universal sodium phosphate buffer (USPB), the Tris-HCl buffer (UTHB), and the specific buffers, were compared for assays of soluble protein, free proline, superoxide radical (), hydrogen peroxide (H₂O₂), and the antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), ascorbate peroxidase (APX), glutathione peroxidase (GPX), and glutathione reductase (GR) in Populus deltoide. Significant differences for protein extraction were detected via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2-DE). Between the two universal extraction buffers, the USPB showed higher efficiency for extraction of soluble protein, CAT, GR, , GPX, SOD, and free proline, while the UTHB had higher efficiency for extraction of APX, POD, and H₂O₂. When compared with the specific buffers, the USPB showed higher extraction efficiency for measurement of soluble protein, CAT, GR, and , parallel extraction efficiency for GPX, SOD, free proline, and H₂O₂, and lower extraction efficiency for APX and POD, whereas the UTHB had higher extraction efficiency for measurement of POD and H₂O₂. Further comparisons proved that 100 mM USPB buffer showed the highest extraction efficiencies. These results indicated that USPB would be suitable and efficient for extraction of soluble protein, CAT, GR, GPX, SOD, H₂O₂, , and free proline.

Keywords:

• antioxidant enzymes
• extraction efficiency
• free proline
• reactive oxygen species
• universal sodium phosphate buffer extraction solution
• universal Tris-HCl buffer extraction solution