Journal: Bio-protocol

Article Title: Advancing 2-DE Techniques: High-Efficiency Protein Extraction From Lupine Roots

doi: 10.21769/BioProtoc.5461

Figure Lengend Snippet: 2-DE SDS-PAGE analysis for proteins isolated using the phenol (A) and modified Tris-EDTA (B) methods. For protein isolation, 1 g of roots was ground in a mortar with liquid nitrogen. Isoelectric focusing was performed according to the manufacturer’s instructions using the ReadyPrep TM 2-D Starter Kit. Electrophoretic separation was carried out at a constant voltage of 70 V for approximately 6 h. The gels were stained with Coomassie Brilliant Blue solution and then incubated in a destaining mixture of 20% (v/v) ethanol and 12% (v/v) acetic acid. The selected spots (marked as 1, 2, and 3) were identified.

Article Snippet: It is recommended to use the ReadyPrep 2-D Starter Kit and ReadyStrip IPG strips from Bio-Rad for sample preparation.

Techniques: SDS Page, Isolation, Modification, Staining, Incubation

Journal: Bio-protocol

Article Title: Advancing 2-DE Techniques: High-Efficiency Protein Extraction From Lupine Roots

doi: 10.21769/BioProtoc.5461

Figure Lengend Snippet: To optimize the protein precipitation procedure, proteins from the extracts were precipitated using a 20% (w/v) TCA/acetone solution (Methods I and II) or a 20% (w/v) TCA/water solution (Methods III and IV). Two precipitation time variants were also tested: 1 h for Methods I and III, and 12 h for Methods II and IV. For isoelectric focusing, 120 μg of protein was applied. Isoelectric focusing was performed according to the manufacturer’s instructions (ReadyPrep TM 2-D Starter Kit). Electrophoretic separation was carried out at a constant voltage of 70 V for approximately 6 h. The gels were stained with Coomassie Brilliant Blue solution and then incubated in a destaining mixture of 20% (v/v) ethanol and 12% (v/v) acetic acid. The selected spots (marked as 1, 2, and 3) were identified.

Article Snippet: It is recommended to use the ReadyPrep 2-D Starter Kit and ReadyStrip IPG strips from Bio-Rad for sample preparation.

Techniques: Staining, Incubation

Journal: Bio-protocol

Article Title: Advancing 2-DE Techniques: High-Efficiency Protein Extraction From Lupine Roots

doi: 10.21769/BioProtoc.5461

Figure Lengend Snippet: 2-DE SDS-PAGE analysis for proteins isolated using the phenol (A) and modified Tris-EDTA (B) methods. For protein isolation, 1 g of roots was ground in a mortar with liquid nitrogen. Isoelectric focusing was performed according to the manufacturer’s instructions using the ReadyPrep TM 2-D Starter Kit. Electrophoretic separation was carried out at a constant voltage of 70 V for approximately 6 h. The gels were stained with Coomassie Brilliant Blue solution and then incubated in a destaining mixture of 20% (v/v) ethanol and 12% (v/v) acetic acid. The selected spots (marked as 1, 2, and 3) were identified.

Article Snippet: ReadyPrep 2-D Starter Kit 2-D Gel Electrophoresis kit (Bio-Rad Laboratories, Inc., catalog number: 1632105) 28.

Techniques: SDS Page, Isolation, Modification, Staining, Incubation

Journal: Bio-protocol

Article Title: Advancing 2-DE Techniques: High-Efficiency Protein Extraction From Lupine Roots

doi: 10.21769/BioProtoc.5461

Figure Lengend Snippet: To optimize the protein precipitation procedure, proteins from the extracts were precipitated using a 20% (w/v) TCA/acetone solution (Methods I and II) or a 20% (w/v) TCA/water solution (Methods III and IV). Two precipitation time variants were also tested: 1 h for Methods I and III, and 12 h for Methods II and IV. For isoelectric focusing, 120 μg of protein was applied. Isoelectric focusing was performed according to the manufacturer’s instructions (ReadyPrep TM 2-D Starter Kit). Electrophoretic separation was carried out at a constant voltage of 70 V for approximately 6 h. The gels were stained with Coomassie Brilliant Blue solution and then incubated in a destaining mixture of 20% (v/v) ethanol and 12% (v/v) acetic acid. The selected spots (marked as 1, 2, and 3) were identified.

Article Snippet: ReadyPrep 2-D Starter Kit 2-D Gel Electrophoresis kit (Bio-Rad Laboratories, Inc., catalog number: 1632105) 28.

Techniques: Staining, Incubation