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Protein Expression Methods
Published in Jay L. Nadeau, Introduction to Experimental Biophysics, 2017
Visualization of total protein content can be achieved using a variety of different staining techniques. The classical protein stain, which is still heavily used, is Coomassie blue. Coomassie has a sensitivity of ~8 ng, and staining of polyacrylamide gels is easy and inexpensive (Figure 5.5). A number of fluorescent dyes for rapidly staining gels with increased sensitivity have been developed, including SYPRO Orange (4–8 ng), SYPRO Red (4–8 ng), Oriole (<1 ng), Krypton (<1 ng), and Flamingo (<1 ng). Many of these dyes can be visualized and documented using a standard UV transilluminator and gel documentation system for the visualization of DNA gels, as discussed in Chapter 2. While these dyes provide increased sensitivity relative to Coomassie, they are also much more expensive. The classic method for visualization of extremely low protein levels in polyacrylamide gels is silver staining, which has a sensitivity rivaling the fluorescent protein dyes. In silver staining, Ag+ ions are allowed to bind to proteins immobilized in the gel. After washing the gel to remove unbound silver ions, the ions are chemically reduced to metallic silver.
Proteomics investigation of molecular mechanisms affected by EnBase culture system in anti-VEGF fab fragment producing E. coli BL21 (DE3)
Published in Preparative Biochemistry and Biotechnology, 2019
Bahareh Azarian, Amin Azimi, Mahboubeh Sepehri, Vahideh Samimi Fam, Faegheh Rezaie, Yeganeh Talebkhan, Vahid Khalaj, Fatemeh Davami
Cell pellets were washed three times with ice cold wash buffer (Tris 3 m mol l−1, sucrose 250 m mol l−1). The cells were lysed with lysis buffer (7 mol l−1 urea, 2 mol l−1 Thiourea, 40 m mol l−1 Tris, 4% CHAPS, 0.2% (w/v) Biolyte 4/7 ampholyte (Bio-Rad Laboratories, Hercules, CA), and 50 m mol l−1 DTT) and sonicated on ice for 5 × 30 s with 15 s of cooling breaks. The supernatant was collected after being centrifuged at 14000 g, 10 min, and 4 °C. 100 µg of lysates were mixed with rehydration buffer (8 mol l−1 Urea, 4% CHAPS, 0.2% Biolyte 4/7, 0.0002% Bromophenol Blue and 50 m mol l−1 DTT) and loaded onto immobilized pH gradient strip, 17 cm, pH 4–7 (Bio-Rad Laboratories). Following the rehydration for 16 hr at 25 °C, the isoelectric focusing (IEF) was performed, using PROTEAN® IEF Cell System (Bio-Rad Laboratories) for a total of 50 kVh at 20 °C. The strips were equilibrated in equilibration buffer 1 (6 m mol l−1 urea, 2% SDS, 20% glycerol, 0.05 M Tris-HCl, and 2% DTT) and equilibration buffer 2 (6 m mol l−1 urea, 2% SDS, 20% glycerol, 0.05 mol l−1 Tris-HCl, and 2.5% Iodoacetamide) each for 20 min. IPG strips were placed on top of the gradient acrylamide gels, 12–16%. Electrophoresis was carried out in two steps: 16 mA for 30 min followed by 24 mA for 5 hr, using PROTEAN® II XL Cells (Bio-Rad Laboratories). To visualize protein spots on gels, silver staining was performed.