Bca protein detection kit

The synthetic recombinant plasmid was transformed into E. coli BL21 (Sangon Biotech Co., Ltd Shanghai China). The transformed E. coli BL21 cells were grown by adding a small amount of Luria–Bertani (LB) in a shaker at 220 rpm and 37 °C and then inoculated into a large bottle of LB (containing 30 μg/ml kanamycin) at a dilution of 1:100. After 3 h, 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) (Solarbio, Beijing, China) was added to induce protein synthesis for 4 h. The bacteria were collected by centrifugation. The pellet was suspended in a binding buffer (0.5 M NaCl, 50 mM Tris, and 5 mM imidazole; pH 8.0) for sonication. Centrifuge the lysate after sonication and resuspend the pellet (in the form of inclusion bodies) in a binding buffer containing 8 M urea, dissolve and centrifuge at 4 °C overnight, and purify the His-tag-containing recombinant protein by affinity chromatography using NiSepharose TM excel (GE). The purified protein was refolded in dialysate (0.25 M NaCl, 50 mM Tris, 0.5 Mm EDTA, 2 mM GSH and 0.2 mM GSSG; pH8.0) at 4 °C, and contained different concentrations of urea (6 M to 0 M in decreasing order of 1 M). Finally, the protein was dialyzed twice in PBS (pH 7.4) to obtain a fully refolded protein, and the protein concentration was measured with the BCA protein detection kit (Takara) [5 (link)].

The total proteins were extracted from the cells using RIPA lysis buffer (Thermo Scientific, United States) containing 1% phenylmethanesulfonyl fluoride on ice. The protein concentration was measured using a BCA protein detection kit (Takara, Tokyo, Japan, T9300A). Lysates (50 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene fluoride membrane, followed by blocking with 5% bovine serum albumin (BSA). The membranes were incubated along with the following primary antibodies: β-actin (Sigma-Aldrich, St Louis, MO, United States, A5441); Anti-ITGB4 antibody (1:1,000, Abcam, ab182120). Afterwards, the membranes were washed three times with TBST (TBS 0.1% Tween-20) and incubated with the HRP-conjugated secondary antibody (ab205718; Abcam) for 1 h at room temperature. GADPH was used as the loading control.