His tag mouse monoclonal antibody

Japanese eel liver cells were used, and the proteins were extracted 24 h after transfection. The overexpressed cells were washed twice with PBS, then lysed by adding Western and IP cell lysates (RIPA, Beyotime, Shanghai, China) for 15 min on ice, and centrifuged at 12,000× g for 5 min, and the supernatant containing the proteins was retained. Then, 2× SDS Protein Sampling Buffer was added to the supernatant, boiled for 15 min, and stored at −80 °C for Western blot analysis. His Tag Mouse Monoclonal Antibody (Product No. AF5060, Beyotime, Shanghai, China) was used as the primary antibody and HRP-labeled Goat Anti-Mouse IgG (Product No. A0216, Beyotime, Shanghai, China) was used as the secondary antibody.

Western Blot experiment was used to detect the binding activity of rAaCrus1 to bacteria. Briefly, the microorganisms (1 × 10⁸ CFU) in a 1.5 mL centrifuge tube were incubated in 200 μL of His-SUMO-AaCrus1(20 μM) by gentle rotation for 30 min at room temperature. The cells were collected and washed three times with 1× Tris Buffered Saline (TBS) and then resuspended. After the centrifugation at 10,000 rpm for 5 min, the bacteria and supernatant were directly loaded on SDS-PAGE for analysis, respectively. rAaCrus1 was the positive control. Finally, proteins were transferred onto a polyvinylidene fluoride membrane (PVDF), which was blocked with 5% skim milk in 1× TBST. The blot was incubated with His-Tag Mouse Monoclonal Antibody (1:1000, Beyotime, Shanghai, China) and a horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1:2000, Beyotime, Shanghai, China), respectively. Detection was completed with the BeyoECL Plus (Beyotime, Shanghai, China), according to the manufacturer’s instructions.

For the detection of recombinant hPH20 expression, samples were harvested by centrifugation (9,000 × g for 10 min at 4°C). The supernatant was mixed with 4 × Protein Native PAGE Loading Buffer (TaKaRa, Dalian, China) and heated at 100°C for 10 min. SDS-PAGE was performed by 10% Bis-Tris Protein Gels in MES running buffer at 100 V. Then, SDS-PAGE gel was electroblotted to PVDF transfer membrane (PerkinElmer, Shanghai, China) using a transfer buffer containing 20% methanol, 15.1 g L⁻¹ glycine, and 3.0 g L⁻¹ Tris. Blotted membrane was blocked with QuickBlock™ Blocking Buffer (Beyotime Biotechnology, Shanghai, China) for 1 h. For immunodetection, His-tag Mouse Monoclonal Antibody (Beyotime Biotechnology, Shanghai, China) diluted 1: 1,000 with TBS (2.4 g L⁻¹, pH 7.6 Tris buffer containing 8.0 g L⁻¹ NaCl) was used as a primary antibody. After washing with TBST (2.4 g L⁻¹, pH 7.6 Tris buffer containing 8.0 g L⁻¹ NaCl and 0.1% Tween-20) for 30 min, a 1:1,000 dilution of the secondary antibody, HRP-labeled Goat Anti-Mouse lgG (H + L) (Beyotime Biotechnology, Shanghai, China) was added and incubated at 4°C with gentle shaking for 1 h. After washing with TBST for 30 min, DAB Horseradish Peroxidase Color Development Kit (Beyotime Biotechnology, Shanghai, China) was used to develop the color on the membrane.