The samples and controls were loaded and separated with reducing SDS-PAGE. Stain free 4–20% gels were used (Bio-rad). The proteins were transferred to 0.45-micron PVDF membranes (Bio-rad) using the Trans-Blot Turbo transfer system (Bio-rad). The blot was blocked using the Western blocker solution (Sigma Aldrich) and incubated in with either anti-Z-domain (2.87 mg/mL) (1:1000) or anti-ABD (1 mg/mL) (3:1000) antibodies, both obtained from Affibody AB followed by incubation with either anti-mouse antibody (1:5000) for anti-Z-domain or anti-rabbit (1:5000) antibody for anti-ABD antibodies, respectively. Both secondary antibodies were HRP-conjugated and visualized by using West Pico Plus HRP substrate (Thermo Fischer) and measured with a ChemidoC XRS image analyzer (Bio-Rad). As positive controls, standards of purified ZHER3_1-ABD (1.97 mg/mL), ZHER3_1-ZHER3_1-ABD (0.77 mg/mL) and ZHER3_1-ABD-ZHER3_1 (1.34 mg/mL) provided by Affibody AB were used with a concentration of 0.01 g/L or stated otherwise.
0.45 micron pvdf membranes
Manufactured by Bio-Rad
The 0.45-micron PVDF membranes are a type of laboratory equipment used for various applications. They are made from polyvinylidene fluoride (PVDF) material and have a pore size of 0.45 microns. These membranes are designed for filtration, separation, and sample preparation purposes.
Automatically generated - may contain errors
2 protocols using 0.45 micron pvdf membranes
1
Western Blot Analysis of Affibody Constructs
2
Western Blot Analysis of Fusion Proteins
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The samples and controls were loaded and separated with reducing SDS-PAGE. Stain free 4-20% gels were used (Bio-rad). The proteins were transferred to 0.45-micron PVDF membranes (Bio-rad) using the Trans-Blot Turbo transfer system (Bio-rad). The blot was blocked using the Western blocker solution (Sigma Aldrich) and incubated in with either anti-Z-domain (2.87 mg/mL) (1:1000) or anti-ABD (1 mg/mL) (3:1000) antibodies, both obtained from A body AB followed by incubation with either antimouse antibody (1:5000) for anti-Z-domain or anti-rabbit (1:5000) antibody for anti-ABD antibodies, respectively. Both secondary antibodies were HRP-conjugated and visualized by using West Pico Plus HRP substrate (Thermo Fischer) and measured with a ChemidoC XRS image analyzer (Bio-Rad). As positive controls, standards of puri ed Z HER3_1 -ABD (1.97 mg/mL), Z HER3_1 -Z HER3_1 -ABD (0.77 mg/mL) and Z HER3_1 -ABD-Z HER3_1 (1.34 mg/mL) provided by A body AB were used with a concentration of 0.01 g/L or stated otherwise.
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