Alkaline phosphatase conjugated goat anti mouse igg h l antibody

Purified recombinant proteins were separated by 12% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) transfer membrane (Immobilon-P, Millipore, USA) and blocked by 0.25% casein containing 1xTBS-T buffer (Tris buffered saline containing Tween 20; 20 mM Tris-Cl pH: 7.8, 0.5 M NaCl, 0.5% Tween 20) for 30 minutes. The membranes were probed with a 1∶50 dilution of monoclonal anti-polyhistidine antibody (Sigma, Germany) for 1.5 hours. Next, the membranes were washed thrice with 1xTBS-T and probed with a 1∶2500 dilution of alkaline phosphatase-conjugated goat anti-mouse IgG (H+L) antibody (Sigma) in 1xTBS. Thereafter, the membranes were washed thrice with 1xTBS-T and 1xTBS and the blot was developed in diethanolamine buffer (10% Diethanolamine, 0.5 mM MgCl2•6H2O, pH: 9.8) containing 4.3% 5-bromo-4-chloro-3-indolyl phosphate (diluted in dimethylacetamide), 4.1% Nitro-BT (diluted in 70% (v/v) dimethylformamide) (Applichem, Germany).

To observe the expression levels, purity and immunoreactivity, proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). The separated proteins were transferred to polyvinylidene difluoride (PVDF) transfer membrane (Immobilon-P, Millipore, MA), blocked by 6.25% non-fat dry milk for 1 h at room temperature. The membranes were probed with a 1/3333 dilution of monoclonal anti-polyhistidine antibody (Sigma-Aldrich, USA) for 1.5 h at room temperature. Then the membranes were probed with a 1/3333 dilution of alkaline phosphatase-conjugated goat anti-mouse IgG (H + L) antibody (Sigma-Aldrich, USA) for 1 h at room temperature. The blot was developed with diethanolamine buffer (10% Diethanolamine, 4 M HCl pH: 9.8, 0.5 mM MgCl2•6H2O) containing 4.3% 5-bromo-4-chloro-3-indolyl phosphate (BCIP) diluted in dimethylacetamide, 4.1% Nitro-BT diluted in 70% (v/v) dimethylformamide (Applichem, Germany).

SDS-PAGE and Western blot to show the recombinant proteins was performed as previously described^{44 (link)–47 (link)}. Separation of recombinant proteins was achieved by 12% SDS-PAGE. Then, the separated proteins were transferred to a polyvinylidene difluoride (PVDF) transfer membrane (Immobilon-P, Millipore, USA). The membranes were blocked with 6.25% non-fat dry milk containing 1xTBS-T buffer (20 mM Tris–Cl pH: 7.8, 0.5 M NaCl, 0.5% Tween 20). Next, the membranes were probed with monoclonal anti-polyhistidine antibody (1:2000) (Sigma, USA) in 1xTBS-T for 1.5 h. Thereafter, the membranes were washed three times with 1xTBS-T and probed with a alkaline phosphatase-conjugated goat anti-mouse IgG (H + L) antibody (1:2500) (Sigma, USA) in 1xTBS-T for 1 h. After incubation, the membranes were washed three times with 1xTBS-T and subsequently with 1xTBS. The blots were developed with diethanolamine buffer (10% Diethanolamine, 0.5 mM MgCl₂-6H₂O, pH: 9.8) containing 4.3% 5-bromo-4-chloro-3- indolyl phosphate (diluted in dimethylacetamide) and 4.1% Nitro-BT (diluted in 70% (v/v) dimethylformamide) (Applichem, Germany).