Mouse anti strepii polyclonal antibody

Protein samples were boiled for 10 to 15 min in SDS loading buffer [100 mM Tris-Cl, pH 6.8, with 2% w/v SDS, 10% w/v glycerol, 0.6 mg·mL⁻¹ bromophenol blue, and 2.5% w/v β-mercaptoethanol]. Proteins were separated by SDS-PAGE (10 or 12%) and electroblotted onto PVDF membranes (0.5 µm, Amersham). Equivalent protein loading was determined by OD₆₀₀ of whole cells, by bicinchoninic acid assay of protein concentration, and by Coomassie blue staining of parallel gels. Alkaline phosphatase (AP)-linked anti-Flag M2 monoclonal antibody (Sigma-Aldrich) and mouse anti-StrepII polyclonal antibody (Qiagen) combined with goat anti-mouse IgG (H + L)-AP-linked antibody (Sigma-Aldrich) were used for immunoblotting (IB). AP activity was detected by chemiluminescence with CDP-Star (Applied Biosystems) and X-ray film (Hyperfilm; Amersham Biosciences).