Alkaline phosphatase linked anti flag m2 monoclonal antibody

Cell pellets were boiled in SDS-reducing buffer for 20 min and centrifuged at 14,000 x g for 10 min at RT to obtain the supernatant. Samples were separated by 12% SDS-PAGE and transferred to PVDF membranes. Affinity tagged proteins were detected on membranes by immunoblotting with alkaline phosphatase-linked anti-Flag M2 monoclonal antibody (Sigma) and with mouse anti-StrepII polyclonal antibody (QIAGEN) followed by goat anti-mouse IgG (whole molecule)-alkaline phosphatase-linked antibody (Sigma). Alkaline phosphatase activity was detected by chemiluminescence using CDP-Star (Applied Biosystems) with X-ray film (Hyperfilm; Amersham Biosciences).

Cell pellets (2 OD₆₀₀ units) were resuspended in 100 μl of 2X SDS-PAGE loading buffer by vigorous pipetting and vortexing and were then boiled for 15 minutes prior to separation by SDS-PAGE analysis. The OD₆₀₀ of the cell culture was measured to normalize the loading amount of total cellular proteins (0.1 units per lane) and equivalent levels of protein loading were confirmed by Coomassie G250 staining of parallel gels. Total cellular proteins were separated on 12% reducing SDS-PAGE gel followed by electroblotting onto polyvinylidene difluoride (PVDF) membranes (Amersham) according to the BioRad standard protocol. Flag-tagged proteins were detected by alkaline phosphatase-linked anti-Flag M2 monoclonal antibody (Sigma-Aldrich). Immunoreactive antigens were detected by chemiluminescence using horseradish peroxidase (HRP) substrate (Immobilon).