Alkaline Phosphatase, Bacterial Antibody (L2)

F201C1B.

Alkaline phosphatase (AP). MAB1012 has a high affinity and recognizes an AP determinant resistant to denaturation by SDS-PAGE. It is therefore ideally suited for sensitive and specific detection of AP fusion proteins by Western Blot analysis of E. coli transformants expressing fusion products.

IgG2a

Monoclonal

Mouse

phoA

Unpurified

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Western Blot at 42-45kDa on SDS-PAGE,reducing gels for natural E. coli alkaline phosphatase monomer. Alkaline phosphatase fusion proteins will vary depending upon the target fusion protein. Immunocytochemistry/Immunofluorescence : reacts with E.coli. AP fusion protein targets in acetone fixed cell preparations. 1:4000, other fixatives or conditions untested. ASSAY: Preparation of E. coli TnphoA transformants: E. coli strain CC118 was transformed with plasmid pGEM-3Z containing TnphoA insertional mutations in the p101 gene of Mycoplasma hyorhinis, which encodes a protein with a typical N-terminal prokaryotic single peptide (Yogev et al. 1991). Identification of fusion protein with MAB1012 transformants: Transformants are grown in 2XYT medium to OD600=0.6. Cells were centrifuged 3 minutes at 10,000 x g, suspended in SDS-PAGE sample buffer, heated at 100C for 5 minutes, frozen and thawed and centrifuged as above at room temperature to remove insoluble material. The sample is applied at 9% to a SDS-PAGE gel, and Western immunoBlot is performed as described (Yogev et al. 1991). Immunoprecipitation: 5ul of antibody per 500ul of lysate in RIPA or 0.5% triton X-100 solutions.