Purification of alkaline phosphatase protein and clarifying of the function of alkaline phosphatase on B lymphocyte

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Alkaline phosphatase (ALP) is a kind of the GPI-anchoring protein expressed on the cell surface. ALP is distributed in intestine, placenta, liver, kidney, bone, and some organs. It is also a fact that ALP is temporarily expressed on the cell surface of B lymphocytes in B cell differentiation. However, the function or role of ALP in B cell differentiation remains still unclear, so that we try to clarify the function of ALP.We tried to purify ALP protein from a Burkitt's lymphoma cell line bearing ALP.To make cell lysate from 1x1010 ALP-expressing cells, Cell Lysis Buffer ＆ Extraction Buffer were mixed and vortex vigously on ice. The cell lysate was purified by Ion-exchanging chromatography, Gel-filtarting chromatography and Hydroxyapatite chromatography. We got 386mg purified protein including ALP protein.In next step, We immunized BALB/c mouse with purified ALP protein or whole cells of ALP expressing cell line to produce anti-human ALP antibody. After 2 or 3 times immunization, spleen cells were fused with NS-1 cell (mouse myeloma cell line). Now We have got 3 candidates which might produce anti ALP antibody and are checking reactivity with ALP expressing cells and other non-ALP expressing cells. We are going to confirm whether the clones actually produce anti ALP antibody.It is one of the most important thing to produce an antibody against human lymphocyte ALP because we think anti ALP antibody makes easier to investigate the function and the structure immunologically and molecular biologically.