IMMUNOAFFINITY CHROMATOGRAPHY OF ALKALINE PHOSPHATASE-TAGGED PROTEINS

1.        Precolumn: (2 ml of Sepharose 4B; Sigma Catalog No. 4B-200)

2.         Immunoaffinity column: [2 ml of monoclonal anti-human placental alkaline phosphatase (clone 8B6)-agarose; Sigma Catolog No. A-2080]

3.         Wash precolumn and immunoaffinity column sequentially:

            a.         10-column volumes of Wash Buffer (20 ml)

b.         5-column volumes of Tris Buffer, pH 8.0 (10 ml)

c.         5-column volumes of Tris Buffer, pH 9.0 (10 ml)

d.         5-column volumes of Triethanolamine Solution (10 ml)

e.         5-column volumes of Wash Buffer (10 ml)

4.         Apply filtered or centrifuged conditioned medium containing the alkaline phosphatase-tagged protein at a rate of 5 column volumes/hr or approximately 10 ml/hr to the precolumn.

5.         Once all of the conditioned medium is loaded onto the immunoaffinity column then disconnect the precolumn and begin washing the immunoaffinity column.

            a.         10-column volumes of Wash Buffer (20 ml)

b.         5-column volumes of Tris Buffer, pH 8.0 (10 ml)

c.         5-column volumes of Tris Buffer, pH 9.0 (10 ml)

[Between each change of buffer, drain the buffer to the level of the resin before adding new buffer]

6.         Elute with 5 column volumes of Triethanolamine Solution into collection tubes containing 0.2 vol of 1 M Tris-Cl, pH 6.7.

7.         Wash and store the column in TSA Solution.

8.         Dialize the eluted sample (approximately 15 ml) in Phosphate Buffered Saline, pH 7.2.  Three changes, 2-liters each.  At least 2 h per change.

9.         Concentrate the sample using Centriprep-10 Concentrators (Amicon, Beverly, MA).  Load the sample and centrifuge at 5 C for 40 minutes.  Remove the filtrate and centrifuge for 10 minutes.  Final volume should be between 0.75 and 1.0 ml.        

10.       All procedures are performed at 5°C.