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288 Tchaga
residue is buried and is not exposed for binding to the adsorbent. In our group,
we have observed that native phosphorylated Stat1 cannot bind to a number of
phosphotyrosine-specific antibodies (unpublished data).
2. Materials
1. Phosphoprotein Enrichment Kit (Clontech Cat. no. 635624). The kit comes with
the following reagents and materials suitable for six purifications.
• Six Phosphoprotein Affinity Columns (1 ml, disposable).
• 220 ml Buffer A (Extraction/Loading Buffer)—Clontech proprietary buffer.
• 45 ml Buffer B (Elution Buffer)—20 mM sodium phosphate, 0.5 M sodium
chloride, pH 7.2.
2. 2-ml microcentrifuge tubes.
3. 5-ml screw-cap centrifuge tubes.
4. pH meter or pH paper.
5. Micropipettor.
6. BCA Protein Assay Reagent Kit (Pierce Biotechnology, Rockford, IL, USA)—
provides a detergent-compatible BCA reagent for quantifying total protein (see
Note 1). Required for tissue extraction:
7. Mortar and Pestle.
8. Alumina (Sigma, St. Louis, MO, USA). The following materials may be required
depending on your purification:
9. Sterile Syringes and syringe filters (0.45 μm) for filtering lysates.
10. Phosphatase Inhibitors (if phosphatase inhibitors are desired).
11. Sodium orthovanadate (1–2 mM).
12. Sodium fluoride (10–50 mM).
13. Gel Filtration Column (for phosphatase inhibitor removal or buffer exchange).
PD-10, (GE Healthcare, Piscataway, NJ, USA).
14. Microconcentrators for sample concentration (optional).
15. Millipore 4-ml centrifugal filter and tube (Millipore) and
16. Millipore 0.5-ml centrifugal filter and tube (Millipore).
3. Methods
The protocol outlined below covers the experimental setup when using
Clontech’s phospho-specific metal ion affinity resin (PMAC) Phosphoproteins
Enrichment Kit (Clontech, Palo Alto, USA).
This kit has been developed with the goal to enrich as great an amount of
phosphorylated proteins in as quick a time as possible, reducing unwanted dephosphorylation
and/or proteolysis by running the purification at 4ºC (Fig. 2).