High-Efficiency Solid-Phase Capture Using Glass Beads Bonded to Microcentrifuge Tubes: Immunoprecipitation of Proteins from Cell Extracts and Assessment of Ras Activation

• Published in: Analytical biochemistry 2002-03, Vol.302 (2), p.298-304
• Main Authors: Chen, Jeffrey C., von Lintig, Friederike C., Jones, Stephen B., Huvar, Ivana, Boss, Gerry R.
• Format: Article
• Language: English
• Subjects: 3T3 Cells - chemistry; Animals; Cell Extracts - chemistry; Dimethyl Suberimidate - chemistry; Guanine Nucleotide Dissociation Inhibitors - analysis; Guanine Nucleotide Dissociation Inhibitors - immunology; HL-60 Cells - chemistry; Humans; Mice; Microspheres; Polypropylenes - chemistry; Precipitin Tests - methods; Proteins - analysis; Proteins - immunology; ras Proteins - analysis; ras Proteins - immunology; rho-Specific Guanine Nucleotide Dissociation Inhibitors; Silanes - chemistry; Staphylococcal Protein A - chemistry; Tumor Cells, Cultured
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.2001.5572

We have bonded glass microbeads (425–600 μm diameter) to the inner walls of polypropylene microcentrifuge tubes. In addition to increasing the surface area of the tubes manyfold, the beads provide surface Si groups which can be reacted with a silane compound such as aminopropyltriethoxysilane, yielding a free amino group. The amino group is reacted with another cross-linking reagent, for example, the homobifunctional compound dimethyl suberimidate, which can form a covalent bond with amine groups of proteins. After binding protein A or G to the dimethyl suberimidate, the beads were used to immunoprecipitate proteins from cell extracts; we show that the protein A/G-coated glass beads yield similar amounts of immunoprecipitated proteins as a standard method using protein A– or G–agarose beads, but with fewer contaminating proteins. In addition, we show that when immunoprecipitating Ras from cell extracts and measuring the amounts of Ras-bound GTP and GDP, the new method yielded higher guanine nucleotide levels than protein G–agarose beads, suggesting that it caused less denaturation of Ras. Because the glass beads are bonded to the walls of the tubes, the immunoprecipitates can be washed rapidly and efficiently, and we show that 20–30 tubes can be washed in 1/10 the time required to wash immunoprecipitates on protein A– or G–agarose beads.