Measuring the absolute GTP binding ability of small G-proteins
Technical Tip from CYTOSKELETON
If you are looking to measure the absolute GTP binding ability of a small G-protein, then first you must treat with EDTA, then perform size exclusion chromatography to remove all nucleotides. Then you can add back tritiated GTP and lock the nucleotide in with MgCl2, followed by additional size exclusion chromatography to remove any free tritiated GTP. Then, count the protein fractions in a scintillation counter. Some researchers have used DEAE discs instead of size exclusion chromatography to measure a small G-protein's GTP binding ability. Please search Methods In Enzymology and GTP binding for these method papers that were published between 1980-2000.