Small G-protein Technical Tips

Tip 1: GTP loading of small G-proteins

Tip 2: Measuring the absolute GTP binding ability of small G-proteins

For more specific technical tips please view our product pages and datasheets. 

Tip 1: GTP loading of small G-proteins

The GTP loading method uses EDTA to remove MgCl₂ and then GTP nucleotide is added to allow the exchange.  Then MgCl₂ is added back to lock in the GTP.  Two datasheets give the experimental details (cat# RH01 and BK100).  The RhoGAP assay biochem kit also provides useful information (cat# BK105). 

Tip 2: Measuring the absolute GTP binding ability of small G-proteins

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.