Member of the Ras superfamily of small GTPases. Similar to most GTPases, H-Ras cycles between GDP- and GTP-bound states; the GTP-bound form engages a set of diverse effectors to regulate cell processes, involved primarily in regulating cell division.1 In particular, H-Ras is active downstream of many growth factor receptors and major effectors include Raf kinase and PI3-kinase.2 Nucleotide cycling is regulated by guanine nucleotide exchange factors (GEFs), guanine dissociation inhibitors (GDIs) and GTPase activating proteins (GAPs).
Protein is > 99.5% pure as determined by densitometry of the final sample submitted to SDS-PAGE and stained with Coomassie blue (Fig. 1). H-Ras was heterologously expressed in E. coli as a histidine-tagged protein and purified by a combination of affinity, ion exchange, and gel exclusion chromatographies. The tag was removed prior to final purification leaving a Gly-Ala dipeptide at the N-terminus followed by residues 1-186 of human H-Ras. The terminal cysteine, which would normally be post-translationally isoprenylated in eukaryotic cells, has been mutated to serine (C186S).
- Wennerberg K, et al., The Ras superfamily at a glance. J Cell Sci 118, 843-846 (2005).
- Castellano E, et al., Functional specificity of Ras isoforms: so similar but so different. Genes Cancer 2(3), 216–231 (2011).
Species: Homo sapiens.
Sequence: Residues 1-186 of H-Ras. The terminal cysteine is mutated to serine (C186S) and the N-terminus retains an Gly-Ala dipeptide remaining from the affinity tag.
Calculated molecular weight: 21,110 g/mol.
Experimental molecular weight: NA.
Extension coefficient: 13,410 M-1cm-1 (assuming all cysteines remain reduced).
Amount: 100 or 500 μg.
Concentration: 1 mg/ml (determined by absorbance at 280 nm).
Buffer: 25 mM Tris pH 7.5, 150 mM NaCl, 5% glycerol and 1 mM DTT.
Form: Frozen liquid.
Storage and stability:
Storage: -80 °C. After initial thaw, aliquot and store at -80 °C until further use.
Stability: Stable for up to 12 months at -80 °C and up to 2 days at 4 °C. Repeated freeze-thaw cycles not recommended.
Notes: Please see accompanying sheet describing activity.