Product Uses Include
The human Rac1 protein has been produced in a bacterial expression system. The protein is supplied as a lyophilized powder. When it is reconstituted in distilled water to 5 mg/ml, the protein is in the following buffer: 50 mM Tris pH 7.5, 0.5 mM MgCl2, 50 mM NaCl, 0.5% sucrose, and 0.1% dextran. Protein concentration is determined by the Precision Red Advanced Protein Assay Reagent, Cat. # ADV02.
The recombinant protein is 22 kDa, consisting of the Rac1 protein plus a histidine tag in the amino-terminus.
For other forms of Rac1 as well as many other purified small G-proteins, see our main small G-protein product page.
Purity is determined by scanning densitometry of proteins on SDS-PAGE gels. His-Rac1 samples are >90% pure.
Figure 1: His-Rac1 protein purity determination. A 10 µg sample of RC01 (His-Rac1 molecular weight approx. 22 kDa) was separated by electrophoresis in a 12% SDS-PAGE system. The protein was stained with Coomassie Blue.
The biological activity of RC01 is determined by its ability to exchange nucleotide. This is tested by a pulldown assay using GST-tagged PAK-PBD beads (Cat. # PAK02) and GTPγS (Cat. # BS01) or GDP loaded His-Rac1. The PAK protein is an effector of Rac1, and will specifically bind to the GTP bound form Rac1. Using this assay, the amount of biologically active GTP-bound Rac1 is determined. Stringent quality control ensures that >80% of the His-Rac1 protein produced is capable of binding GTP.
Kulkarni, S., Goll, D. E. and Fox, J. E. (2002). Calpain cleaves RhoA generating a dominant-negative form that inhibits integrin-induced actin filament assembly and cell spreading. J. Biol. Chem. 277, 24435-24441.