We determined the percentage of HaloTag® Standard Protein, which was comprised of the 34kDa HaloTag® protein and 26kDa GST protein, cleaved by quantitating and comparing the bands of uncleaved HaloTag® Standard Protein in each experimental reaction to those present in the water control reactions (Figure 1). Bands were quantitated using Image Lab™ Software. The presence of NaCl (>10mM), imidazole (>5mM), glutathione (>1mM) and MgCl2 (>1mM) decreased ProTEV Plus cleavage efficiency to less than 50%. ProTEV Plus cleavage efficiency remained at ≥94% in the presence of Tween™ 20, Triton™ X-100, IGEPAL®, glycerol, urea, Protease Inhibitor Cocktail and guanidine for all concentrations tested (Figure 2).
Figure 1. ProTEV Plus digestions containing NaCl, imidazole, glutathione or MgCl2 resulted in a variable amount of cleavage inhibition. HT-GST protein (10µg) was digested with 5 units of ProTEV Plus in 50µl reactions for 30 minutes at 30°C. Reactions were separated using 4–20% SDS-PAGE at 200V for 1 hour. Complete inhibition controls (+) contained 5% SDS, and no-inhibition controls (–) contained water.
Figure 2. Percent cleavage of HaloTag® Standard Protein using ProTEV Plus in the presence of various compounds. Percent cleavage was calculated by Image Lab™ Software by comparing the bands of the partially digested HaloTag® Standard Protein to the band of undigested HaloTag® Standard Protein. Panel A. Imidazole, NaCl, Glutathione and MgCl2. Panel B. Protease Inhibitors, Urea, Guanidine, IGEPAL®, Triton™ X, Glycerol, Tween™ 20, SDS and DTT.