PEG conjugation

For PEG conjugations, the solution of his-G3C-GFP protein was incubated with an excess of maleimide PEG20K or maleimide PEG40K at 4°C overnight in 20 mM phosphate buffer (pH 7.4), 150 mM NaCl, and 1 mM tris(2-carboxyethyl)phosphine. To remove unconjugated PEG, the his-tagged protein solution was incubated with nickel-nitrilotriacetic acid beads for 1 h. The beads were washed three times in HEPES buffer (25 mM HEPES and 150 mM NaCl (pH 7.4)) followed by elution of the bound protein in HEPES buffer containing 200 mM imidazole. PEG-conjugated protein was separated from unconjugated protein by size-exclusion chromatography using a Sepharose G75 column. Purified PEG20K-GFP and PEG40K-GFP were stored as small aliquots at −80°C.