sentence: Immediately after elution , 10 mM DTT was added to each fraction and NaCl concentration was adjusted to 1 M. HIV-1 , BIV and EIAV INs produced to carry cleavable N-terminal His - tags were digested with thrombin ( 3 NIH units of thrombin per mg of His - tagged IN ) for 3 h at 25 ° C in the presence of 20 mM β - mercaptoethanol or HRV14 3C protease ( 20 μ g of protease per mg of His - tagged IN ) for 6 – 12 h at 4 ° C in the presence of 20 mM DTT ; the tag-free INs were then purified by chromatography on Heparin sepharose as above.Wild type and D366N LEDGF were made as previously described ( 24,25,31 ) .