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  • FAQ: What cleavage reagent should be used for IPL? What is the efficiency of cleavage and IPL if I use MESNA or DTT?

    The thiol reagent used to induce cleavage during the standard IMPACT purification, typically 1,4-dithiothreitol (DTT), lowers the proficiency of the ligation reaction [Evans, T. et al. (1998) Protein Sci.7,2256-2264; Ghosh, I. et al.(2011). Site-specific protein labeling by intein-mediated protein ligation. Methods Mol Biol. 705:87-107.]. Use of 2-mercaptoethanesulfonic acid (MESNA) or thiophenol results in greater than 90% ligation proficiencies. We recommend the use of 2-mercaptoethanesulfonic acid, sodium salt (MESNA) (SIGMA Chemical Co., Cat.No. M-1511) because it is an odorless compound. We recommend the use of pTXB1 or pTXB3 for IPL as the Sce VMA intein in pTYB vectors does not cleave well with MESNA, resulting in lower yields of the target protein.

    In some cases where cleavage is inefficient you may try DTT; in this case try and do ligation as soon as possible as the thioester bond is not as stable as when MESNA is used. [Ghosh, I. et al. (2011). Site-specific protein labeling by intein-mediated protein ligation. Methods Mol Biol. 705:87-107. PubMed ID: 21125382]