What cleavage reagent should be used for IPL?
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 et al.,(1998)Protein Sci.7,2256-2264]. 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.
