Protocol for IPL
A typical lPL reaction can be carried out by mixing two reactants at 4°C – 25°C at pH 8 - 9. One of the components should have a final concentration of at least 0.5 - 1 mM. Regarding ligation of a peptide to a protein, for greater extent of ligation, we use the protein at 1 -10 uM with 0.5 - 1 mM peptide; we add the two components in the presence of 0.1 M Tris pH 8.5 and 10mM MESNA and let the reaction go overnight at 4°C or one hour at room temperature.
The following protocol illustrates a typical labeling experiment. Mix 4 µL of L-[35S]-cysteine (11.0 mCi/mL, Perkin Elmer, cat# NEG-022T) or 4 µL of peptide with an N-terminal cysteine (10 mM) with a 36 µL aliquot of protein freshly isolated using the IMPACT system. Incubate the reaction solution at 4°C overnight. To examine the labeled or ligated protein, add 20 µL of 3X SDS-PAGE sample buffer (with DTT) to the protein sample and boil for 10 minutes. Analysis of the sample can then be performed using SDS-PAGE.