The possibilities for ligation problems are: 1. Peptide does not possess a N-terminal cysteine or the sulfhydryl group is oxidized. 2. The peptide solution may be very acidic and cause the pH of the reaction to drop significantly. Check the pH of your peptide solution. If the pH is below 6 dissolve the peptide in 1 M Tris (pH 9.0). 3. Peptide preparation contains impurities. Purify the peptide. We purify the peptide using a Vydac semi-preparative C18 column for reverse phase purification using a 180 min linear gradient, 10%-100% B with a flow rate of 2 ml per minute. Buffer A 0.1% TFA/ H2O (V/V), and buffer B being 0.1% TFA/60% CH3CN/40% H2O (V/V/V). 4. Concentration of peptide is incorrect. 5. Peptide or ligation product is insoluble. 6. The carrier protein has lost its ligation capabilities due to repeated freeze-thaw cycles or long term storage at -20°C. Use the control peptide, PBI, to test ligation.