Protocol+for+conjugating+peptides+to+Tentagel+beads

Protocol for conjugating peptides to Tentagel beads


 * Solubilize the tentagel resin in acetonitrile (most likely done in a syringe).
 * Weigh out enough peptide to have 0.03 mmol peptide/(100 mg tentagel resin) for the reaction.
 * Solubilize the peptide with H20 and acetonitrile
 * 50% H20 and 50% acetonitrile (200 uL and 200 uL) may be sufficient to solubilize the peptide
 * Resuspend resin in 50% H20 and 50% acetonitrile for 5 min with shaking.
 * Add peptide solution to resin solution.
 * Measure the pH and adjust the pH to a value close to 6.5 using 10% triethylamine in acetonitrilie.
 * approximately 50 uL of the 10% TEA may be required
 * May want to spin samples down and nanodrop the supernatent to determine the initial concentration of unconjugated peptide.
 * Incubate overnight at room temperature in a rotisserie or other mixing device in a round bottom tube to prevent the peptides from becoming trapped in the narrow point of a pointed tube.
 * Wash the beads in 50% HPLC A buffer (0.1% TFA in H20) and 50% HPLC B buffer (0.1% TFA in 90% acetonitrile in H20) to remove unconjugated peptide. This can be done by loading the resin into the back of a syringe. Adding the buffer, and pushing this buffer out of the syringe. More buffer can be sucked back up into the syringe to wash the beads again. The beads should be washed for a total of about 5-7 times. The flow-through of the wash should probably be saved since this contains peptide that may be valuable.
 * Store the remaining beads at 4 C (may want to dry the beads 1st).