topic

Ti-IMAC protocol

description

Reagents and materials

  1. 80% ACN with 6% TFA (!!make fresh everyday!!)
  2. 80% ACN
  3. 80% ACN with 0.5M glycolic acid (or lactic acid)
  4. 50% ACN with 1% ammonium hydroxide (make fresh)
  5. Ti-IMAC beads (MR-TIM005from ReSyn Biosciences)
  6. magnet

Note: Use 50 to 100 µL of Ti_IMAC beads for every 1 mg of peptide. 50 µL will be cleaner, but 100 µL will have higher phosphopeptide yield.

 

Procedure

  1. Wash beads three times with 1 mL 80% ACN with 6% TFA
  2. Resuspend sample in 1 mL 80% ACN with 6% TFA and combine with beads
  3. Vortex 20 minutes at ambient temperature
  4. Wash beads three times with 1 mL 80% ACN with 6% TFA
  5. Wash beads once with 1 mL 80% ACN
  6. Wash with 1 mL 80% ACN with 0.5 M glycolic acid
  7. Wash beads one times with 1 mL 80% ACN
  8. Elute two times with ~300 µL 50% ACN with 1% ammonium hydroxide
  9. Dry and desalt