IMAC

Solutions

  • H2O
  • 40 mM EDTA (pH 8.0)
  • 100 mM FeCl3
  • preparation buffer: 1:1:1 (v:v:v) solution of acetonitrile/methanol/0.01% acetic acid
  • loading/wash buffer: 80% acetonitrile/0.1% TFA
  • elution buffer: 50% acetonitrile 50% 1:20 ammonia in H2O (pH 12)
  • acidification buffer: 10% formic acid
  • storage buffer: 30% ethanol

Destaining solution

  • 125 mL methanol
  • 100 mL water
  • 25 mL acetic acid


Procedure

Prepare Resin

  1. Wash beads three times with 1 mL H2O
  2. Incubate beads with 1 mL 40 mM EDTA (pH 8.0) for 30 min with shaking
  3. Wash beads three times with 1 mL H2O
  4. Incubate beads with 1 mL 100 mM FeCl3 for 30 min with shaking
  5. Resuspend beads in preparation buffer
  6. Wash three times with 1 mL loading/wash buffer

Phosphopeptide enrichment

  1. Resuspend beads in loading/wash buffer
  2. Add resuspended beads to sample
    • Sample should be desalted peptides dried in speed vac or lyophilizer
    • You may want to set aside some of your sample for analysis without phosphopeptide enrichment
  3. Incubate sample and beads for 30 minutes with shaking
  4. Wash three times with 1 mL loading/wash buffer
  5. Remove liquid
  6. Add 100 μL elution buffer and incubate for 1 minute
  7. Collect eluted peptides and acidify immediately with acidification buffer (~35 μL)
  8. Concentrate in speed vac (don’t dry to completion) and resuspend in your MS loading buffer (e.g. 0.01% formic acid)
  9. Sample is ready for analysis

Clean beads

  1. Wash beads two times with 1 mL H2O
  2. Incubate beads with 40 mM EDTA (pH 8.0) for 5 min
  3. Wash beads two times with 1 mL H2O
  4. Resuspend beads in storage buffer and store at 4 °C

This method was adapted from methods described by Ficaro et al.