Protocol for GL spin columns

Buffer A: 80% MeCN, 1% TFA
Buffer B: 80% MeCN, 1% TFA, 1M Glycolic Acid
Buffer C: 20% MeCN, 5% aqueous ammonia

**Be sure to check tubes after each spin to make sure that entire volume has passed through spin column.

** Buffer B is only used for complex lysates and typically not from gel-band analysis or single protein solution samples. Buffer B will increase the specificity of the enrichment, but does result in a slightly lower total absolute amount of phosphorylated peptides. If you choose to use Buffer B, it can replace steps “a” and b” in the “wash” portion of either protocol.

For 600ug capacity tips:

  1. Bring samples up in 150uL of Buffer A.
  2. Pre-Elute
    • Add 100 uL Buffer C, centrifuge (1000 x g, 2min)
  3. Condition
    • Add 100 uL Buffer A, centrifuge (3000 x g, 2min)
    •  Add 100 uL Buffer A, centrifuge (3000 x g, 2min)
    • Remove the 200 uL of effluent from the waste fluid tube.
  4.  Bind
    • Add sample in 150uL Buffer A, centrifuge (1000 x g, 10 min)
    • Re-apply flowthrough, centrifuge (1000 x g, 10 min)
  5. Wash
    • Add 100 uL Buffer A, centrifuge (1000 x g, 2min)
    • Add 100 uL Buffer A, centrifuge (1000 x g, 2min)
    • Add 100 uL Buffer A, centrifuge (1000 x g, 2min)
    • Add 100 uL Buffer A, centrifuge (1000 x g, 2min)
    • Switch to new collection tube (1.5mL Epi)
  6. Elute
    • Add 100 uL Buffer C, centrifuge (1000 x g, 5min) 
    • Add 100 uL Buffer C, centrifuge (1000 x g, 5min) 
  7. Acidify
    • Add ~6 uL of neat formic acid to elution fraction and check pH < 4.
  8. Freeze and Lyophilize.

For 200ug capacity tips (exact protocol from GL Sciences):

  1. Bring samples up in 65 uL of Buffer A.
  2. Condition
    • Add 20 uL Buffer A, centrifuge (3000 x g, 2min)
    • Add 20 uL Buffer A, centrifuge (3000 x g, 2min)
    • Remove the 40 uL of effluent from the waste fluid tube.
  3. Bind
    • Add sample in 65 uL Buffer A, centrifuge (1000 x g, 10 min)
    • Re-apply flow through, centrifuge (1000 x g, 10 min)
  4. Wash
    • Add 20 uL Buffer A, centrifuge (3000 x g, 2min)
    • Add 20 uL Buffer A, centrifuge (3000 x g, 2min)
    • Add 20 uL Buffer A, centrifuge (3000 x g, 2min)
    • Add 20 uL Buffer A, centrifuge (3000 x g, 2min)
    • Switch to new collection tube (1.5mL Epi)
  5. Elute
    • Add 50 uL Buffer C, centrifuge (1000 x g, 5min)
    • Add 50 uL Buffer C, centrifuge (1000 x g, 5min)
  6. Acidify
    • Add ~3 uL of neat formic acid to elution fraction and check pH < 4.
  7. Freeze and Lyophilize.