topic

Tissue Lysis and digestion protocol

description
  1. Resuspend tissue in 6M GnHCl 100 mM tris pH 8
  2. Probe sonicate until fully homogenized (excess material can be frozen here)
  3. QS with MeOH to 90% [MeOH]
  4. Centrifuge 9,000x g for 5 min
  5. Decant (remove excess supernatant with pipette)
  6. Air dry 5 min
  7. Resuspend in 8M urea & 100 mM Tris (should be <3 mg/ml)Probe sonicate until resuspended
    • Probe sonicate until resuspended
  8. Quantify protein by BCA assay (or method of choice)
  9. Add 10 mM TCEP, 40 mM chloroacetamide
  10. Add LysC 50:1, incubate 4 hrs
  11. QS with 100 mM tris to 2 M urea
  12. Add trypsin 50:1, incubate overnight
  13. Add TFA to pH ~1
  14. Centrifuge 9,000x g for 5 min

 

Desalting (≤300 µg)

10 mg Strata X, 1 mL volume (reversed phase solid phase extraction cartridge)

Note: Use gravity or a vacuum manifold

  1. 1 mL 100% ACN
  2. Fill cartridge with 0.2% FA
  3. Load sample
  4. Fill cartridge with 0.2 % FA
  5. Elute into clean tube with 300 µl 80% ACN 0.2% TFA.
  6. Dry down reconstitute in 0.2% FA.

 

Desalting (≤1 mg)

30 mg Strata X, 3 mL volume (reversed phase solid phase extraction cartridge)

Note: Use gravity or a vacuum manifold

  1. 3 mL 100% ACN
  2. Fill cartridge with 0.2% FA
  3. Load sample
  4. Fill cartridge with 0.2 % FA
  5. Elute into clean tube with 900 µl 80% ACN 0.2% TFA.
  6. Dry down reconstitute in 0.2% FA.

 

Desalting (≤5 mg)

60 mg Strata X, 3 mL volume (reversed phase solid phase extraction cartridge)

Note: Use gravity or a vacuum manifold

  1. 3 mL 100% ACN
  2. Fill cartridge with 0.2% FA
  3. Load sample
  4. Fill cartridge with 0.2 % FA
  5. Elute into clean tube with 1.5 mL 80% ACN 0.2% TFA.
  6. Dry down reconstitute in 0.2% FA.

 

Desalting (≤10 mg)

100 mg Strata X, 3 mL volume (reversed phase solid phase extraction cartridge)

Note: Use gravity or a vacuum manifold

  1. 3 mL 100% ACN
  2. Fill cartridge with 0.2% FA
  3. Load sample
  4. Fill cartridge with 0.2 % FA
  5. Elute into tube with 2 ml 80% ACN 0.2% TFA.
  6. Dry down reconstitute in 0.2% FA.

 

Peptide concentration

Resuspend sample in 0.2% FA, targeting ~2 µg/µL

Use Peptide colorimetric assay for peptide quantitation.