Protein ID Solutions Sample Prep Guide
95
Peptide Cleanup with Agilent C18 Spin Tubes
9
Troubleshooting
Problem
Cause
Solution
Poor or incomplete
sample binding
High pH, lack of
ion-pairing agents
Ensure TFA was added to sample.
Sample contains
organic solvent
Dry sample and resuspend in 20 µL 0.1% to 0.5% TFA.
Sample not sufficiently
hydrophobic to bind C18
resin
None
Resin became dry
before sample addition
Ensure resin does not dry during activation and equilibration; keep resin
in equilibration/wash solution until sample addition.
Poor or incomplete
sample recovery
Highly hydrophobic
sample
Use 70% ACN/0.1% TFA for elution.
Sample loss due to
nonspecific binding
Nonspecific binding of peptides to plastics can cause significant sample
loss at very low peptide concentrations.
Minimize contact with plastics and storage at low peptide quantities
(that is,
300 fmol).
Peptide Cleanup C18 Spin Tubes are not recommended for routine use at
total peptide quantities of
300 fmol as special handling may be
required.
Detection limits of
application
Ensure sample is within the detection limit of the specific downstream
application.
Note: Limits vary considerably based on application and
instrumentation.