A STAGE Tip Procedure for Desalting and Purification of Peptides

Abstract

Source:
Tia Rizakos¹, Jennifer Geddes-McAlister<sup>1

1</sup>Molecular and Cellular Biology Department, University of Guelph.

This video demonstrates peptide purification through the STAGE Tip procedure. A miniature column containing resin beads bonded to long hydrocarbon chains facilitates hydrophobic interaction-based purification. Upon loading a pre-digested peptide sample into the column, the peptide fragments bind to the resin beads, allowing impurities to pass through. An elution buffer that disrupts the hydrophobic interaction is applied to elute the peptide fragments.

Protocol

1. Purification and Desalting of Peptides

1. Pack three layers of C18 resin in a 200 μl pipette tip for each peptide sample to construct a STop And Go Extraction (STAGE)-tip.
2. Add 1/10 stopping solution (20% acetonitrile [ACN)] and 6% trifluoroacetic acid [TFA]) to samples.
3. Centrifuge samples for pellet precipitation at 13,200 rpm for 10 min.
4. Transfer the supernatant to a new 2 mL tube and discard the pellet.
5. Wash stage tips with 100 μl of 100% ACN.
6. Centrifuge STAGE tips at 1000 x g for 2 min.
   NOTE: Centrifuge STAGE tips until all liquid has run throug....