A STAGE Tip Procedure for Desalting and Purification of Peptides
Transcript
Take a STop And Go Extraction or STAGE Tip, a miniaturized purification column.
The tip contains small silica beads bonded to long hydrocarbon chains for hydrophobic interaction-based peptide purification.
Wash with buffers containing a high percentage of acetonitrile, removing resin impurities.
Introduce a binding buffer and centrifuge, equilibrating the column for optimal peptide binding.
Load a pre-digested peptide sample obtained from differentiated neutrophil-like cells, containing peptide fragments and impurities, including salts.
Centrifuge to pass the sample through the column. The peptide fragments bind to the hydrocarbon chains via hydrophobic interactions, while the salts are removed.
Add the binding buffer and centrifuge, removing the remaining impurities.
Introduce the high acetonitrile-containing buffer for elution.
Using a syringe, pass the buffer through the column. Acetonitrile binds to the hydrocarbon chains, displacing the peptide fragments that elute with the buffer.
Perform proteomic profiling to identify the purified peptides.
