Peptide Purification: An RP-HPLC-based Technique to Extract Peptides from Digested Protein Lysates
Transcrição
To isolate and purify peptides, begin with a suspension of protein lysate containing a mixture of digested peptides.
Treat the mixture with trifluoroacetic acid or TFA-based reagent. This anionic or negatively charged reagent forms an ion-pair complex with basic or positively charged residues on peptides, thus masking their charge and improving peptide hydrophobicity.
Now, assemble a reversed-phase high-performance liquid chromatography, or RP-HPLC, column containing a porous silica-based stationary phase, immobilized with non-polar hydrophobic ligands. Add a peptide-compatible equilibration buffer to the column to prepare the stationary phase before sample application.
Now, load the acidified sample into the column. Within the column, the hydrophobic peptides selectively adsorb to the hydrophobic ligands of the stationary phase. Run a wash buffer through the column to elute any unbound or unwanted particles.
Next, pass a less polar organic elution buffer, with high hydrophobicity, through the column. Owing to their selective affinity towards the elution buffer, the bound peptides desorb from the column matrix.
Collect the peptide-containing eluate from the column. Lyophilize the eluate to remove the water content and obtain a dry powder of pure and stable peptides.
