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Experimental procedure for ion-exchange chromatography
Both peptides contain arginine and lysine that make the peptide positively charged. Thus the peptides can be separated by Cation exchange chromatography using a low pH buffer ( pH = 6)
Step 1: making 3 L of a 200 mM buffer at your desired pH.
Making MES buffer:
Molar mass: 195.2 g/mol
117.12 g MES (free acid) is dissolved in 2000 mL of ddH2O
pH is adjusted to the desired value with 10 N NaOH/ HCl
Volume is then made up to 3000 ml
Step 2: explaining what type of ion-exchange resin you will run the column to collect your samples
Cation exchange resin is required to separate the peptides. At low pH and low ion concentration, peptides will attach to the column (with the negatively charged matrix). Then elution with a low salt buffer ( 400mM NaCl) will elute peptide 1 as it has low positive charge ( one R and one K amino acid). A high salt buffer (1M NaCl) will completely elute the peptide 2 as it has high positive charge (two R amino acid)....
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