CM-HA Resin Protocol Guide · Application note

Lysozyme on CM-HA Resin

Basic protein (pI about 11, 14.3 kDa) · step elution with sodium phosphate

Note 1.0.0 Updated 2026-11-12 Preliminary data

Summary

Lysozyme bound almost completely to CM-HA Resin under the conditions below. Very little protein came through during loading.
Bound lysozyme eluted at the first phosphate step (50 mM) with 150 mM NaCl present in the buffers.
Use this as a starting method for other basic proteins, then adjust NaCl and the phosphate steps.

Conditions

Item	Setting
Column	5 mL, packed with CM-HA Resin (R&D batch)
Buffer A	A-II: 5 mM sodium phosphate, 150 mM NaCl, 0.5 mM CaCl₂, pH 6.8 (recipe)
Buffer B	500 mM sodium phosphate, pH 6.8
Sample	Lysozyme dissolved in buffer A, 2.5 mL injection
Flow rate	1 mL/min (5 min residence time)
Steps	Equilibrate and load in A → wash with A → 50 mM → 150 mM → 300 mM phosphate (each about 25 mL) → strip with B
Detection	A280 of collected fractions

Results

Step	Share of recovered lysozyme	Observation
Load and wash (buffer A)	about 2 %	Almost no unbound protein
50 mM phosphate	about 90 %	Sharp peak shortly after the step reached the column, with a tail through the end of the step
150 / 300 mM phosphate	about 7 %	Small amounts only

Shares are calculated from the A280 of the collected fractions in one run. They describe where lysozyme elutes, not the binding capacity.

Tips

NaCl controls retention of basic proteins. Basic groups bind to the phosphate sites like a cation exchanger, so NaCl weakens binding. If your protein elutes too early, lower the NaCl in buffer A; if it binds too tightly, raise it.
Always dissolve or exchange the sample into buffer A before loading.
Degas buffer A and keep it at room temperature. Air in the flow path shows up as flat, square UV signals that are not protein.
For quantitation, collect and measure all fractions including the strip.

Data status

This note is based on a single run (November 2026) with an R&D batch of CM-HA Resin. Recovery, dynamic binding capacity and run-to-run repeatability will be added after further runs, and the note version will go up when they are.

Version	Date	Changes
1.0.0	2026-11-12	First release: conditions and elution position from one run.