Troubleshooting protein recovery issues

Are you frustrated that you aren’t getting the amount of pure protein that you expected? Don’t worry!

For example, if your protein recovery is higher than expected, it could be caused by proteins coeluting with other substances.

If you are experiencing poor binding, it’s possible that your sample has the wrong pH.

Maybe your recovery is lower than expected, which could mean the protein is being degraded by proteases.

Other common reasons for protein recovery issues, and how to fix them, are summarized by Cytiva R&D protein purification experts in the following tables.

 

PROTEIN RECOVERY IS HIGHER THAN EXPECTED – POSSIBLE CAUSES AND REMEDIES

Possible cause	Remedy
Proteins coeluting with other substances	Optimize running conditions to improve resolution Check buffer conditions used for assay before and after run Check selection of resin
Cross-contamination from a previous run on the same column	Clean using recommended procedures If purifying several antibodies, use a column packed with MabSelect PrismA™. (NaOH CIP* can be used)

 

POOR BINDING OF PROTEIN – POSSIBLE CAUSES AND REMEDIES

Possible cause	Remedy
Sample has wrong pH or buffer conditions incorrect	Use a desalting column packed with Sephadex™ G-25 to transfer sample into correct buffer
Column not equilibrated sufficiently in buffer	Repeat or prolong equilibration step until conductivity and/or pH are constant
Microbial growth has occurred in column	Clean according to cleaning procedures and store in 20% ethanol when not in use
Metal ion stripping from IMAC* resin	Use a desalting column packed with Sephadex™ G-25 to remove metal ion stripping agents from sample or use a column packed with Ni Sepharose™ excel resin (e.g. HisTrap™ excel)
Binding capacity of resin is exceeded	Pack a larger column If using a HiTrap™ column, connect up to three columns in series

 

ACTIVITY IS HIGHER THAN EXPECTED – POSSIBLE CAUSES AND REMEDIES

Possible cause	Remedy
Different assay conditions used before and after chromatography step	Use same conditions for all assays
Inhibitors removed during separations	Use a desalting column packed with Sephadex™ G-25/dialyze original sample before measuring activity, because cell lysates/ extracts often contain low molecular weight substances that can affect activity

 

PROTEIN RECOVERY IS LOWER THAN EXPECTED – POSSIBLE CAUSES AND REMEDIES

Possible cause	Remedy
Protein degraded by proteases	Add protease inhibitors to sample and buffers to prevent proteolytic digestion Run sample through a resin such as Benzamidine Sepharose™ 4 Fast Flow (high sub) to remove trypsin-like serine proteases
Protein adsorbed to filter during sample preparation	Use another type of filter with low protein binding (e.g., Protein Prep for ÄKTA™ systems)
Proteins precipitated	HIC: Check salt conditions; adjust to improve solubility IEX: Check pH and salt conditions; adjust to improve solubility
Hydrophobic interactions are occurring	IEX: Add denaturing agents, polarity-reducing agents, or detergents. Add 10% ethylene glycol to running buffer to prevent hydrophobic interactions SEC, AC*: Use denaturing agents, polarity-reducing agents, or detergents
Nonspecific adsorption to resin	IEX: Reduce salt concentration to minimize hydrophobic interaction. Add suitable detergent or organic solvent (e.g., 5% isopropanol) SEC: Increase salt concentration in the buffer, up to 300 mM sodium chloride
Proteins not eluting	HIC: Consider use of additives to reduce hydrophobic interactions, or use a less hydrophobic resin AC: If using competitive elution, increase concentration of competitor (e.g., imidazole) in elution buffer

 

ACTIVITY IS LOW, BUT RECOVERY IS NORMAL – POSSIBLE CAUSES AND REMEDIES

Possible cause	Remedy
Protein might be unstable or inactive in buffer	Determine pH and salt stability of protein Include additives to stabilize protein of interest
Enzyme separated from co-factor or other necessary component	Test by pooling aliquots from fractions and repeating assay

 

 

View more chromatography troubleshooting tips here

* SEC = size exclusion chromatography,
IEX = ion exchange chromatography,
HIC = hydrophobic interaction chromatography,
AC = affinity chromatography,
IMAC = immobilized metal ion affinity chromatography,
CIP= cleaning in place