GE Healthcare Life Sciences is now Cytiva - Find out more

November 21, 2017

Troubleshooting protein recovery issues

By Séverine Lebarq, Global Product Marketing Manager, Protein Purification at GE Healthcare

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 GE R&D protein purification experts in the following tables.

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 from several sources or batches, use a column packed with MabSelect SuRe™. (NaOH CIP* can be used)
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
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
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., Whatman™ SPARTAN™ syringe filters)

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
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

Watch out for our upcoming article about protein purification troubleshooting regarding column inspection.

Can’t wait for the next post? Download our Protein purification troubleshooting guide

This guide helps you quickly identify root causes for many of the common issues encountered during protein purification. It gives practical advice on small changes that you can make to fix these problems.

* 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