Troubleshooting protein recovery issues

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)

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

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

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

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