Affinity Chromatography - Department of Molecular and Cellular ...

Situation Cause Remedy
Protein does not bind Sample has not been filtered properly. Clean the column, filter the sample
or elute as expected.
and repeat.
Sample has altered during storage. Prepare fresh samples.
Sample has wrong pH or buffer Use a desalting column to transfer sample into
conditions are incorrect. the correct buffer (see page 134).
Solutions have wrong pH.
Calibrate pH meter, prepare new solutions and
try again.
The column is not equilibrated Repeat or prolong the equilibration step.
sufficiently in the buffer.
Proteins or lipids have
Clean and regenerate the column or use a
precipitated on the column.
new column.
Column is overloaded with sample. Decrease the sample load.
Microbial growth has occurred Microbial growth rarely occurs in columns
in the column.
during use, but, to prevent infection of packed
columns, store in 20% ethanol when possible.
Precipitation of protein in the Clean the column, exchange or clean the filter
column filter and/ or at the top or use a new column.
of the bed.
Low recovery of activity, but Protein may be unstable or Determine the pH and salt stability of the
normal recovery of protein. inactive in the elution buffer. protein.
Collect fractions into neutralization buffer such
as 1 M Tris-HCl, pH 9 (60–200 µl per fraction).
Enzyme separated from
Test by pooling aliquots from the fractions and
co-factor or similar.
repeating the assay.
Lower yield than expected. Protein may have been Add protease inhibitors to the sample and
degraded by proteases.
buffers to prevent proteolytic digestion.
Run sample through a medium such as
Benzamidine 4 Fast Flow (high sub) to remove
serine proteases.
Adsorption to filter during
Use another type of filter.
sample preparation.
Sample precipitates.
May be caused by removal of salts or
unsuitable buffer conditions.
Hydrophobic proteins.
Use chaotropic agents, polarity reducing agents
Protein is still attached to ligand. or detergents.
More activity is Different assay conditions have Use the same assay conditions for all the
recovered than was been used before and after assays in the purification scheme.
applied to the column. the chromatographic step.
Removal of inhibitors during
separation.
Reduced or poor flow Presence of lipoproteins or Remove lipoproteins and aggregrates during
through the column. protein aggregates. sample preparation (see Appendix 1).
Protein precipitation in the
Modify the eluent to maintain stability.
column caused by removal
of stabilizing agents during
fractionation.
Clogged column filter.
Replace the filter or use a new column.
Always filter samples and buffer before use.
Clogged end-piece or
Remove and clean or use a new column.
adaptor or tubing.
Precipitated proteins.
Clean the column using recommended methods
or use a new column.
Bed compressed.
Repack the column, if possible, or use a
new column.
Microbial growth.
Microbial growth rarely occurs in columns
during use, but, to prevent infection of packed
columns, store in 20% ethanol when possible.
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