What should I do if a portion of the recombinant protein passes through a TALON CellThru column without binding to the column?
What should I do if a portion of the recombinant protein passes through a TALON CellThru column without binding to the column?
If a fraction of the his-tagged protein present in the cell extract fails to adsorb to a TALON CellThru column, several explanations may account for this observation:
1. Some types of recombinant proteins may interact with the proteins embedded in the cell membrane. Also, in crude protein lysates, some cellular compartments may not be completely broken down. When an aliquot of crude lysate is run on an SDS-protein gel, all membranes are solubilized, and cellular compartments are broken down by SDS. That explains why all recombinant proteins present in the cells can be seen on an SDS-protein gel.
2. When a crude lysate is run over a TALON CellThru column, all membranes and unbroken cellular compartments pass through the column. If a recombinant protein strongly interacts with membrane proteins or if it is contained within unbroken compartments, it will not be adsorbed by TALON CellThru, and instead will appear in the wash fractions.
3. However, if a crude lysate is centrifuged to bring down all debris, and then this clarified lysate is loaded onto a standard TALON or Ni-NTA column, no protein will be observed in the wash fractions. This is good, but there will be very little recombinant protein retained on the column! Why? Because it was all pulled down during the centrifugation step, along with pieces of membrane and unbroken compartments.
Recommended solutions:
TALON CellThru will actually improve yields of proteins that may interact with membrane proteins or are compartmentalized. Try the following:
1. Run the lysate through the column several times.
2. Use denaturing conditions.
3. Use nonionic detergents for protein extraction.