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Immobilized Phospholipid Chromatography 301
therefore recommended to wash the column before commencing the separation,
with 100% Buffer C until a stable baseline is reached followed by re-equilibration
in Buffer A conditions.
4. The blank run may need to be repeated two to three times to ensure proper
equilibration, particularly for a newly purchased column or if the column has been
stored for a long time.
5. The addition of detergent to the mobile phase may be varied depending on
the separation efficiency. The detergent, chaotrope additives, and phospholipids
present in the collected fractions may affect typical methods such as the BCA
method in determining the protein content. The detergent, chaotropes, and lipidcompatible
methods (such as DC/RC protein kit from Bio-Rad or 2D protein Quant
kit from Amersham Bioscience, Piscataway, NJ, USA) are required to accurately
determine the amount of proteins. Compatibility of detergent to further 1D or 2D
gel electrophoresis also needs to be considered for testing the purity of protein.
6. Removal of detergent from the collected fraction may be required to recover the
activity of membrane proteins, which can be achieved by the selective adsorption
of the detergent to hydrophobic substrates of Bio-Beads.
7. Column regeneration is typically achieved by continued washing with the starting
or running buffer. However, because of the hydrophobic nature of membrane
proteins, the binding of membrane proteins to the lipid ligands may be very
strong. Hence, high stringency wash buffers are necessary to completely remove
the residual bound membrane proteins.
References
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