1. 5th Summary- Magnetic Nanoparticles based separation of Proteins Dr. Bansal Vibha
February 21-28, 2014
Proteins are macromolecules that consist of up to 20 different amino acids. They play key functions in
the living system such as carrying oxygen, controlling the sugar levels in the blood, and defending
against foreign cells, pathogens, and bacteria. Isolation of proteins may have different objectives such as
catalyst usage, therapeutics, dietary supplements and structure studies. Affinity chromatography, a
technique used for the purification of a biomolecule depending on its biological function, is used to
separate proteins according to the interaction between the protein(s) and a specific ligand.
Nanoparticles are more efficient due to their superior qualities such as higher surface to volume ratio,
efficient dispersibility without internal diffusional limitations. Magnetic nanoparticles (MNPs) are used in
affinity chromatography since they are fast, scalable, easily automated, easy to separate from other
suspended solids, combined with affinity binding, and reduce the pretreatment and chromatography
stages into a single step isolation. Tissue plasminogen activator (tPA), is an enzyme found in endothelial
cells involved in the breakdown of blood clots by catalyzing plasminogen to plasmin, an enzyme
responsible for clot breakdown. To obtain the pure protein, the MNPs are first prepared and bound to
tPA. Then several washes are performed, which removes all the nonspecific bound proteins. An elution,
a technique used to separate tPA from MNPs by lowering its pH, is then carried out. This step will result
in pure proteins. Finally the absorbance of these samples and blanks can be read to know if proteins are
present by running the affinity chromatography, and to know if there was any enzyme activity by
running the casein zymography. According to the absorbance of each sample, a Chromatogram can then
be prepared, which is a pattern formed on the adsorbent medium by the sheets of samples separated
by chromatography. These separations indicate enzyme activity and can easily be seen in the gel. The
results of the gels indicated the presence of large amounts of protein in the load and spent, but none in
neither of the eluates. Zymography can be used for peptidase investigation, and for identifications and
characterizations in biological living systems. For instance, it could be used to detect low levels or
absence of thrombin, the active form of prothrombin, which converts fibrinogen to fibrin so essential for
blood coagulation.