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.