Proteins perform many functions in living organisms and differ based on their amino acid sequence. Affinity chromatography uses the interaction between proteins and ligands to separate proteins, and magnetic nanoparticles can be used to make this process faster and more efficient. In this experiment, magnetic nanoparticles were prepared and bound to tissue plasminogen activator to isolate it using affinity chromatography, washing and eluting steps. Absorbance readings would then indicate if proteins were successfully isolated.

1. 5th
Summary- Protein Isolation and PAGE Electrophoresis Dr. Vibha Bansal
February 21 & 28, 2014
Proteins are macromolecules that consist of one or more chains of
amino acids. They perform a vast array of functions within living
organisms, including catalyzing metabolic reactions, replicating
DNA, responding to stimuli, and transporting molecules from one
location to another. Proteins differ from one another primarily in
their sequence of amino acids, which is dictated by the nucleotide
sequence of their genes, and which usually results in folding of the
protein into a specific three-dimensional structure that determines
its activity. Proteins can be arranged into primary, secondary,
tertiary and quaternary varying in their shape. 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 proteins
and a specific ligand. Magnetic nanoparticles are used in affinity
chromatography since they are fast, scalable, easily automated,
easy to separate from other suspended solids, and combined with
affinity binding, reduces the pretreatment and chromatography
stages into a single step isolation. Tissue plasminogen activator is
an enzyme involved in the breakdown of blood clots by catalyzing
plasminogen to plasmin. In the experiment, we prepared the
magnetic nanoparticles in order to bind it to the tissue plasminogen
activator. Then we washed the magnetic nanoparticles to remove
the nonspecific bound proteins. We eluted the liquid to separate
the tissue plasminogen activator from the magnetic nanoparticles.
Finally the absorbance of these samples and blanks can be read
to know if proteins are present and prepare a chromatogram.