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Lecture

Protein Purification Methods

In course

CH-319: Experimental biochemistry and biophysics

A 7-week long (4+8 h) experiment where you plan and construct a fluorescent sensor protein starting from DNA bricks. The protein will be expressed in and purified from E.coli, characterized by biochemical and spectroscopic methods. Your report will be in ...

Description

This lecture covers the purification of expressed proteins, including topics such as transformation into E. coli, lysis of bacteria, immobilized metal-ion chromatography (IMAC), analysis of purified proteins, and the fluorescent protein database.

Instructor

Rudolf Hovius

Official source

Related concepts (16)

Fusion protein

Fusion proteins or chimeric (kī-ˈmir-ik) proteins (literally, made of parts from different sources) are proteins created through the joining of two or more genes that originally coded for separate proteins. Translation of this fusion gene results in a single or multiple polypeptides with functional properties derived from each of the original proteins. Recombinant fusion proteins are created artificially by recombinant DNA technology for use in biological research or therapeutics.

Green fluorescent protein

The green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. The label GFP traditionally refers to the protein first isolated from the jellyfish Aequorea victoria and is sometimes called avGFP. However, GFPs have been found in other organisms including corals, sea anemones, zoanithids, copepods and lancelets. The GFP from A. victoria has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm.

Protein purification

Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms. Protein purification is vital for the specification of the function, structure and interactions of the protein of interest. The purification process may separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins.

Protein

Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, responding to stimuli, providing structure to cells and organisms, 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 protein folding into a specific 3D structure that determines its activity.

Transformation matrix

In linear algebra, linear transformations can be represented by matrices. If is a linear transformation mapping to and is a column vector with entries, then for some matrix , called the transformation matrix of . Note that has rows and columns, whereas the transformation is from to . There are alternative expressions of transformation matrices involving row vectors that are preferred by some authors. Matrices allow arbitrary linear transformations to be displayed in a consistent format, suitable for computation.

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Explores protein composition, synthesis, purification, and analysis techniques for understanding protein structure and function.

Protein Expression & Purification

Explores protein expression, purification, and gel filtration for isolating protein complexes.

Proteomics: Post-Translational Modifications and Interactions