Tools - Electrophoresis and Chromatography

To study proteins and their functions, one must first Produce, Extract, and Purify the protein. 

    Produce - tissue rich in protein / over-expression using cultured cells (see below)

    Extract - cell disruption followed by centrifugation

       Purification - take advantages of differences in solubility, charge, size, and specificity.

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Column chromatography - Separation by charge / size / or affinity

• a matrix is in a cylindrical holder
• buffer flows through the matrix
• fractions are collected
• separation of biomolecules

Ion-exchange chromatography

• proteins have charges due to amino acid side groups
• bind to charged column matrix depending on their charge at a particular pH
• anionic--negatively charged: phosphocellulose, heparin sepharose, S-sepharose
• cationic--positively charged: DEAE-sepharose, Q-sepharose
• elute bound proteins from column based on charge and displacement by salt or pH

High Performance Liquid Chromatography (HPLC)

• gravity flow very slow--depends on size and amount of liquid at the top
• HPLC used high pressure to force liquid through
• special matrixes and columns
• fast and sometimes better resolution

Affinity Chromatography - use of "tagged" proteins to create affinity site - sep. by specificity

column matrix has a ligand that specifically binds a protein e.g., ATP-agarose specialty affinity columns for binding recombinant proteins with certain "tags" 6xHis added at N or C terminus--binds Ni++ column other types of "tags"--chitin, glutathione S-transferase (GST).....

from Qiagen website

Gel Filtration - separation by size

• separates on the basis of size, not charge
• porous beads--think of golf balls
• small molecules go into the holes and get trapped temporarily
• large molecules are too large to enter the holes and pass on by
• exclusion size--depends on the size of the holes

• how long the molecules get trapped determines elution order
• large out first > medium > small out last
• choose the size of matrix for the separation needed

• method of separation of charged molecules
• movement of charge particles in an electrical field
• electrophoretic mobility reflects both charge and size/shape
• Many kinds of electrophoresis: usually have a solid medium e.g., paper, gel, TLC plate
• gel electrophoresis used to separate large molecules: DNA, RNA, protein
• a "gel" is like very stiff JELLO
• acrylamide forms polymers that can be cross-linked to varying degrees in a chemical process
• the cross-linking determines the size of "holes" that the molecules pass through

• apply electrical current across the medium, Anode (+) and Cathode (-)
• positively charged molecules move to the Cathode
• negatively charged molecules move to the Anode
• proteins depending on their charge will move in either direction,

• SDS is a detergent--amphipathic, has both hydrophobic and hydrophilic characteristics
• hydrophobic tail of SDS interacts with hydrophobic side chains of amino acids
• number of SDS molecules bound is proportional to the number of amino acids
• 1 molecule of SDS bound per ~2 amino acids
• SDS overwhelms any inherent charges and effectively turns the protein into a polyelectrolyte
• SDS also disrupts tertiary structure
• b-mercaptoethanol is also included to reduce disulfide bonds and destroy intra-chain cross-linking
• protein mobility in SDS PAGE is proportional to protein size