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

Electrophoresis

• 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

The following is after the Amersham Biosciences Web Site: In SDS polyacrylamide gel electrophoresis (SDS-PAGE) separations, migration is determined not by intrinsic electric charge of polypeptides but by molecular weight. Sodium dodecylsulphate (SDS) is an anionic detergent that denatures proteins by wrapping the hydrophobic tail around the polypeptide backbone. For almost all proteins, SDS binds at a ratio of approximately 1.4 g SDS per gram of protein, thus conferring a net negative charge to the polypeptide in proportion to its length. The SDS also disrupts hydrogen bonds, blocks hydrophobic interactions, and substantially unfolds the protein molecules, minimizing differences in molecular form by eliminating the tertiary and secondary structures. The proteins can be totally unfolded when a reducing agent such as dithiothreitol (DTT) is employed. DTT cleaves any disulphide bonds between cysteine residues. The SDS-denatured and reduced polypeptides are flexible rods with uniform negative charge per unit length. Thus, because molecular weight is essentially a linear function of peptide chain length, in sieving gels the proteins separate by molecular weight. There are two types of buffer systems used in protein gel electrophoresis: continuous and discontinuous.

A continuous system uses only one buffer for the tanks and the gel. In a discontinuous system, first developed by Ornstein (1964) and Davis (1964), a nonrestrictive large-pore gel called a stacking gel is layered on top of a separating (resolving) gel. The two gel layers are each made with a different buffer, and the tank buffers differ from the gel buffers.

In a discontinuous system, protein mobility — a quantitative measure of the migration rate of a charged species in an electric field — is intermediate between the mobility of the buffer ion of the same charge (usually negative) in the stacking gel (leading ion) and the mobility of the buffer ion in the upper tank (trailing ion). When electrophoresis is started, the ions and the proteins begin migrating into the stacking gel. The proteins concentrate in a very thin zone, called the stack, between the leading ion and the trailing ion. The proteins continue to migrate in the stack until they reach the separating gel. At that point, due to a pH or an ion change, the proteins become the trailing ion and "unstack" as they separate on the gel. Although a continuous system is slightly easier to set up than a discontinuous system and tends to have fewer sample precipitation and aggregation problems, much greater resolution can be obtained with a discontinuous system. Only minimal concentration of the sample takes place with continuous gels, and proteins form zones nearly as broad as the height of the original samples in the sample wells, resulting in much lower resolution.  The discontinuous Laemmli system (Laemmli, 1970), a denaturing modification of Ornstein (1964) and Davis (1964), is the most widely used system for research protein electrophoresis today. The resolution in a Laemmli gel is excellent because the treated peptides are concentrated in a stacking zone before entering the separating gel.

Caution:

Acrylamide is a neurotoxin and should be handled with care.