MCDB separating proteins

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MCDB separating proteins
2014-02-02 14:18:05

laboratory methods.
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  1. Key to Purification: Find out What Makes the Target Protein Special
    • Size: Size exclusion chromatography
    • Charge: Ion exchange chromatography
    • Ligands: Affinity chromatography
    • Hydrophobicity: Hydrophobic chromatography
  2. Key to Purification: Find out What Makes the Target Protein Special
    • Size: Size exclusion chromatography
    • Charge: Ion exchange chromatography
    • Ligands: Affinity chromatography
    • Hydrophobicity: Hydrophobic chromatography
  3. Separation by charge:
    Ion-exchange chromatography exploits differences in the sign and magnitude of the net electric charge or proteins at a given pH.

    • a. The column matrix is a synthetic polymer (resin) containing bound charged groups; those with bound anionic groups are called cation exchangers, and those with bound cationic groups are called anion exchangers. The affinity of each protein for the charged groups on the column is affected by the
    • pH(which determines the ionization state of the molecule) and the concentration
    • of competing free salt ions in the surrounding solution. Separation can be
    • optimized by gradually changing the pH and/or salt concentration of the mobile
    • phase so as to create a pH or salt gradient. In cation-exchange chromatography,
    • the solid matrix has negatively charged groups. In the mobile phase, proteins
    • with a net positive charge migrate through the matrix more slowly than those with a net negative charge, because the migration of the former is retarded more by interaction with the stationary phase.

    b. Proteins move through the column at rates determined by their net charge at the pH being used. With cation exchangers, proteins with a more negative net charge move faster and elute earlier.

    c. In ion-exchange columns, the expansion of the protein band in the mobile phase the protein solution) is caused both by separation of proteins with different properties and by diffusional spreading.
  4. Size-Exclusion chromatography (SEC):
    • Size-Exclusion chromatography (SEC): separates proteins according to size.
    • -Contrary to intuition, large proteins elute earlier than small proteins on a SEC column.
    • -Protein mixture is added to column containing cross-linked polymer, beads with engineered pores of a particular size. Protein molecules separate by size; larger molecules pass more freely, appearing in the earlier fractions.
    • -SEC can also be used to determine the approx. molecular weight and size of a protein.
  5. Separation by Affinity chromatography:
    • Separation by Affinity chromatography: based on binding affinity.
    • The beads in the column have a covalently attached chemical group called a ligand – a group or molecule that binds to a macromolecule such as a protein. When a protein mixture is added to the column, any protein with affinity for this ligand binds to the beads; its migration through the matrix is retarded.
    • After the proteins that do not bind are washed through the column, the bound protein is eluted by a solution containing either a high concentration of salt or free ligand.
    • Salt weakens the binding of the protein to the immobilized ligand attached to the beads, releasing the protein from the matrix; the protein product that elutes from the column is often bound to the ligand used to elute it.
  6. Isoelectric focusing
    • Isoelectric focusing is a procedure used to determine the isoelectric point (pI) of a protein.
    • A protein sample may be applied to one end of a gel strip with an immobilized pH gradient. Or, a protein sample in a solution of ampholytes may be used to rehydrate a dehydrated gel strip. Each protein migrates until it reaches the pH that matches its pI.
    • An electric field is applied.
    • After staining, proteins are shown to be distributed along the pH gradient according to their pI values.
  7. Two-dimensional PAGE or two-dimensional
    • Two-dimensional PAGE or two-dimensional electrophoresis permits the resolution of complex mixtures of proteins. This process separates proteins of identical molecular weight that differ in pI, or proteins with similar pI values but differ in molecular weights.
    • a. Separate proteins in first dimension on gel strip with isoelectric
    • focusing.

    • b. Then, separate proteins in second dimension on SDS-polyacrylamide
    • gel.

    • c. The 2D gels vertical axis decreases in molecular weight from top to bottom and the horizontal axis decreases in pI from left to right. Vert. separation reflects differences in molecular weight. Hor. Separation reflects
    • differences in pI.

    d. Proteins in spot can be identified by mass spectrometry.

    e. Ideally: each spot on the 2D-gel corresponds to a single protein.

    • f. Reality: Spot may contain multiple, similar proteins (isoforms
    • etc.).
    • Purification
    • progress can be monitored by SDS-PAGE: An electrophoretic method commonly
    • employed for estimation of purity and molecular weight.

    • a. SDS (sodium dodecyl sulfate): denatures proteins (binding partially
    • unfolds proteins) and masks individual net charge of protein (the bound SDS
    • contributes a large net negative charge).

    • i. This renders the charge of the protein insignificant and conferring
    • on each protein a similar charge-to-mass ratio.

    • ii. Electrophoresis in the presence of SDS therefore separates proteins
    • almost exclusively on the basis of mass (molecular weight), with smaller
    • poly-peptides migrating more rapidly.

    • iii. When compared with the positions to which proteins of known
    • molecular weight migrate in the gel, the position of an unidentified protein
    • can provide a good approximation of its molecular weight.

    b. SDS-PAGE reveals: size of proteins and purity.
  9. What does SDS actually do in gel
    A protein will bind about 1.4 times its weight of SDS, nearly one molecule of SDS for each amino acid residue. The bound SDS contributes a large net negative charge, rendering the intrinsic charge of the protein insignificant and conferring on each protein a similar charge-to-mass ratio. In addition, SDS binding partially unfolds proteins, such that most SDS-bound proteins assume a similar rodlike shape.
  10. What methods are used for the two dimensions of 2-dimensional
    electrophoresis? What order are they used in?
    The methods used for the two dimensions of the 2-dimensional electrophoresis are isoelectric focusing and SDS-polyacrylamide gel.
  11. Pay attention to disulfide bonds in proteins: how
    they can be broken (be able to name something that breaks them), and what they
    form between (be careful!). What levels of protein structure contain them?
    • Breaking disulfide bonds in proteins:  Two common methods are illustrated. Oxidation of a cystine residue with performic acid produces two cysteic acid residues. Reduction by dithiothreitol (DTT) (or β-mercap-toethanol) to form Cys residues must be followed by further modification of the reactive —SH groups to prevent re-formation of the disulfide bond Carboxymethylation by iodoacetate serves this purpose.
    • Disulfide bonds are contained in the tertiary and quaternary structure of proteins.