Summary of the Biochemistry book, Stryer et al,
Production and design of biomolecules
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Chapter 3 Exploring proteins and proteomes
Protein play a crucial role in nearly all biological processes – in catalysis, signal transmission,
and structural support. This remarkable range of functions arises from the existence of
thousands of proteins, each folded into a distinctive three-dimensional structure that
enables it to interact with one or more of a highly diverse array of molecules.
Proteins can be separated from one another on the basis of solubility, size, charge and
binding ability.
Mass spectrometry provides a powerful method for determining the mass and sequence of
a protein.
Antibodies are choice probes for locating proteins in vivo and measuring their quantities.
Monoclonal antibodies, able to recognize specific proteins, can be obtained in large
amounts and used to detect and quantify the protein both in isolation and in cells.
Salting-out: Most proteins are less soluble at high salt concentrations. It can be used to
fraction proteins.
Dialysis: Proteins can be separated from small molecules such as salt through a
semipermeable membrane. Molecules having dimensions significantly greater than the pore
diameter are retained inside the dialysis bag.
Gel filtration chromatography: Is a more discriminating separation technique based on size,
also known as molecular exclusion chromatography. The sample is applied to the top of a
column consisting of porous beads made of an insoluble but highly hydrated polymer. Small
molecules can enter beads, but large ones cannot. The result is that small molecules are
distributed in the aqueous solution both inside the beads and between them, whereas large
molecules are located only in the solution between the beads. Large molecules flow more
rapidly through is column and emerge first because a smaller volume is accessible to them.
Ion exchange chromatography: To obtain a protein of high purity one chromatography step
is usually not sufficient. If a protein has a net positive charge at pH 7, it will usually bind to a
column of beads containing carboxylate groups, where negative proteins will not. The
bound protein is eluted with a salt buffer, where proteins that have a low density of net
,positive charge will tend to emerge first, followed by those having a higher charge density.
This is also called cation exchange to indicate that positively charged groups will bind to the
anionic beads. Positively charged groups (cation) can be separated with negative
carboxylmethulcellulse (CM-cellulose) and negative charged groups (anion) can be
separated with positive DEAE-cellulose.
Affinity chromatography: is another powerful means of purifying proteins that is highly
selective for the protein of interest. In general it can be effectively used to isolate a protein
that recognizes group X by (1) covalent attaching X or derivative of it to a column; (2) adding
a mixture of proteins to this column, which is then washed with buffer to remove unbound
proteins; and (3) eluting the desired protein by adding a high concentration of a soluble
form of X or altering the conditions to decrease binding affinity.
High performance liquid chromatography (HPLC): is an enhanced version of the column
techniques already discussed. The column materials are much more finely divided and, as a
consequence, possess more interaction sites and thus greater resoling power. Because of
the finer material, pressure must be applied to the column to obtain adequate flow rates.
The net result is both high resolution and rapid separation. The detector measures the
absorbance of the eluate at a particular wavelengths, resulting in a profile where the
measured proteins are showed.
SDS is an anionic detergent than disrupts nearly all noncovalent interactions in native
proteins.
Beta-mercaptoetethanol or dithiothreitol is added to reduce disulphide bonds.
Small proteins move rapidly through the gel, whereas large proteins stay a the top, near the
point of application of the mixture.
Isoelectric focusing: proteins can also be separated electrophoretically on the basis of their
relative contents of acidic and basic residues. The pI of a protein is the pH at which its net
charge is zero. Each protein will move until it reaches a position in the gel at which the pH is
equal to the pI of the protein.
Two dimensional electrophoresis: is where isoelectric focusing can be combined with SDS-
PAGE to obtain very high resolution separation. First isoelectric focusing is done and then
the proteins are separated on size.
Several important conclusions can be drawn from the preceding equation for centrifugation:
- The sedimentation velocity of a particle depends in part on its mass. A more massive
particle sediments more rapidly than does a less massive particle of the same shape
and density.
- Shape, too, influences the sedimentation velocity because it affects the viscous drag.
The frictional coefficient f of a compact particle is smaller than that of an extended
particle of the same mass. Hence, elongated particles sediment more slowly than do
spherical ones of the same mass.
- A dense particle moves more rapidly than does a less dense one because the
opposing buoyant force is smaller for the denser particle.
- The sedimentation velocity also depends on the density of the solution.
, Problems with native purification is that you often require a large amount of tissue to obtain
a sufficient amount of protein for analytical study. However, due to recombinant technology
there are created some significant advantages:
1. Proteins can be expressed in large quantities.
2. Affinity tags can be fused to proteins. For affinity chromatography.
3. Proteins with modified primary structures can be readily generated.
Advantages in the field of immunology have enabled the use of antibodies as critical
reagents for exploring the functions of proteins within the cell.
An antibody (also called an immunoglobulin, Ig) is itself a protein and have specific and high
affinity for the antigens that elicited their synthesis. An antibody recognizes a specific group
or cluster of amino acids on the target molecule.
Polyclonal antibodies are derived from multiple antibody-producing
cell populations. The heterogeneity of polyclonal antibodies can be
advantageous for certain applications, such as the detection of a
protein of low abundance, because each protein molecule can be
bound by more than one antibody at multiple distinct antigenic sites.
Monoclonal antibodies are all identical, produced by clones of a single
antibody-producing cell. They recognize one specific epitope. While
polyclonal antibodies are heterogeneous mixtures of antibodies, each
specific for one of the various epitopes on an antigen.
An antibody that is specific for the protein of interest is called the
primary antibody. A second antibody is called the secondary antibody
and is specific for the primary antibody. Typically, the secondary
antibody is fused to an enzyme that produces a chemiluminescent or
coloured product or contains a fluorescent tag, enabling the identification and
quantification of the protein of interest.
Mass spectrometry enables the highly precise and sensitive measurement of the atomic
composition of a particular molecule, or analyte, without prior knowledge of its identity. It
operate by converting analyte molecules into gaseous, charged forms (gas-hase ions).
Through the application of electrostatic potentials, the ratio of the mass of each ion to tis
charge (the mass-to-charge ratio, or m/z) can be measured.
The amino acid sequence of a protein provides valuable information.
1. The sequence of a protein of interest can be compared with all other known
sequences to ascertain whether significant similarities exist.
2. Comparison of sequences of the same protein in different species yields a wealth of
information about evolutionary pathways.
3. Amino acid sequences can be searched for the presence of internal repeats. Such
repeats can reveal the history of an individual protein itself.
4. Many proteins contain amino acid sequences that serve as signals designating their
destinations or controlling their processing.
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