Chapter 3 - Exploring proteins and proteomes Flashcards
Proteins can be separated from one another on the basis of
solubility, size, charge, and binding ability.
Genomes
The complete DNA base sequences.
Proteome
The entire complement of proteins that is or can be expressed by a cell, tissue, or organism.
The proteome is much _______ than the genome.
larger
The purification process of proteins is an essential first step in
understanding their function.
Purification should yield a sample containing
only one type of molecule.
Assay
An analytical procedure to detect and quantify cellular process or metabolic reactions.
A positive result on the assay indicates
that the protein is present.
Specific activity
the ratio of enzyme activity to the amount of protein in the mixture.
Ideally, the specific activity will ______ as the purification proceeds.
rise
Common protein purification techniques
- salting out
- dialysis
- gel-filtration chromatography
- ion-exchange chromatography
- affinity chromatography
- HPLC
Salting out
Most proteins are less soluble at high salt concentrations and can be precipitated out of solution.
Dialysis
Separating proteins from small molecules through a semipermeable membrane.
In IEC, to force the migration of analytes, mobile phases consist of
buffers, often with increasing ionic strength (e.g. a higher concentration of NaCl).
Affinity chromatography is
a separation method based on a specific binding interaction between an immobilized ligand and its binding partner.
Transcription factors
proteins that regulate gene expression by binding to specific DNA sequences.
What is the absorbance wavelength of a peptide bond?
220 nm.
Velocity of a protein in an electric field equation
v = Ez/f
E = electric field strength
z = charge
f = frictional coefficient
In gel electrophoresis, small molecules will
move faster through the gel.
The isoelectric point (pI) of a protein is the
pH at which its net charge is zero.
Isoelectric focusing
A method of separating proteins according to their isoelectric point.
SDS-PAGE
sodium dodecyl sulfate - polyacrylamide gel electrophoresis
In 2-D electrophoresis, proteins are separated vertically by _______ and horizontally by ________.
mass; isoelectric point
sediment coefficient equation
s = m(1 - vp)/f
m = mass
v = partial specific volume
p = density of the medium
f = frictional coefficient
Sediment coefficients are usually expressed in
Svedberg units (S)
The smaller the S value, the
slower a molecule moves in a centrifugal field.
The sedimentation technique for determining mass is
very accurate.
Antibody
A protein synthesized by vertebrates in response to the presence of a foreign substance.
Antigen
A foreign substance in the body which results in the production of antibodies.
Epitope
The part of the antigen molecule to which the antibody attaches itself.
The specificity of the antibody-antigen interaction is a consequence of
the shape complementarity between the two surfaces.
Polyclonal antibodies
A heterogeneous mix of antibodies, derived from the immune response of many different B-cells.
ELISA stands for
Enzyme-linked immunosorbent assay
Indirect ELISA is used to detect
the presence of antibody.
Sandwich ELISA is used to detect
the presence of antigen.
Fluorescent markers provide a powerful means of
examining proteins in their biological context.
Synthetic peptides can serve as antigens to
stimulate the formation of specific antibodies.
Synthetic peptides can be used to isolate receptors for
many hormones and other signal molecules.
Solid phase peptide synthesis involves
the successive addition of protected amino acid derivatives to a growing peptide chain immobilized on a solid phase.
3 components in x-ray crystallographic analysis.
- protein crystal
- source of x-rays
- detector
3 basic principles underlying x-ray diffraction
- Electrons scatter x-rays
- The scattered waves recombine
- The way waves recombine depends only on the atomic arrangement
NOESY stands for
nuclear Overhauser enhancement spectroscopy
