Lecture 20 - Methods of protein analysis I Flashcards
Gel electrophoresis can be used to
determine the relative amount of different proteins in a sample
In gel electrophoresis, small molecules
readily move through the porous gel
In gel electrophoresis, proteins migrate from the
negative to the positive electrodes
SDS-PAGE stands for
sodium dodecyl sulfate polyacrylamide gel electrophoresis
A discontinuous buffer system contains (3)
- Tank buffer (Tris-Gly pH 8.3)
- Stacking buffer (Tris-HCl pH 6.8)
- Resolving buffer (Tris-HCl pH 8.8)
A stacking gel forces
the proteins to organize into a narrow band at the start of the resolving gel
In SDS-PAGE, proteins that differ by in mass by about ____ can be distinguished
2%
SDS-PAGE can be used to examine the efficacy of a protein purification protocol by
analyzing each fraction during purification and seeing the bands present
Isoelectric focusing is a method that is used to separate proteins based on
their isoelectric point
In isoelectric focusing, a protein stops moving in the gel when
it reaches the pH in the gel that is equal to its pI
Protein concentration can be determined by (4)
- UV absorption
- Lowry (Folin-Ciocalteau) method
- Bradford protein assay
- Bicinchoninic Acid (BCA) method
To follow the purification of a protein, we must also know the
enzyme activity in our sample
Knowing the enzyme activity and the protein concentration, we can calculate the
specific activity
Specific activity is the ratio of
enzyme activity to protein concentration
Enzyme activity will ________ as the purification proceeds
decrease
During purification, the specific activity of the protein will
increase
The high specificity of antibodies for their target proteins allows us to (3)
- Tag a specific protein
- Make drugs
- Image molecular targets in the body
An antibody (also called an immunoglobulin, Ig) is
a protein, which is synthesized by animals in response to the presence of a foreign substance (antigen)
Antibodies 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 called an antigenic determinant or epitope
The specificity of the antibody-antigen interaction is a consequence of the
shape complementarity between the two surfaces
Immunological methods depend on the ability to generate
antibodies to a specific antigen
Polyclonal antibodies are derived from
multiple antibody-producing cell populations
Most antigens have _______ epitopes
several
Polyclonal antibodies are
heterogeneous mixtures of antibodies, each specific for one of the various epitopes on an antigen
Monoclonal antibodies are
all identical, produced by clones of a single antibody-producing cell and recognize one specific epitope
Is it usually more advantageous to work with polyclonal or monoclonal antibodies?
monoclonal
ELISA stands for
enzyme-linked immunosorbent assay
ELISA is used to
detect the amount of protein or other antigen present in a biological sample
Indirect ELISA is used to
detect the presence of antibody
Sandwich ELISA is used to
detect antigen rather than antibody
Western blotting allows for detection of
a protein in a complex mixture
In western blotting, antibodies can be labeled with (3)
- Radioactive label
- Fluorescent label
- Enzyme that generates a colored product
Cells can be stained with fluorescence-labeled antibodies to reveal
the location of a protein of interest
Immunoprecipitation (IP) used
antibodies to “pull” a protein out of a solution
