Analysis of Proteins and Nucleic Acids Flashcards
What are antibodies composed of?
modular domains
What is the structure of antibodies?
modular domain structure - 3 domains
What do antibodies contain?
heavy and light chains (2 each) that are each composed of alternative gene segments encoding parts of the individual domains
What are antibodies produced by?
B cells
What are the functions of antibodies?
- localize proteins in a cell
- protein purification
- diagnostically
- inhibitors of protein activity
What is the function of T cells?
produce similar proteins to distinguish self from non-self in immune surveillance - New FDA treatment involves genetic engineering of T cells to fight cancer
How are proteins denatured?
by boiling in Sodium dodecyl sulfate (SDS) and beta-mercapto-ethanol
What is Sodium dodecyl sulface (SDS)?
an ionic detergent
What is beta-mercapto-ethanol
a reducing agent
What happens to the structure of denatured proteins?
loses all 2’, 3’, and 4’ structures and disulfide bonds
What is the function of SDS?
coats proteins uniformly with negative charge, and thus the amount of SDS is proportional to the length of the protein
T or F: SDS-coated proteins have roughly equal mass to charge ratios
true
What is PAGE?
SDS-Polyacrylamide Gel Electrophoresis (PAGE)
How do proteins migrate in PAGE?
negatively charged, SDS-coated proteins will migrate to the positive electrode
What is the rate at which molecules move in PAGE?
at a rate according to the mass:charge
How do SDS and BME interact with proteins?
completely denature proteins and give them all a similar mass:charge
What will the gel matrix determine?
the rate of migration with the rate being inversely proportional to the mass
How are proteins separated?
by size
How are proteins first separated in immunoblotting?
separated by SDS-PAGE (1D or 2D)
How are proteins blotted in immunoblotting?
transferred by electrophoresis to a membrane
What happens to the antibody after it recognizes the protein of interest (the primary antibody, 1)?
incubated with the blot, and excess unbound antibody is washed away
What happens to the blot after it is recognized?
incubated with an antibody (secondary, 2) that recognizes the constant fragment (Fc) of the 1 antibody
What happens to the 2 antibody?
linked to an enzyme and produces a fluorescent of chemiluminescent reaction in the presence of the appropriate substrate
How are acidic and basic groups charged?
charged at pH above (acidic) or below (basic) their pKa
T or F: uncharged proteins will migrate in an electric field?
false, charged proteins
How do proteins move in a pH gradient?
proteins will migrate until they have no net charge will stop; this is the isoelectric point
What is the first step in two-dimension gel-electrophoresis?
proteins are separated according to their isoelectric points in a tube containing a pH gradient across which a charge is applied
When do proteins stop migrating in gel electrophoresis?
no net charge and then no longer will move in the electric field
How do charged molecules move in agarose gel electrophoresis?
move in an electric field toward the oppositely charged pole; in the gel apparatus a buffer conducts the current
How are DNA and RNA charged?
uniformly charged, so mass ratio is constant. Hence, they will migrate at same rate in electric field
Why do gel matrix allows separation on size?
due to the pore size of matrix
What sizes alter rate of movement?
any topology, closed circle, supercoiled circle, secondary structures of ssDNA, or RNA)
What analysis can be done in denaturing conditions?
ssDNA and RNA analysis
What allows DNA to move in an electric field?
negative charge of DNA and RNA
What are used to separate DNA molecules?
Agarose (for DNA > 500bp) or polyacrylamide (for DNA < 1000bp) matrices are used to separate DNA molecules
How does linear DNA migrate?
uniformly based on size (distance traveled is inversely proportional to its size
What is done before gel electrophoresis can perform on DNA?
to determine their size, DNA molecules are first cleave to make them linear
How is DNA vizualized
with a stain
What protects the host genome from foreign DNA?
restriction-modification systems
What does bacteria produce?
produce a restriction endonuclease also must produce a DNA modification enzyme (a methylase) to protect their own DNA from digestion
What does methylation in the recognition sequence prevent?
prevent the binding of the restriction enzyme to the sequence, thereby preventing cleavage
How do restriction enzymes bind to DNA?
as dimers
What is the function of restriction enzyme dimers?
recognize palindromic DNA sequences (two half sites) and cleave DNA symmetrically on both strands of the DNA
What produces restriction enzyme?
produced by bacteria protect against invading DNA (e.g. phage infection)
What does the length of recognition sequence determine?
the approximate cleavage frequency
What does DNA base composition affect?
affect frequency of cleavage (e.g. AT- rich versus GC-rich DNA
What is the frequency of a specific sequence in DNA?
f = (0.25)^n; n = length of sequence
Is sequence composition random?
not random; some sequences are more or less abundant than expected on random basis
What is the function of an electrophoretic mobility shift assay (EMSA)?
used to determine the interaction between protein and DNA molecules
What is a Non-denaturing gel?
usually agarose, may be polyacrylamide
What is an example of a gel-shift?
a DNA molecular ( the probe) moves more slowly when bound to a protein. As more protein is added, a greater proportion of DNA moves at a slower rate through the gel. Testing different protein concentrations allows the binding kinetics of the protein to be determined
What are the four steps of Chromatin Immunoprecipitation (ChIP)
- treat live cells with chemicals (e.g. HCHO) to cross link proteins to DNA
- fragment DNA randomly
- use antibodies to immunoprecipitate specific protein along with DNA to which it binds
- reverse crosslinking to remove protein and purify DNA
How are DNA identified in ChIP?
by PCR (ChiP), hybridization (ChIP-chip), or high-throughput sequencing (ChIP-seq)
