Lecture 20 - Biochemical Techniques Flashcards
what kind of physical/chemical characteristics can be exploited to learn more about molecules(proteins)?
- size
- charge
- specificity
- solubility
why do we want to purify proteins?
- to understand the structure
- to understand the mechanisms of action
- to detect protein modifications
- to identify protein binding partners
def: rupturing the cell to release the internal contents
homogenization
what kind of cells need to be broken to get enough protein
- tissue from an organ
- tissue culture cells
- yeast cells
- bacteria cells
what do we want our cell homogenate to have?
- disrupted the plasma membrane
- released soluble proteins
- maintain the integrity of organelles
Lysis buffer needs to help break apart the cell, but not all the ___________
organelles
what is frequently added to lysis buffers to prevent protein degradation
protease inhibitor
what is the disadvantage of using harsh lysis buffers, like amphipathic detergents?
they can denature the protein
what are the two common techniques to fractionate a sample?
- differential centrigfugation
- density gradient centrifugation
different __________ __________ separate organelles or macromolecules
sedimentation rates
def: separation of cell organelles or other particles of different size and density by their different rates of sedimentation in a centrifugal field
differential centrifugation
T or F: differential centrifugation does not yield a pure sample, so further steps are required
true
def: a measure of how rapidly the particle sediments when subjected to centrifugation, expressed in terms of Svedberg units
sedimentation coefficient
def: more contaminants can be removed using this kind of centrifugation, samples are placed on top of a gradient, centrifugation causes particles to move into discrete zones, based on the density of the gradient and the density of the particle/organelle
density gradient centrifugation
dense particles move into the gradient ______ __________ than less dense
more rapidly
if you want a specific protein, what do you need to know?
you need to know its properties to be able to identify a specific protein
def: a procedure where a mixture of dissolved components is fractioned as it moves through some type of porous matrix (usually in a column)
chromatogaphy
differential and density gradient centrifugation give us relatively pure peroxisomes, but if we want to further purify them, what do we do?
use in enzyme assays
what are the 3 types of chromatography?
- ion-exchange chromatography
- gel filtration chromatography
- affinity chromatography
def: relies on the differences in surface charge of the macromolecule, beads carry a weak (+) or (-) charge, charge proteins interact with beads to varying degrees, and proteins can be eluted using solutions at characteristic pH and salt concentrations
ion-exchange chromatography
proteins can be eluted, come out the bottom of the column, by doing what?
washing the column with different pH buffer
what happens as the pH is lowered in ion-exchange chromatography?
- negatively charge amino acids become neutral
- positively charged amino acids become more abundant
def: relies on difference in the size and shape of the macromolecules, beads have different size pores that allow proteins to percolate through them, smaller proteins enter all the pores and take longer to get through the column, larger proteins pass through the column more rapidly
gel filtration chromatography
def: relies on highly specific interactions between a ligand/substrate/antibody and the target, beads have a specific molecule attached to their surface and preferentially bind to the protein being purified, proteins are unbound and purified using salt, a change in pH , or mild detergents
affinity chromatography
def: when antibodies are used in affinity chromatography
immunoprecipitation
T or F: affinity chromatography usually results in near total purification of the desired molecule in a single step
true
T or F: if you have a catalase specific antibody, you can purify catalase
true
def: group of techniques that use an electric field to separate charged molecules
electrophoreisis
what are the most commonly used support media for electrophoresis?
polyacrylamide or agarose
what is electrophoresis useful for?
characterizing a mixture of soluble proteins
what does how quickly molecules move during electrophoresis depend on?
- shape
- charge
- size
what are detergents used for?
- isolate and analyze membrane proteins
- solubilize, purify, and reconstitute functional membrane protein systems
how are detergents able to disrupt the lipid bilayer?
- they are amphipathic
- disrupt the hydrophobic interactions, dissolving the lipids
prior to electrophoresis, insoluble and soluble proteins are usually exposed to high heat and SDS, which _______ _________ and _________ _______ associations
protein-protein, protein-lipid
how does SDS bind?
ratio of 1 SDS molecule per 2 amino acid residues
disulfide bonde reducing agents are also often added to the buffer, like:
- beta-mecratoethanol
- dithiothreitol (DTT)
what happens if SDS and DTT are boiled?
eliminates the shape and charge component, therefore proteins can only be separated based on their size
when proteins are loaded on a polyacrylamide gell and the electric potential is applied, what happens to the negatively charged proteins/SDS?
they run toward to positively charged bottom go the gell
the ________ the polypeptide the __________ it moves
smaller, faster
when does the gel stop?
when the smallest proteins reach the bottom
def: separates proteins in two dimensions, first by charge and then by size
two-dimensional SDS-PAGE
what can 2D-gels be useful for?
resolving subtle differences in various isoforms of proteins
def: uses magnetic and electric fields to separate molecules on charge and mass?
mass spectrometry
what is the first step of mass spec?
proteins are digested by a protease to break them into smaller peptides
def: proteins are transferred to a membrane and bound to specific antibodies
western blotting or immunoblotting
what are the two regions on antibodies?
- they have constant regions that are the same for all antibodies
- variable regions that are identical to each other but unique for each antibody
def: represent a mixture of antibodies produced by group of clonal related B cells
polyclonal antibodies
def: grown from single cells (cell culture)
monoclonal antibodies
def: fuse a lymphocyte to an immortalized myeloma cell
hybridomas
what do hybridomas do?
can produce a single antibody type and secrete it in to the culture media for collection
def: combining the resolving power gel electrophoresis and the specificity of antibodies
western blotting (immunoblotting)
what happens to solubilized mixtures of proteins?
- separated by SDS-PAGE
- transferred to membrane using electric current
- incubated with a primary antibody wash, then add a second antibody
- react with a substrate and image
def: the entire hereditary information in the form of DNA. This includes both the genes and the non-coding sequences of the DNA
genome
def: the entire complement of RNA expressed by a genome in a cell tissue, or organism. This will vary depending on the cell’s physiological state
transcriptome
def: the entire complement of proteins expressed by a genome in a cell, tissue, or organism. This will vary depending on the cell’s physiological state
proteome
what can the term proteome be used to describe?
all the proteins a cell CAN produce OR all the proteins a cell DOES produce
T or F: the transcriptome and proteome are larger than the genome in eukaryotes
true, because there are more RNA and proteins than genes due to alternate splicing of genes and post translation modifications
def: field that merges computer science with biology to address questions about DNA and gene expression
bioinformatics
def: can be used to see if tow proteins associate in vitro, often done using affinity chromatography
co-immunoprecipitaton assays
what’s the key difference between hub proteins and non-hub proteins?
hub proteins are more likely to be essential for cellular survival
hub proteins have:
- several binding interfaces, binding different proteins at the same time
- a single binding interface, which is capable of binding several different proteins at different times
def: in vivo technique to find where a protein binds within the genome of a cell
chromatin immunoprecipitation (CHIP)
what biochemical techniques can be used to determine protein structure?
- x-ray crystallography
- nuclear magnetic resonance spectroscopy
- cryo-electron microscopy
