Biology Techniques Flashcards
(27 cards)
SDS-PAGE (SDS-Polycrylamide gel electrophoresis)
matrix for migration = highly cross-linked gel
proteins are treated with:
- SDS (gives negative charge, denaturing and solubilizing agent
- B-mercaptoethanol (reducing agent that breaks S-S bonds)
application of an electric field
proteins migrate through gel - separation by molecular mass (larger place higher than smaller)
2-Dimensional Gel Electrophoresis (A+B)
A. proteins are treated with solubilizing agent, B-mercatoethanol and urea to denature them, BUT intrinsic proteins charge is unchanged
- given that every protein has isoelectric point (pH where net charge is zero), proteins migrate through a polyacrylamide gel with pH gradient
- each protein migrates until it reaches its isoelectric point (net charge equal zero the protein is no more pulled by electric field)
B. SDS-Page
- migration now applied with 90° angle in respect to direction of isoelectric point
- separation of proteins by size and charge
Western Blotting
- transfer all proteins (from SDS-Page) onto a sheet of Nitrocellulose paper (using electricity)
- put sheet in solution containing specific type of antibody labeled with radioactive isotope or fluorescent dye
- protein of interest is detected
Mass Spectrometry
useful to identify unknown protein after purification techniques
explore the principle that charged particles have very precise dynamics when subjected to electrical and magnetic fields in a vacuum
- in order to get the mass/charge ration
determine the exact mass of proteins derived from purification techniques
Peptide Mass Fingerprinting
type of mass spectrometry
- unknown protein of interest is cleaved into short peptides using proteases
- peptides are ionized
- migration through electrical field towards detection plates gives mass/charge ration
- precise masses are compared to genomic/proteomic databases = process allows to trace back the amino acid sequence of protein
Immunoprecipitation (variation of affinity chromatography)
- heavy beads are coated with specific antibodies that recognize the protein of interest
- solution containing beads-antibodies added at top of protein extract
- binding occurs
- low speed centrifugation is applied
- supernatant is discarded while pellet is collected
used to purify small amount of enzymes from cell extracts to carry out enzymatic activity analysis
epitope tag - can be added to protein of interest, tag is an epitope for specific antibody
- makes immunoprecipitation faster
Co-Immunoprecipitation
used to identify proteins that bind tightly one to another
useful for characterizing biological role of an unknown protein
- target protein is immune-precipitated from lysate using antibodies coupled to beads
- partner proteins also precipitate and can be characterized through mass fingerprinting
Protein Structure
X-ray crystallography - the study of X-ray diffraction through the sample nuclei
- it implies having a crystalline sample that can be obtained as a result of pressure, pH and salt concentration applied to sample
NMR spectroscopy - exploits spins of electrons
- it does not require a crystalline sample
Gene Knockout
reverse genetics - proceeding from genes to mutation
used to find out function of a gene, by observing the effects of not having the gene
gene of interest is completely inactivated/deleted (both copies) in ‘knockout animals’
DNA Microarray
snapshot of gene expression
- mRNAs from a cell are converted to cDNAs through reverse transcriptase
- cDNAs are fluorescently labelled
- then left to hybridize to the fragments bound to microarray - automated fluorescence microscope determines which mRNAs were present in original sample
different variants can be used to compare gene expression in 2 samples or for comparative genomic hybridization
cluster analysis - used to identify sets of genes that are coordinately regulated
- genes that have same expression pattern are likely to be involved in common pathways or processes
FACS (fluorescence-activated cell sorting)
used to sort a heterogeneous mixture of biological cells into 2 or more containers, one cell at a time
- based upon specific light scattering and fluorescent characteristic of each cell
- antibody, coupled to fluorescent dye, labels specific cells of interest
- mixed cell suspension enters the system, fluorescence of droplets measured
- droplets containing a single fluorescent cell (negative charge)
- droplets containing a single NON fluorescent cell (positive charge)
- electric field deflects droplets into collecting tubes according to charge
- occasional cell clumps are left uncharged and discarded into waste containers
Centrifugation
separation of samples done according to size/density
- cell homogenate (variety of membrane-enclosed organelles) inserted into rotors create huge centrifugal forces
- sample starts to sediment at bottom of tube - the smaller the sub cellular compartment, the higher the centrifugal force required to make it sediment
- progressive higher speeds of centrifuge used to separate various cell organelles one another
Chromatography
process used for protein purification
- solution of proteins passes through a column containing a porous solid matrix
- proteins are slowed down to different extents by their interaction with the matrix
- collected separately as they get out the column
ion exchange - beads carrying a positive or negative charge
- purification done by charge
gel filtration - smaller molecules go through canaliculi of beads
- while big molecules go faster
- purification done by size
affinity - takes advantage of the ligand-receptor binding
- it sorts proteins that can bind to specific ligand
- specific process to purify proteins
Pulse and Chase
used to examine a cellular process occurring over time by exposing the cells to a labelled compound for short period of time (usually 5 mins)
- successively to the same UNLABELLED compound
in this way it’s possible to follow the fate of a certain protein when introducing radioactive AAs
FRET (fluorescence resonance energy transfer)
used to find out whether ONE PROTEIN and ANOTHER ONE interact with each other
- 2 proteins of interest are produced as fusion proteins attached to different color variant of GFP
- if there is interaction (when excited, one fluorochrome can transfer energy to the other)
- illumination at excitation lambda of the first, production lambda of second - if NO interaction (illumination at excitation lambda of the first, fluorescence of first, illumination at excitation lambda of second, fluorescence of second)
Gene Knockdown - RNA interference (RNAi)
NON-Coding RNAs are used to reduce gene expression
PROs:
- less time consuming
- cheaper
CONs:
- it can affect other genes related to target one (off-targeting)
- does not work for all genes
- results are not a proof of function as strong as one coming from knockout experiments
miRNA (microRNA)
- encoded in human genome
- cropped inside nucleus
- translocation into cytosol and degradation on one strand (Dicer)
- miRNA and proteins form upon base pair there is degradation of mRNA
siRNA (small interfering RNA)
- comes from viruses and transposable elements
- double-stranded RNA is cleaved by Dicer into fragments (siRNA) which are still double-stranded at this point
- one strand is degraded after binding to RISC proteins
- single-stranded RNA attached to RISC attaches complementary nucleotides on mRNA
- matching is usually exact = rapid degradation
Recombinant DNA Technology
- Restriction nucleases - cut DNA double helix at short DNA sequence
- gel-electrophoresis - separates fragments by size, DNA bonds made visible by ethidium bromide
- DNA cloning - used to make identical copies of DNA sequence isolated from rest of genome
- cDNA synthesis
- PCR (polymerase chain reaction)
- DNA sequencing - dideoxysequencing - Sanger’s method
- ChIP (chromatin immunoprecipitation)
- Souther Blot - analog to Western Blot but used for identification of DNA
- Norther Blot - analog to Southern Blot but used for identification of RNA
Gene expression profiling
measurement of expression of thousands of genes at once to create a global picture of cellular function
SILAC (stable isotope labelling with AAs in culture)
technique based on mass spectrometry that detects differences in protein abundance among samples using non-radioactive isotopic labelling
popular method for quantitative proteomics
another way of determining absolute abundance is by introducing a known peptide with known quantity in the mass spec analysis
- peptide can be used as a meter to compare quantity between peptides of interest (spiking)
TAP (tagging in yeast)
used to determine absolute abundance of proteins
small tag is inserted in every gene (can be recognized by antibodies
Western Blots used to show how much of protein is being expressed
RNA sequencing
used to reveal presence and quantity of RNA in a sample at given moment
used to determine intron/exon boundaries
Ribosome Profiling
used to determine which mRNAs are actively being translated
global snapshot of all active ribosomes in a cell at given time
output is speed of translation of mRNA at given time
GFP Tagging
GFP is added to protein of interest
localization - by looking at where fluorescence is, it is possible to identify where the protein of interest is
Microscopy
- cell diameter is 10-20 micrometers
- cells are translucent and colorless
- internal features can only be seen through contrast provided by stains
radiation cannot be used to probe structural details much smaller than its own lambda
light microscopy - uses visible light combined with a system of lenses that magnify a small specimen
fluorescence microscopy - absorption of lambda 1, emission of lambda 2
antibodies (indirect immunocytochemistry
- primary antibody binds to molecule of interest
- secondary antibodies bind to one primary antibody
CONS
- incompatible with life because membrane is made permeable to antibody through small punctures
quantum dots
- one way to detect changes in concentration and localization of molecule inside LIVING cells
- tiny particles of cadmium selenide are coupled to antibodies that bind our protein of interest
- they are all excited by same blue light
- larger dots –> increase lambda
GFP
- other way to detect dynamic phenomena
- GFP was isolated from jelly fish
- it is encoded by a single gene that can be cloned and introduced into cells of other species
