Chapter 4-Nucleic Acids And Protein Methods Flashcards
Function of the Y2H system
To test the interaction between 2 proteins through the downstream activation of a reporter gene
Advantages of Y2H system
- Relatively fast and easy method to screen for protein-protein interactions
- Requires little hands-on time and technical skill
- Can be scaled up by screening yeast libraries of tagged prey proteins against a single bait, allowing thousands of potential interactions to be screened rapidly
Limitations of Y2H system
- High rates of false positives and false negatives
- Tagged proteins may not fold correctly and may not bind to their targets
- Interaction must occur in the nucleus for the reporter gene to be activated
Function of Y1H system
Tests for protein-DNA interaction
In the Y1H system, which transcriptional domain is the protein bound to?
Activating domain
Advantages of Y1H system
- Able to detect protein-DNA interactions that are not actually activating transcription e.g. DNA repair proteins and repressor proteins
- Compatible with existing libraries
- Can detect isoform specific interactions, detection of highly specific interactions
Disadvantages of Y1H system
- No information on the functional consequences of DNA-protein interaction
- Rates of false-positive results are high
- Improper protein folding may occur resulting in loss of interactions
Function of EMSA
Tests for DNA-protein interaction (need to know DNA sequence)
Advantages of EMSA
- Highly sensitive, assay can be performed with small protein and nucleic acid concentrations and small sample volumes
- Assay works well with both highly purified and crude cell extracts
- A wide range of nucleic acid sizes are compatible with the assay
Function of DNA footprinting
- to study DNA-protein interactions (need to know general protein-binding region)
Function of ChIP
To study protein-DNA interactions (when you don’t know which part of the DNA the protein of interest binds to)
3 types of protein purification chromatography
Affinity, ion-exchange, gel-filtration
How does affinity chromatography work?
- separation method based on specific binding interaction between an immobilised ligand and its binding partner on the protein
Advantages of affinity chromatography
- Cheap
- High specificity
- High degree of purity of protein
How does ion-exchange chromatography work?
- separates proteins based on total charge
How does gel filtration chromatography work?
- separates proteins based on size through filtration through a gel
What is the principle behind isolation of nucleic acids using spin columns?
- nucleic acids bind to silica under certain conditions but not others
Conditions for the use of mini prep kits
- small scale preparations of plasmids with yields of 2-5ug of DNA/RNA
Conditions for use of maxi prep kits
- large scale preparation of plasmids with yields of 500ug-2mg DNA/RNA
- more time consuming and expensive
How to obtain plasmids and not genomic DNA?
- perform alkaline lysis: shorter stretches of DNA like plasmids renature following denaturation much faster than longer stretches of DNA like gDNA
What are the components in a PCR mix?
- buffer
- MgCl2
- forward and reverse primers
- template DNA
- dNTPs
- taq polymerase
Purpose of qPCR
- relative quantification of the increase in amt of cDNA as it is amplified
- measures amt of amplified product at each stage during PCR cycle
What is the dye used in qPCR and what does it bind to?
- SYBR green; dsDNA
What is the Ct in qPCR?
- fractional PCR cycle no. at which fluorescence is greater than the threshold
How to calculate relative amt of DNA in qPCR using Ct?
- 2^n where n is the difference in Ct number
Purpose of Sanger sequencing
- to determine the nucleotide sequence in DNA
Difference between dideoxynucleotides (ddNTPs),and deoxyribonucleotide triphosphates (dNTPs)
ddNTPs contain a hydrogen group on the 3’ carbon instead of a hydroxyl group
Components in a Sanger sequencing master mix
- template
- forward and reverse primers
- excess dNTPs
- DNA polymerase
- small amounts of ddATP/ddTTP/ddCTP/ddGTP
Advantages of Next-Gen Sequencing compared to Sanger sequencing
- Ability to sequence many copies of genes simultaneously
- Lower cost
- Able to sequence samples with low input amounts of starting material
Purpose of microarrays
- to determine which genes are expressed in different cells
Purpose of RNA-seq
- measures gene expression across the entire transcriptome using NGS
Purpose of BCA assay
- for quantitation of proteins
What’s the wavelength used for BCA assay?
562 nm
Purpose of Bradford assay
- for quantification of proteins
Wavelength for absorbance for Bradford assay
595 nm
Why is polyacrylamide suitable for SDS-PAGE?
- chemically inert, electrically neutral, hydrophilic and transparent for optical detection
Substrate of HRP
Luminol
Why is a loading control needed?
- to demonstrate that there was equal protein loaded in the sample
Function of X-ray crystallography
- determine protein structure
- to produce well-ordered crystals that is lacking in contaminants
- proteins of any size can be precipitated
When does crystallisation occur?
- protein concentration is greater than its solubility limit and the protein is in a supersaturated state
Conditions for protein crystallization
- at least 97% protein purity
- pH should correspond to pI since solubility of protein is lowest here
- suitable conc of protein
- temperature
- precipitants (increase ionic strength and lowers solubility of protein to enable ppt)
Bragg’s law
- when x-rays are scattered from a crystal lattice, peaks of scattered intensity are observed which correspond to the following conditions: angle of i=angle of scattering, path length diff is equal to an integer number of wavelengths
- calculate details about protein structure
What is Nuclear Magnetic Resonance?
- physical phenomenon which occurs when the nuclei of certain atoms are immersed in a static magnetic field and exposed to a radio frequency energy source
- conducted in solution
What is NMR suitable for?
- small proteins (25kDa max, around 220aa)
What is nuclear spin?
- fundamental nuclear property
- comes in multiples of 1/2
- individual unpaired protons, electrons and neutrons possess nuclear spins of 1/2
- In 1H atom, proton has a nuclear spin of 1/2
- 1H, 13C most commonly used in NMR
What is resonance in NMR?
- nuclei absorb energy of radio waves that enable a change in the angle of nuclear spin
Range of magnetic field strength of NMR
- 200-900 MHz
Define chemical shift
- frequency of absorption for a nucleus of interest relative to that of the molecular standard
- serves as a guide to analyse organic chemical structures
- molecular standard for 1H and 13C is TMS bc of molecular symmetry: all 12 H absorb at the same frequency and all 4 C absorb at the same frequency
How to understand the number of signals in NMR spectra?
- corresponds to the number of diff protons in molecule
- a set of protons in the same magnetic environment are equivalent and will give a single signal
How to understand position of signals? (Chemical shift)
- reveals what kind of proton gave that signal
- dependent on nuclear shielding
- with high nuclear shielding, less resonant energy is required to flip nucleus hence low chem shift
How to understand splitting in NMR?
- due to nuclear spin interactions of one nucleus with one or more nearby nuclei through spin spin coupling
- provides info on how many H neighbours (split into N+1 peaks where N=no. of H on adjacent atom)
Which group has no signal splitting?
- OH bc of H20 interference
Cryo-EM
- generally >200 kDa, sometimes 80 kDa
