biotechnology

Question 1

classical competition binding assay

Answer 1

• You need a receptor, a suspected ligand which is labelled radioactively or fluorescently and an antagonist
  1. Start by adding just the agonist (labelled) do a filtration and measure the fluorescence  this is 100% receptor ‘saturation’ with your ligand
  2. Then start adding antagonist
  3. At some point 50% of all receptors have bound the agonist (labelled) and 50% have bound the unlabelled antagonist  IC50

Question 2

scintillation proximity assay

Answer 2

• No need for antagonist
• You need a SPA bead filled with a scintillant that emits light upon beta-radiation from tritium (H3)
  1. The SPA bead contains the scintillant and on its outside membrane it has a receptor
  2. You add a tritium-labelled ligand
  3. When it binds to the receptor the scintillant in the bead stars to emit light which can be measured
• Still not ideal since you need to attach a label to the ligand which in some cases might block the binding site for its receptor giving false negatives

Question 3

fluorescent resonance energy transfer (fred)

Answer 3

• In FRET the binding of a ligand to its receptor indirectly emits radiation which can be measured
• Both the receptor and ligand need to be labelled
• You irradiate the receptor with 440nm wavelength if there is no interaction 480nm are reflected –> if there is an interaction this reflection changes
• Based on proximity not PPI

Question 4

Surface plasmon resonance

Answer 4

1. You bind a receptor to a sensor chip covered in a gold surface
2. A microfluidics device is applied across the surface of the chip on which the receptor is placed
3. In the fluid is the analyte (the suspected ligand)
4. On the other side of the gold film a light source shines polarized light at the chip at a specific angle called the resonance angle
   • At this angle the electrons in the gold start to resonate – the electrons are now referred to as surface plasmons – they are highly sensitive to their surrounding environment in this state
   • The resonating of the electrons results in a decrease in the energy of the reflected light which can be measured  the dip in energy can be used to assess the surface of the chip (has the ligand bound yes or no?)
5. When the ligand binds to its receptor the dip in energy of the reflection (resonance) angle of the light changes
   - SPR can also be used t assess the binding affinity of the system
   • If the change in intensity is measured over time we can determine the time it takes for the ligand to bind and dissociate which gives an indication of binding affinity

Question 5

western blotting

Answer 5

• Need to know what to look for
• Depends on specific antibodies that you make for proteins or protein modifications that you want to detect
  1. First prepare sample and perform gel electrophoresis to separate the proteins based on mass
  2. Add a primary and secondary (labelled) antibody against the protein or specifically against a phosphorylated form of your protein of interest and see if its there

Question 6

phosphoproteomics - untargetted assay

Answer 6

• No idea of what the protein of interest is involved in you are at step 1
  1. Cell lysis
  2. Protein digestion (trypsin)  you want to know which of all the proteins are phosphorylated when you add a ligand to your receptor of interest
  3. Phosphoprotein enrichment (to discriminate between phosphorylated and not)
  • Column chromatography: IMAC, MOAC, SCX or antibodies
  4. To identify the proteins you use a MS and asses the peaks
  5. After identification you can look at the sequences of the proteins to find phosphorylation sites (tyrosine, serine and threonine)

Question 7

phosphoproteinenrichment - MOAC

Answer 7

• To capture the phosphopeptides using MOAC you use titanium oxide TiO2
  • You do this in a pipette tip only need 20microliters of digested peptides in loading buffer
  • The proteins that make it through the TiO2 are non-phosphopeptides (low pH)
  • The ones that stay in the TiO2 are phosphopeptides (high pH)
• IMAC is the same principle however Fe3+ is used

Question 8

LC-MSMS

Answer 8

1. liquid chromotograph separates the peptides based on mass and affinity for mobile and stationary phase
2. MS ionises the peptides to give them a charge
3. they are accelerated and go into a charged room where depending on their mass they will bend and fall on a detector which can use the bend to identify them
   - MSMS is just doing it twice to get specific reading

Question 9

protein oxidation

Answer 9

• There is no antibodies that can be used to measure the products of protein oxidation and you cant use MS
• Oxidation of macromolecules is complex and many modifications can be made:
  • Direct oxidation of amino acids
  • Oxidative cleavage of protein backbone
  • Lipid peroxidation
  • Sugar oxidation
• All modification in the end lead to the formation of protein carbonyls  these can be detected by DNP which complexes together  this complex can be detected by a antibody and thus western blotting can be used

Question 10

MaMTH

Answer 10

• Used to solve the proximity FRED issue
  1. Tag 2 membrane-bound proteins with half ubiquitin (cub-TF and nub)
  2. If they interact the ubiquitin becomes whole
  3. DUB is recruited and cleaves of the TF from the Cub
  4. TF goes to the vector containing reporter gene
  5. Binds to promotor and causes expression of reporter gene (GFP or luciferase)

Question 11

pull-down assay

Answer 11

1. bind a ligand to an agarose bead
2. the bait protein will bind to this ligand and bead
3. wash away all other proteins
4. use this complex to find the prey protein in a new protein sample (to find new PPI)
5. wash away other proteins
6. western blot

Question 12

immunoprecipitation

Answer 12

1. attach magnetic beads to an antibody
2. add protein sample
3. use magnet to pull out the bead-antibody-protein complex
4. to identify the proteins doe AP assay

Question 13

affinity purification assay

Answer 13

1. Do a pull down or immunoprecipitation assay to collect proteins of interest
2. Do a western blot to separate them
3. To identify them digest them using trypsin
4. Then use the peptides to do a LC-MS/MS
5. Use database to identify the peaks

Question 14

CRISPR/CAS9 in bacteria

Answer 14

• Many bacteria harbour RNA-guided adaptive immune systems encoded by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and accompanying CRISPR-associated (Cas) proteins
• The CRISPR-Cas systems rely on a library of small CRISPR RNAs (crRNAs) transcribed from CRISPR loci, together with Cas proteins, for sequence-specific detection and silencing/destroying of foreign nucleic acids
• The CRISPR-Cas machinery targets invasive non-self DNA via base pairing with the crRNA guide sequence, leading to Cas protein-mediated DNA cleavage
• CRISPR-Cas adaptive immunity comprises three distinct stages
  1. Spacer acquisition  a short protospacer sequence from an infecting mobile element is incorporated into the CRISPR array as a new spacer. Such acquired spacer sequences serve as a genetic record of prior infections.
   The protospacers are selected for based on PAMs (protospacer adjacent motifs), since bacteria don’t have these bacterial DNA is not at risk
  2. CRISPR-Cas expression  also known as CRISPR RNA (crRNA) biogenesis, the CRISPR array is transcribed into a long precursor (pre-crRNA) and subsequently processed by endonucleolytic cleavage into mature crRNAs that consist of a single spacer surrounded by partial CRISPR repeat sequences on one or both sides
  3. DNA interference ¬ mature crRNAs assemble with Cas proteins into surveillance complexes that target DNA for degradation, thereby preventing the propagation of viruses and plasmids

Question 15

CRISPR/Cas gene editing

Answer 15

• Done in germ cells or stem cells never in somatic cells

1. Researchers create a small piece of RNA with a short “guide” sequence that attaches (binds) to a specific target sequence of DNA in a genome.
2. The RNA also binds to the Cas9 enzyme.
3. As in bacteria, the modified RNA is used to recognize the DNA sequence, and the Cas9 enzyme cuts the DNA at the targeted location.
4. Once the DNA is cut, researchers use the cell’s own DNA repair machinery to add or delete pieces of genetic material, or to make changes to the DNA by replacing an existing segment with a customized DNA sequence

• If the goal is knocking out a gene no donor DNA is used and the cell has to use NHEJ which creates indels (insertion/deletions) the goal is a frameshift mutation –> low efficacy (1/10)
• if the goal is fixing a mutation donor DNA is supplied and the goal is that HR is used to incorporate the donor DNA to fix a gene –> lower efficacy (1/100)