Restriction Endonucleases
- a.k.a. restriction enzymes
- Methalated sequences on host bacteria DNA prevent cutting of host DNA
What is TBE Buffer?
- This is the buffer, fills the pores in the arose gel, so that DNA fragments can diffuse through
- You add this before you place your samples into the wells
What is Loading dye? (what is it made of and what does it do?)
- Glycerol: to make the sample sink to the bottom of the well
- Bromophenol Blue: this enables us to see the sample, and tell when it is reaching the end of the arose gel, so we know when to stop electrophoresis
Cutting sequence of Alu 1
AG CT
TC GA
Cutting sequence of Taq 1
T CGA
AGC T
Cutting sequence of EcoR1
G AATTC
CTTAA G
Cutting sequence of Not 1
GC GGCCGC
CGCCGG CG
Ethidium Bromide: what does it do, what colour is it?
- Used to visualise the DNA
- when bound between bases, it takes on a pink-orange fluoresce under UV light
Transaminases: cofactor? what do they do?
- Pyridoxial phosphate: from Vitamin 6
- Move amino groups from amino acid and oxo acids
What was the transaminase reaction we did? And with what enzyme?
- Glutamate (A.A.) + Pyruvate (keto.a.) –> Alanine (A.A.) + Oxoglutarate (keto.a.)
- Glutamate amino transferase
Glutamate Dehydrogenase: cofactor? what does it do?
- Catalyses oxidative deamination of glutamate into oxoglutarate
- NAD+ and NADP+ as a cofactor
What does aminopeptidase do?
- Cleaves the amino acid off the N terminus
What does carboxypeptidase do?
- cleaves the amino acid off the C terminus
2,4,-DNP
- Stains keto/oxo acids yellow
Ninhydrin + Heat
- Stains Amino Acids purple
Km
- Measure of affinity of an enzyme for its substrate
- [S] that half saturates the enzyme
- Half Vmax
- Smaller Km means larger affinity
- units mmol
Vmax
- Max velocity an average enzyme can obtain
- units µmol/min
Kcat
- Catalytic constant - turnover number
- I.e. # of substrate molecules transformed per molecule of enzyme per second
Competitive Inhibitor
- Inhibitor and substrate similar structure
- Both compete for same active site
- Km increases, Vmax remains the same
Uncompetitive Inhibitor
- When the substrate binds, this causes the inhibitors binding site to appear
- Can only bind to ESC
- Vmax is dramatically reduced - not much free enzyme to catalyse
- Km - minimally reduced
