What is the colour change that Coomassie blue undergoes?
Green-brown (free) → Blue (protein-bound)
What is the abdorbance of protein-bound Coomassie blue?
What is the absorbance of NADH?
What reaction in LDH assay is driven in practice?
- Lactate + NADH → Pyruvate + NAD+
- Much greater maximum rate of reaction
What is SDS?
Sodium dodecyl sulfate
What is the purpose of SDS?
1. Denatures proteins (unfold + dissociates subunits)
2. Shields charged proteins
What stain is used to detect LDH in non-denaturing PAGE?
Tetrazolium → Formazan (purple)
How are large volumes of antibodies produced?
Fusion of B-cells with myeloma cells.
Why is the relationship between protein concentration and absorbance not linear?
Coomassie blue doesn't bind to protein with 1:1 stoichiometry.
What is a katal?
What are the ways that a mixture of proteins can be characterised further?
1. Testing for specific activity of enzymes
2. Antibodies and ELISA
3. Purification of proteins by chromatography
What additional properties are revealed by non-denaturing PAGE?
1. Preserves multimeric proteins
2. Preserves activity of proteins (e.g. allows for activity of specific enzymes to be tested)
3. Reveals information on charge but not size
What is the P:O ratio?
Number of phosphates esterified per atom of O
What is state 3?
- Pi and ADP present, so oxidative phosphorylation is occuring.
- Fast rate of O2 usage
What is state 4?
- Pi and ATP present, so oxidative phosphorylation is not.
- Slow rate of O2 usage.
What is the respiratory control ratio?
Rate of O2 usage (State 3)/Rate of O2 usage (state4)
What is the H+ equivalent of the P:O ratio?
P:O = H+:O/H+:P
What is the purpose of adding malate + glutamate?
Provides substrates for the malate-glutamate shuttle which is used to carry electrone into mitochondria to generate NADH.
What is the meaning of the respiratory control ratio?
The higher the ratio, the less leaky the mitochondria, the more efficient it is.
What factors affect state 4 O2 consumption rate?
H+:O ratio. The higher the H+:O, the greater the PMF, the smaller the state 4 O2 consumption rate.
What are the effects of PMF on rate of respiration?
PMF inhibits respiration by inhibiting the ETC.
What are the possible explanations for why ATP produced may be lower than theoretical?
1. PMF insufficient to phosphorylate all ADP to ATP
2. ATPases and other enzymes present may have broken down some ATP
What are the effects of high levels of Ca2+ in the matrix?
Causes activation of permeability transition pores that cause mitochondrial swelling
What drives Ca2+ uptake into the mitochondria?
Why does Ca2+ entry stimulate respiration?
1. Ca2+ entry decreases PMF
2. Ca2+ entry causes permeability transition pores to appear that eliminate PMF due to being permeable to H+ ions
Why does high levels of Ca2+ inhibit PMF?
Causes outer mitochondrial membrane to burst and dissipates cytochrome C.
How does arsenate stimulate respiration?
1. Substitutes Pi in ATP synathase, forming ADP-As
2. Hydrolyses, allowing re-synthesis and thus dissipates PMF
Where do different substrates feed into the ETC?
Malate + Glutamate: Complex I (NADH)
Succinate: Complex II (FADH2)
Ascorbate + TMPD: Cytochrome c
What did the 2 bands in the undigested plasmid correspond to?
1. Supercoiled plasmid (closer to well)
2. Relaxed plasmid
What are the effects of adding bacteriostatic and bacteriolytic agent?
Bacteriolytic effects inhibited due to
What are the reasons for not obtaining any PCR product during PCR experiment?
1. DNA may be damaged
2. Primers may not be complementary to DNA
3. Primers may anneal to DNA too weakly, resulting in melting when temperature raised to working temperature of Taq polymerase
4. The buffer composition needs to be correct; i.e. there needs to be sufficient Mg2+ in the mixture (as it is cofactor for Taq polymerase)
5. Temperature too high and may denature enzyme
6. Temperature may not be high enough to melt DNA
7. Formation of secondary hair-pin loops in DNA
What is the purpose of adding RNAse to preparation?
To break down RNA
What is the purpose of adding EDTA to the preparation?
To cellate Mg2+ and prevent DNAse from breaking down plasmid DNA
What is the purpose of adding SDS to the preparation?
Lyse bacterial cells open
What is the purpose of adding NaOH to the preparation?
What is the purpose of adding high [salt] solution to the preparation?
Precipitates out cellular debris and long genomic DNA
What are the melting temperatures of primer pairs?
A/T = 2°C
C/G = 4°C
Why are primers with lots of CG pairs bad?
1. Induces formation of secondary DNA hair-pin structures
2. High annealing temperatures