Practicals (key information)

Question 1

What is the colour change that Coomassie blue undergoes?

Answer 1

Green-brown (free) → Blue (protein-bound)

Question 2

What is the abdorbance of protein-bound Coomassie blue?

Answer 2

595 nm

Question 3

What is the absorbance of NADH?

Answer 3

340 nm

Question 4

What reaction in LDH assay is driven in practice?

Answer 4

• Lactate + NADH → Pyruvate + NAD⁺
• Much greater maximum rate of reaction

Question 5

What is SDS?

Answer 5

Sodium dodecyl sulfate

Question 6

What is the purpose of SDS?

Answer 6

1. Denatures proteins (unfold + dissociates subunits)
2. Shields charged proteins

Question 7

What stain is used to detect LDH in non-denaturing PAGE?

Answer 7

Tetrazolium → Formazan (purple)

Question 8

How are large volumes of antibodies produced?

Answer 8

Fusion of B-cells with myeloma cells.

Question 9

Why is the relationship between protein concentration and absorbance not linear?

Answer 9

Coomassie blue doesn’t bind to protein with 1:1 stoichiometry.

Question 10

What is a katal?

Answer 10

Mol s^-1

Question 11

What are the ways that a mixture of proteins can be characterised further?

Answer 11

1. Testing for specific activity of enzymes
2. Antibodies and ELISA
3. Purification of proteins by chromatography

Question 12

What additional properties are revealed by non-denaturing PAGE?

Answer 12

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

Question 13

What is the P:O ratio?

Answer 13

Number of phosphates esterified per atom of O

Question 14

What is state 3?

Answer 14

• Pi and ADP present, so oxidative phosphorylation is occuring.
• Fast rate of O₂ usage

Question 15

What is state 4?

Answer 15

• Pi and ATP present, so oxidative phosphorylation is not.
• Slow rate of O2 usage.

Question 16

What is the respiratory control ratio?

Answer 16

Rate of O₂ usage (State 3)/Rate of O₂ usage (state4)

Question 17

What is the H⁺ equivalent of the P:O ratio?

Answer 17

P:O = H⁺:O/H⁺:P

Question 18

What is the purpose of adding malate + glutamate?

Answer 18

Provides substrates for the malate-glutamate shuttle which is used to carry electrone into mitochondria to generate NADH.

Question 19

What is the meaning of the respiratory control ratio?

Answer 19

The higher the ratio, the less leaky the mitochondria, the more efficient it is.

Question 20

What factors affect state 4 O₂ consumption rate?

Answer 20

H⁺:O ratio. The higher the H⁺:O, the greater the PMF, the smaller the state 4 O₂ consumption rate.

Question 21

What are the effects of PMF on rate of respiration?

Answer 21

PMF inhibits respiration by inhibiting the ETC.

Question 22

What are the possible explanations for why ATP produced may be lower than theoretical?

Answer 22

1. PMF insufficient to phosphorylate all ADP to ATP
2. ATPases and other enzymes present may have broken down some ATP

Question 23

What are the effects of high levels of Ca²⁺ in the matrix?

Answer 23

Causes activation of permeability transition pores that cause mitochondrial swelling

Question 24

What drives Ca²⁺ uptake into the mitochondria?

Answer 24

PMF

Question 25

Why does Ca²⁺ entry stimulate respiration?

Answer 25

1. Ca²⁺ entry decreases PMF 2. Ca²⁺ entry causes permeability transition pores to appear that eliminate PMF due to being permeable to H⁺ ions

Question 26

Why does high levels of Ca²⁺ inhibit PMF?

Answer 26

Causes outer mitochondrial membrane to burst and dissipates cytochrome C.

Question 27

How does arsenate stimulate respiration?

Answer 27

1. Substitutes Pi in ATP synathase, forming ADP-As 2. Hydrolyses, allowing re-synthesis and thus dissipates PMF

Question 28

Where do different substrates feed into the ETC?

Answer 28

Malate + Glutamate: Complex I (NADH) Succinate: Complex II (FADH₂) Ascorbate + TMPD: Cytochrome c

Question 29

What did the 2 bands in the undigested plasmid correspond to?

Answer 29

1. Supercoiled plasmid (closer to well) 2. Relaxed plasmid

Question 30

What are the effects of adding bacteriostatic and bacteriolytic agent?

Answer 30

Bacteriolytic effects inhibited due to

Question 31

What are the reasons for not obtaining any PCR product during PCR experiment?

Answer 31

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

Question 32

What is the purpose of adding RNAse to preparation?

Answer 32

To break down RNA

Question 33

What is the purpose of adding EDTA to the preparation?

Answer 33

To cellate Mg²⁺ and prevent DNAse from breaking down plasmid DNA

Question 34

What is the purpose of adding SDS to the preparation?

Answer 34

Lyse bacterial cells open

Question 35

What is the purpose of adding NaOH to the preparation?

Answer 35

Denatures dsDNA

Question 36

What is the purpose of adding high [salt] solution to the preparation?

Answer 36

Precipitates out cellular debris and long genomic DNA

Question 37

What are the melting temperatures of primer pairs?

Answer 37

A/T = 2°C C/G = 4°C

Question 38

Why are primers with lots of CG pairs bad?

Answer 38

1. Induces formation of secondary DNA hair-pin structures 2. High annealing temperatures