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Practicals Flashcards

1
Q

What is an assay

A

An indirect method for quantifying a substance or activity of biochemical interest

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2
Q

Why is it known as the Bradford Assay

A

It uses the dye Coomassie Brilliant Blue, which was introduced by Marion Bradford

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3
Q

What are the colours associated with Coomassie Brilliant Blue

A

Green-brown when free in acid solution

Becomes blue when bound to a protein

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4
Q

What does LDH do

How can it’s activity be measured

A

Catalysed conversion of lactate to pyruvate using NAD as a H+ receptor

By following production of NADH

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5
Q

Which fixes conducting medium is used for electrophoresis of proteins

How is it prepared

A

A porous medium of aqueous polyacrylamide

Dissolving acrylamide and a cross linking agent

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6
Q

How do you vary the pore size of polyacrylamide gel

A

Modify the acrylamide monomer concentration during preparation

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7
Q

What happens to proteins in the presence of sodium dodecyl sulphate (SDS)

A

Proteins are completely unfolded

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8
Q

Why can inherent differences in charge due to amino acid side chains obscured in presence of SDS

A

Most proteins bind equal amounts of anionic dodecyl sulphate per gram So behave as polyanions with a constant ratio of negative charge to mass

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9
Q

Most proteins bind what amount of anionic dodecyl sulphate per gram

A

1.4g per gram of protein

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10
Q

Why can multimeric proteins be dissociated into their subunits by SDS

A

Disulphide bonds have been previously reduced by mercaptoethanol

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11
Q

During electrophoresis with polyacrylamide gel that contains SDS proteins move at speeds determined by what

A

The size of their SDS-protein complex

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12
Q

What is an isozyme

A

An enzyme that can exist in more than one molecular form that is usually tissue-specific

AKA isoenzyme

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13
Q

How many isoenzymes exist of LDH

Why

A

Five

because it has four sub units and can be encoded by 2 different genes (A and B) so 5 combinations of A and B are possible

ie. A4, A3B1, A2B2, A1B3, B4

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14
Q

How would you find the origin of an LDH isoenzyme in a diseased serum

A

Compare the activity forms in the gel with that of the LDH from a variety of rat tissue extract by subjecting them to non-denaturing PAGE

Stain the gel with a substrate mixture that turns purple in regions where LDH is found

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15
Q

Why is the Bradford assay not appropriate for diagnosis of a mild heart attack

A

It will not give specific results for the specific isozyme of LDH in the serum

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16
Q

What is an antibody comprised of

A

They are white shaped molecules, comprising four chains, two light and 2 heavy

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17
Q

Describe the light and heavy chain components of an antibody

A

Light: Contains one part of Ig domains

Heavy: Contains two pairs of Ig domains connected by a flexible linker

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18
Q

How are the light and heavy chains of an antibody linked together

A

By a disulphide bond

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19
Q

True or false:

Each arm of an antibody will bind to a different antigenic component

A

False

both find the same antigenic component

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20
Q

What produces antibodies

When are the antigen binding sites produced

A

β cells

During β cell maturation

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21
Q

Which three genes are used in a heavy chain locus

What about the light chain

A

Heavy:
V(variable)
D (diversity)
J (joining)

Light: only V and J

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22
Q

What is somatic recombination

A

Within each cell the V, D, J DNA sequences are randomly recombined during β cell maturation to generate a single V-D-J (or V-J) DNA sequence to form the variable arms of the antibody Y

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23
Q

What is an epitope

A

An antigenic determinant in a protein

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24
Q

What happens if a random beta-cell Happens to express an antibody that binds to an antigen of interest

A

The beta-cell will be stimulated to differentiate and multiply, producing a clone of beta cells, each secreting the same specific antibody against the antigen

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25
Q

Can more than one beta-cell be activated by an antigen

A

Yes

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26
Q

The blood from an immunised animal will generally contain a mixture of different antibodies to an antigen. Why is this and what is it called?

A

It is common for more than one beta-cell to be activated by an antigen

This mixture is known as a polyclonal antibody

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27
Q

How do you produce a mono monoclonal antibody

A

Beta cells must be removed from the spleen of an immunised animal and are fused with cells from a myeloma to produce hybrid cells that grow like cancer cells but produce large amounts of antibody

These antibodies are tested for antigen binding and the cell that produces the tightest binding antibody can then be selected and used to generate a cell line that manufactures large amounts of antibody.

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28
Q

Which are more expensive to produce:

Monoclonal antibodies or polyclonal antibodies

A

Monoclonal

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29
Q

True or false:

Monoclonal antibody is can be produced in unlimited quantities

A

True: parent hybridomas can grow indefinitely

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30
Q

What does ELISA stand for

A

Enzyme linked immunosorbent assay

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31
Q

How many wells are usually in the plastic plates used for ELISA tests

A

96 or 384

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32
Q

What do portable glucose testing kits use

How do they work

A

A disposable mini electrode that contains the fungal enzyme glucose oxidase and an electron carrier, ferrocene

When a sample is applied, any glucose present is oxidised by the enzyme but the enzyme is then diverted from its normal task of delivering the electrons to oxygen by ferrocene, which instead conduct the electrons to the mini electrode. This registers a current, proportional to the amount of glucose in a sample

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33
Q

How to work out P:O ratio in state 3

A

nmol of ADP added
——————————-
nmol of O consumed in state 3

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34
Q

What should be the P:O ratio be for

a) succinate
b) glutamate and malate

A

a) 1.5

b) 2.5

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35
Q

How can a theoretical P:O ratio be calculated

A

The number of protons injected during electron transport (H+:O)
—————————————-
The number of protons used to synthesise each ATP ( H+:P)

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36
Q

What is the number of protons pumped out of every oxygen atom reduced by a pair of electrons for NADH and succinate

A

NADH: 10
Succinate: 6

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37
Q

How do you compare the respiratory control ratio of a and B

A

Divide the gradient of a by the gradient of b

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38
Q

What are the values for the respiratory control ratios of the following:
A) glutamate + malate
B) succinate
C) ascorbate + TMPD

A

A) 3-8
B) 2-5
C) 1-2

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39
Q

Why might respiratory control ratios decrease overtime

A

The more mitochondria has been ill treated the more the membranes become leaky

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40
Q

In the mitochondria and metabolism practical, substrates with a higher proton:O ratio will have a ____ state 4 rate

A

Lower

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41
Q

What is DNP

A

An uncoupler - dinitrophenol

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42
Q

Describe an experiment to test the effects of inhibitors on mitochondria and metabolism

A 
Wash vessel with water and ethanol
Add medium and mitochondria and put lid on
Add glutamate and malate
Add excess of ADP
Add rotenone
Add succinate
Add antimycin 
Add ascorbate and TMPD 
Add cyanide

Give some time between each addition to record the change in O2 usage

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43
Q

Why are glutamate and malate added first to the mitochondrial mixture when testing the effects of inhibitors

A

This produces NADH for oxidation by complex one

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44
Q

what does rotenone do

So what will happen to O2 consumption after its addition to mitochondria

What is added after this? Why?

A

Inhibits complex 1

It will dramatically decrease

Succinate as it is a substrate for complex 2

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45
Q

What will happen to O2 consumption when succinate is added to mitochondrial mixture after rotenone is added?

A

Drastically increases

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46
Q

What is antimycin

A

Inhibitor of complex three

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47
Q

How are ascorbate and TMPD related to oxidative phosphorylation

A

This forms a non-physiological substrate complex IV

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48
Q

What happens to oxygen consumption of mitochondria if cyanide is added

What would you expect to happen

A

Consumption decreases but does not stop

This is because non-enzymatic auto oxidation of ascorbate occurs

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49
Q

What does cyanide do

A

Inhibits complex four by poisoning cytochrome oxidase

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50
Q

Difference between serum and plasma

A

Plasma contains all the clotting factors

Both contain no cells or platelets

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51
Q

How do you get rid of cells from blood

A

Centrifuge

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52
Q

Why may absorption not literally relate to protein concentration when using Coomassie blue dye

What must be used because of this

What cannot be done

Why is any value only an estimate

A

The binding of the die is more complex than a 1:1 stoichiometry between dye and protein

A standard curve

You cannot extrapolate be on the highest value

This die binds to arginine and arginine content per molecule can vary

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53
Q

Some asses cannot differentiate between different proteins why would these be useful in clinical practice

A

If you are interested in work there is protein in urine as an indicator of kidney function

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54
Q

What is the unit of Katal?

What is it used to show

A

Moles per second

Efficiency of enzyme

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55
Q

Why would one bother to convert the activity data to katals per mg protein

A

To present in standard form so you. An compare efficiency of 2 different enzymes found from different assays

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56
Q

How to calculate Mr from SDS PAGE

A

Find Rf (migration distance of protein/ migration distance of dye)

Plot graph of Rf (x) vs Mr (y)

Estimate Mr from calibration curve

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57
Q

Why might SDS page not be able to inform you of the true Mr of a native protein

A

Proteins composed of different subunits of different sizes give to bands and you do not know which ratios of the two bands comprise the protein

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58
Q

Give three ways that non-denaturing PAGE reveals different properties of proteins compared to SDS page

A

1) Preserves multimeric structure and S – S bonds
2) activity of protein is retained and activity can be revealed if there is an appropriate way of converting product of enzyme activity into a coloured substance
3) I’m It does not necessarily reveal the Ama of the multimer since separation is based partly but not only on size

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59
Q

Why can you not observe NADH absorbance in gel

A

One cannot use UV light through a gel and cannot place a detector under each band

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60
Q

Which is more specific,: Bradford assay or Eliza

A

ELISA

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61
Q

How can you measure ATP production

A

Luciferin

In the presence of ATP, oxygen and luciferase, Luciferin undergoes a multistep oxidative decarboxylation to oxyluciferin to produce light

Use a standard curve of the light emitted

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62
Q

Why would ATP decrease if DNP is added

A

Not only is ATP not produced but the current ATP is hydrolysed

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63
Q

What does oligomycin do

A

Inhibits ATP synthase but also inhibits hydrolysis of ATP by DNP

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64
Q

How does calcium affect mitochondria

How can this be monitored

A

High external concentrations of calcium result in calcium uptake, stimulating formation of the mitochondrial permeability transition core which causes swelling as salt and water enters and eventually swelling ruptures the outer membrane and release cytochrome c

Decreased mitochondrial light scattering and thus decreased absorbance

65
Q

What does cyclosporine do

What else has a similar effect

A

Blocks permeability transition pore (but Ca can overcome cyclosporine at high concentrations)

ATP also blocks pore

uncouplers

66
Q

How do uncouples prevent calcium induced pore formation in mitochondria

A

They eliminate the PMF and therefore prevent calcium uptake into the mitochondria which is normally driven by the membrane potential

67
Q

What will you see in the oxygen electrode when calcium is added to mitochondria

What happens if you add cytochrome c

A

First succinate must be added to stimulate respiration

There is an initial stimulation of respiration by calcium due to the increased membrane potential
Followed by an inhibition of respiration by high calcium due to loss of cytochrome c

Addition of XS cytochrome C then stimulates respiration because it is able to replace the lost cytochrome c

68
Q

What happens if you add XS cytochrome c before the addition of calcium to the mitochondria

A

Nothing

The cytochrome C cannot cross the intact outer membrane

69
Q

How does the addition of hexokinase affect the rate of respiration

A

Stimulates

It removes ATP and supplies ADP for ATP synthesis

The stimulation of ATP synthase allows protons back into the mitochondria and decreases the PMF, making the respiratory chain work faster

70
Q

do oligomycin and DNP have the same effect on O2 consumption

A

Oligomycin inhibits the ATP synthase by blocking protons through the synthase. This means the PMF increases back to state 4 level which inhibits respiratory chain and reduces oxygen consumption

DNP equilibrates H+ across the membrane, reducing PMF to 0, stimulating O2 consumption

71
Q

In a medium of mitochondria and glutamate and malate only why does ADP failed to stimulate respiration

What happens if you add arsenate after adding ADP?

A

There is no phosphate in the medium to act as a co-substrate for ATP synthesis

Respiration increases as arsenate can be a substitute for phosphate, allowing the formation of ADP – As (an analog of ATP)

72
Q

Why is the rate of respiration after arsenate is added faster than if you added phosphate to a medium of mitochondria, glutamate + malate and ADP?

A

ADP-As Readily hydrolyses in water, recycling ADP and allowing apparently uncoupled respiration

73
Q

You have a solution of mitochondria, glutamate+malate, ADP and arsenate.
ADP-As has been formed. What happens if you add phosphate?

A

Phosphate will outcompete arsenate

This does not happen immediately because the ADP has to be converted to ATP before state three will slow down state 4.

74
Q

How does DNP affect O2 consumption when added to a mitochondrial solution with ADP and arsenate

A

Would act immediately To block ADP-As synthesis so O2 consumption would rapidly increase

75
Q

Design an experiment to distinguish between a toxin that inhibits the respiratory chain, one that inhibits ATP formation or one that uncouples the mitochondria

A

1) Incubate mitochondria with glutamate and mallet then add ADP to start state 3
2) add mystery toxin
3) If it inhibits respiratory chain or ATP formation, oxygen consumption will be inhibited. An uncoupler will increase O2 consumption
4) if it is one of the former 2 responses, add an uncoupler (DNP)
5) If toxin is ATP formation inhibitor, uncoupling will stimulate rate again by removing PMF. If toxin is an respiratory chain inhibitor, uncouple it will have no effect on the rate as the respiratory chain is inhibited

76
Q

How would you verify a mystery toxin is an uncoupler in mystery toxin experiment

A

Add the mystery toxin in state 4 and see an increase in rate

77
Q

What is the respiratory control ratio (give the equation)

A

The rate of mitochondrial respiration in the presence of Pi and ADP (state 3)
—————————————————
Rate of respiration in the presence of Pi and ATP (state 4)

78
Q

What is state 3

A

The rate of respiration of mitochondria in the presence of Pi and ADP

(When the system is at full pelt)

79
Q

What is state 4

A

When all ADP is used up

80
Q

What are the units you need to know

A 
a= 10^-18
f= 10^-15
p=10^-12
n=10^-9
μ=10^-6
m=10^-3
k=10^3
M=10^6
G=10^9
T=10^12
P=10^15
E=10^18
81
Q

How to work out how often an enzyme will be expected to arise in a genome

What to be careful of

A

How many base you pairs is it? 4^x

Be careful of their being an unspecified base pair (this can be anything) so it would be 4^(x-1)

82
Q

Describe the method for alkaline lysis mini prep

Why is this done?

A
  1. Add SDS, RNase, and NaOH to bacteria
  2. Neutralise the substance
  3. Add buffer (K acetate/ acetic acid) - this causes cell debris and chromosomal DNA to precipitate while plasmid DNA reanneals and stays in solution
83
Q

Why are SDS, RNase and NaOH added to bacteria during alkaline lysis prep

A

SDS denatures lipid membranes

NaOH denatures dsDNA

RNase digests RNA

84
Q

What happens to the plasmid after it is purified by alkaline lysis

A

Digested with restriction enzymes and the DNA fragments are separated by electrophoresis

85
Q

What is used to replicate DNA in PCR

A

DNA polymerase

86
Q

Why is it better to use Taq in PCR than just adding new DNA polymerase after each heating

A

Adding DNA polymerase is expensive and tedious

87
Q

How did the primers in PCR replication differ from primers in Actual DNA replication in the cell

A

PCR- DNA primers used

Cell- RNA primers used

88
Q

U replaces which nuclear tide in RNA

Is U purine or pyramidine

A

T

Pyramidine

89
Q

Why are DNA sequences sometimes added to the end

Which end

A

So the amplify target sequence can be cleaned to an appropriate factor
this sequence is cleaved by restriction endonucleases

5’

90
Q

What does Chelex do

A

Binds metal ions that inhibit PCR

91
Q

Describe the method for PCR

A

Place sample in a solution containing Chelex

Lyse cells by boiling and centrifuge to remove cell debris

Mix with Taq pol, oligonucleotide primers, the 4 deoxy riboNTPs and MgCl2 (the Taq co factor)

Begin temperature cyclinh

92
Q

What is the best and quickest way to measure the size of a DNA molecule

How does this work

A

Gel electrophoresis

DNA is covered in negative charges so will migrate towards the positive electrode

Smaller fragments thread their weight through the sievelike gel matrix more rapidly than larger fragments

93
Q

Describe the method of DNA gel electrophoresis

A

The DNA samples are loaded into Wells at one end of the gel

The gel is then run by applying an electrical field

The dye, ethidium bromide, is added and slots in between DNA base pairs

94
Q

Why is GelRead better than ethidium bromide

A

Both fluoresce under UV but GelRead is: Less toxic

More sensitive

95
Q

Which wavelength do all nuclear acid bases absorb light strongly at

A

260nm

96
Q

What should the ratio of absorbance 260:280nm be

A

2

97
Q

Why would the growth of E. coli without antibiotics be slow in the first 60 minutes

What happens after this

Why does growth eventually stop

A

This is the lag phase as the bacteria adapt themselves to the growth conditions

After this they enter the exponential phase during which they double in number approximately once every 30 minutes

They have reached a gross something factor such as depletion of an essential nutrient and/or the formation of an inhibitory product such as an organic acid

98
Q

What is the effect of chloraphenicol on E. coli

A

It is a bacterial static antibiotic

It inhibits translation by preventing protein chain elongation by inhibiting peptidyl transferase activity of the bacterial ribosome

Hence absorbance of bacteria will not change as the bacteria as a present but just not growing

99
Q

Which enzymes promote chloraphenicol resistance

How does this work

A

CAT (Chloraphenicol acetyltransferase)

Add an acetyl group to the antibiotic, preventing it from binding to the bacterial ribosome

100
Q

Why does penicillin act different to chloraphenicol

A

It is a bacteriolytic agent

It is an irreversible inhibitor of transpeptidase preventing bacterial cell wall synthesis. It does lead to cell lysis.

101
Q

Why might absorbance of bacteria decrease slower when chloramphenicol and ampicillin are added together

A

The bacteriolytic effects of ampicillin I slowed down by chloramphenicol restricting translation and therefore the ability for the cells to divide

Thus cell lysis takes longer

102
Q

Why would a restriction enzyme with a recognition site of 5bp (GANTC) be useful when mapping plasmids of 3kbp

A

As N is unspecified, such sites will arise every 4^4 bp=256bp

Therefore this enzyme is not useful as it cuts too frequently

103
Q

How useful is a restriction enzyme with the recognition sequence of eight base pairs in mapping a plasmid of 5kbp

A

4^8 = every 65.5kbp

This is too high so is unlikely to have any restriction sites

104
Q

Why do bacteria restriction enzymes not cut their own DNA

A

Bacteria use methyltransferase to methylate A and C residues within recognition sites

105
Q

What is the target of DNA methyl transferases

A

Cytosine

106
Q

If you had a large plasmid or DNA fragment, what are the problems with this and what methods could you use to produce a restriction map(3)?

A

Suggestion by multiple restriction enzymes often generates too many bands

This can be circumvented by isolating the individual bands from a single digest and then digesting the bands with a second enzyme

Alternatively a southern blot of a multiple digest can be probed with individual radiolabel fragments from a single digest

Or

107
Q

If you perform n cycles of PCR how many double-stranded molecules are made

How many strands are what you want

A

2^n

(2^n)-2n

108
Q

What does dimorphic mean

A

Present in some people and not others

109
Q

Why do we obtain a 150 bp fragment of DNA using primers that are designed to amplify the Alu insert when there is no insert present?

A

The Primers did not immediately flank the insert but I separated in the gene by 150 base pairs

Otherwise the product would be too small to distinguish

110
Q

Suggest five reasons why a PCR experiment may fail

A
  1. The DNA quality and amount: you need DNA intact without any contaminants that might interfere with PCR
  2. Primer problems: Primers need to be at least 15 nucleotides along with a high enough percentage of GC content to increase melting temperature without creating problems of secondary structure
  3. Cycling parameters
  4. Buffer composition
  5. Type of thermostable polymerase
111
Q

What are important qualities a primer the PCR should contain (5)

A

At least 15 nucleotides long

High percentage of GC content

No repeat motifs

Should not self associate

Both primers should have the same annealing temperature

112
Q

What is important about the buffer composition during PCR

A

Needs to have access magnesium over dNTP as magnesium complexed with dNTPs is essential for dNTP incorporation

Mg2+ is also a cofactor first Taq and increases melting point of DNA

113
Q

What are the results of the following in PCR

1) too little Mg2+
2) high Mg2+
3) too high Mg2+

A

1) no product
2) more and more non specific bands
3) no PCR product

114
Q

Why might you get too many PCR products

How to fix it

A

Primers anneal to several sites on DNA so need to increase stringency

Increasing annealing temperature reduces primer binding to non-target sites

Optimise magnesium

115
Q

What does the hardy Weinberg equation assume (5)

A

In a large population, mating is random, there are no mutations that affect allele frequencies, no migration between populations, no selection, and all genotypes produce with equal success

116
Q

True or false

P 53 has a high concentration in the nucleus usually

A

False

Usually made on demand

117
Q

What balances the translation and destruction of P 53

How does it work

What happens if the cell is stressed

A

A negative feedback loop governed by MDM2 (a product of p53)

MDM2 binds to p53 to allow proteolytic activity

P 53 can be phosphorylated so MDM2 cannot find and P 53 concentration rapidly increases

118
Q

How is TP53 different to P53

A

TP53 is the gene

P53 is the product

119
Q

Dark Giemsa bands represent more condensed AT rich regions whereas light bands mark less condensed GC rich region. Which region contains more genes (3)

A

Heterochromatin stain more darkly then regions of euchromatin

Large sections of constitutive heterochromatin play structural roles for example forming contromeres and these reasons sequences tent be richer in AT nuclear tides

Deamination create a mutual bias that turn methylated C into T so there is a pressure to maintain GC content of gene rich regions

Gene rich regions are regularly transcribed and therefore usually located in euchromatin

120
Q

What do the colourings of Dark Giemsa bands mean?

A

Dark Giemsa bands represent more condensed AT rich regions whereas light bands mark less condensed GC rich region

121
Q

Give the different classes it chromosome morphology

A

Telocentric: no p arm, only q arms

Acrocentric: very short p arm, normal q

Submetacentric: short p - half way between acro and meta

Metacentric: p and q almost equal

122
Q

How to work out if a chromosome is gene rich or poor?

A 
# of coding genes 
————————— x 1 million 
# of base pairs

(X by 1 million to convert to Mb)
Then compare to rest of genome (5.7genes/Mb)

123
Q

What kind of process could cause blocks of a chromosome to appear in a different order or even swap orientation

A

Double-stranded breaks, after which the fragments have been re-assembled in a different order

This could occur because of NHEJ

124
Q

How would you know if a DNA break is paracentric

A

If the centromere isnt included

125
Q

What does synteny do

A

Compares the genome of one species to that of another

Green Lines on the diagrams will link conserved genes
Black lines connect blocks that are the same orientation
Brown lines rink regions that possess opposite orientations

126
Q

As far as synteny is concerned, which genome is most similar to human chromosome 17: chicken, chimpanzee, gorilla

A

All genetic material from chromosome 17 appears on a single chimpanzee chromosome, although much of it has been fragmented and reassembled

The gene order in human DNA is spread between two gorilla chromosomes but the blocks are not much altered

In the chicken the gene sequence is much more fragmented – it is shared between four chromosomes and has undergone multiple rearrangements

Gorilla>chimpanzee> chicken

127
Q

What are paralogs

A

Genes that share high similarity

Eg a-Hb and f-Hb (fetal and adult Hb paralogs)

128
Q

Give the key for the exon sequence you in bio Informatics

A

Empty box= 5’-3’ untranslated region
Filled box: exon
V shaped connecting line: intron

129
Q

What is the difference between a missense valiant and synonymous variant

A

Synonymous mutation is change the code on sequence but do not alter the amino acid

Missense mutations code for different amino acids

130
Q

What causes Li Fraumeni syndrome

What does this mean for inheritance

A

A single germline mutation in p53 So
It is autosomal dominant

CHEK2 on chromosome 22

131
Q

If there is a mutation in both p53, does it make a difference from 1

A

Yes

Different rumours arise

132
Q

What does CHEK2 also do

A

Phosphorylates P 53 to prevent MDM2 ubiquitin ligase binding, allowing [p53] to rise

133
Q

How does LFS change between genders

A

Penetrance by age 50 is higher in females than males

134
Q

Describe the DBD blue β sandwich

A

Four strands, all connected to the neighbour in an anti-parallel fashion

135
Q

Describe the DBD red β sandwich

A

Seven strands, mostly antiparallel, but two strands (two and three) have a parallel connection

136
Q

What feature Of the DNA target doesnt Arg-273 interact with

A

The positively charged side-chain of Arg makes favourable electrostatic contact with two negatively charged phosphate groups on the DNA backbone

137
Q

What kind of interaction is Arg 248 Involved in and what feature of DNA does it target

What is this called

A

The side chain of arginine appears to hydrogen bond with a water molecule located in the minor groove of the DNA target

Intern the water molecule forms a hydrogen bond with a specific A base on the P 53 sequence

Water mediated minor groove interaction

138
Q

Why does phosphorylation of Thr-18 weaken the interaction between P 53 and MDM2

A

Phosphorylation of the hydroxyl group in this Thr sidechain will introduce a larger group that this is more full negative charges. The interface between this portion of P 53 and MDM2 is mostly nonpolar so introducing such a large amount of negative charge is highly unfavourable

139
Q

Explain why pharmaceutical companies might be interested in developing nutlins as anti-cancer drugs

A

Cancers that still possess unmutated P 53 deactivate it by up regulating MDM2 or knocking out the MDM2 inhibitor

Nutlin drugs act as re-activators of wild type p53

140
Q

Why might completely knocking out MDM2 be negative

A

This may give gain-of-function P 53 mutants free reign resulting in tumour information

141
Q

Stretches of GC rich self complimentarity are more problematic for PCR than AT which regions. Why

A

GC rich dimer regions have high melting points and are therefore had to disrupt which would exacerbate problems due to incorrect annealing in a PCR experiment

142
Q

How do you do a southern blot

A

1) Take purified DNA and cleave it using DNase
2) gel electrophoresis
3) expose gel to a filter for a while and fragments will transfer
4) expose the filter to a radio labelled DNA strand that is complimentary to the desired strand
5) expose the filter to an x Ray

143
Q

What are southern, western and northern blots used for

A

Southern: DNA
Northern: RNA
Western: Protein

144
Q

What is the beer lambert law

A

Absorbance= molar absorption/ extinction coefficient x molar conc x optical path length

145
Q

In what ways does non-denaturing PAGE technique reveal different properties of proteins compared to the SDS PAGE technique and why? (3)

A

Preserves the multimeric structure of proteins and S-S bonds

Because the protein is native it allows the activity of the protein to be retained and this activity to be revealed if there is an appropriate way of converting the product of enzyme activity into coloured substance

However it does not necessarily reveal Mr of the multimer since separation is based partly but not only on size

146
Q

Describe method for ELISA

A

Coat plate with abundant amounts of antigen
Wash
Add new antibody at different concentrations
Incubate then wash
Add enzyme linked antibody that was raised against the original antibody
Add a substrate that will change colour when added to the enzyme

147
Q

How does affinity chromatography work

A

Column material contains a molecule that specifically binds to the protein of interest

When the sample is passed through the column and washed only the protein of interest will bind - other proteins are washed away

Your acquired protein can be a looted from the column by using competitive ligand

148
Q

Describe ion exchange chromatography

A

Use the column of charge material

Proteins bind to column with different degrees depending on their charge

Proteins are eluted with increasing salt which disrupts electrostatic interactions

Highly charged proteins will elute at high salt concentrations

149
Q

What does gel filtration chromatography assess

A

Size - smaller take longer

150
Q

Which method is used to asses how much protein

Which protein?

Quantifying levels of a specific protein?

A

Bradford assay

Gel electrophoresis (SDS PAGE)

ELISA

151
Q

Give the 5 gels and their use in MIMS

A

Agarose: separates DNA fragments according to size

Formaldehyde agarose gel: RNA denaturing agent during agarose gel electrophoresis. Formaldehyde inhibits RNase to maintain RNA integrity

SDS PAGE: gel electrophoresis- protein separation by mass, uses polyacrylamide gel

Native PAGE: separates acidic, water soluble and membrane proteins in polyacrylamide gradient gel. No charge used so proteins are separated by charge

8M Urea polyacrylamide denaturing gel: denatures 2nd DNA and RNA structures and is used for separation by weight in a polyacrylamide gel

152
Q

How to reach Katals from absorbance

A

Conc change per sec=

absorbance change per sec
—————————————-
Coefficient x path length

153
Q

How to work out specific activity of enzyme

A

Katals
———————
Protein amount

154
Q

What is PMF=?

A

ΔΨ - (2.303RT/F)Δph

155
Q

How many ATP produced for each of the following

a) 1 molecule of glucose
b) glucose via anaerobic glycolysis
c) glucose via aerobic glycolysis
d) 1 acetyl CoA

A

a) 30 (5 from glycolysis, 25 from TCA cycle (2 pyruvate per 1 glucose)
b) 2 ATP
c) 5 ATP (2 ATP for breakdown and 1 NADH per Glyceraldehyde 3 phosphate - but energy is lost getting NADH out of Mt so the 2NADH only =1.5 ATP each)
d) 10 (3NADH+ 1 ATP + 1 FADH2)

156
Q

Describe the method for Bradford assay

A

Pour acrylamide solution on separating gel. Allow to polymerise

Load samples and Mr markers into gel and apply current

157
Q

How do you match acrylamide concentration to protein

A

Using a higher acrylamide concentration produces a gel with a smaller mesh size suitable for the separation of small proteins.

158
Q

What may a band in PCR <100bp represent

Often v faint

A

Primer dimer
Primers that are not used up dimerise and ethidium bromide can intercalated - there is no PCR product corresponding to this band

MIMS (12 decks)

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