BS1030: Biochemistry > Practicals > Flashcards

Practicals Flashcards

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1
Q

Which tracks are:

  • sample cleaved with DNase
  • undigested sample
  • sample cleaved with Pvu II
  • size markers

and why?

A
  1. undigested sample because it hasn’t moved very far due to the large molecular mass
  2. sample cleaved with Pvu II because this endonuclease cleaves the DNA in two places becuse it is specific
  3. sample cleaved with DNase becuase this endonuclease is non-specific so cleaves the DNA in a number of places. No bands are present because the molecular weights of each fragment are so small
  4. size markers.
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2
Q

How can you calculate the length of a DNA fragment in base pairs?

A
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3
Q

What does the gradient of the Lineweaver-Burk plot represent?

A
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4
Q

What is the role of ethidium bromide in PAGE?

A

It acts as a fluroescent tag

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5
Q

Why is there a second peak at 410nm in the elution profile of MetHb and GFP?

A

It is the ‘foothills’ of GFP absorbance that will peak at 490nm, since GFP has some absorbance at 410nm

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6
Q

How do you calculate turnover number?

A
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7
Q

What is the specificity of micrococcal nuclease?

A

Micrococcal nucelase demonstrates 30-fold preference for cleavage to the 5’ side of A or T rather than at G or C.

It preferentially cuts linker DNA (i.e. stretches without nucleosomes) to protect nucleosomal DNA.

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8
Q

What does the x-intercept represent on a Lineweaver-Burk plot?

A
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9
Q

What is the role of micrococcal nuclease digestion in the separation of histones?

A

It digests insoluble chromatin into insoluble oligonucleosomes

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10
Q

How would you calculate the amount of MetHb applied to the column?

A
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11
Q

Name two factors which affect the rate at which proteins move through a polyacrylamide gel?

A
  1. Molecular weight
  2. Presence of charged side chains
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12
Q

Why is DNA a polyanion?

A

Its phosphate groups have a high pK

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13
Q

What determines whether ion-exchange materials are anionic or cationic?

A

The nature of the ion they attract

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14
Q

How can you tell the difference between a digested and undigested sample on a polyacrylamide gel?

A

Undigested DNA is represented by a sharp band

Digested DNA is represented by a smear on the gel

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15
Q

How would you calculate the amount of MetHb recovered from the column?

A
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16
Q

What is the structure of genomic DNA in eukaryotes?

A
  • Compacted into chromatin which contains a nucleosome
  • Nucleosome consits of eight positively charged, small proteins (histones) and a piece of DNA wrapped around the histone octamer
  • Histone octamer consits of two dimers of the proteins H2A and H2B and two dimers of H3 and H4
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17
Q

How is activity of beta-galactosidase measured?

A

By measuring ONP production from the hydrolysis of the artifical substrate ONPG

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18
Q

What is the first step when separating histone proteins?

A

Incubation and washing with a buffer to break the cell membranes and denature proteins not involved with histones

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19
Q

What is an endonuclease?

A

An enzyme that hydrolyses the phosphodiester backbone of DNA in interior positions

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20
Q

Which of the following methods can be used to elute proteins from an anionic exchange column?

a) Decrease the ionic strength of the mobile phase.
b) Increase the ionic strength of the mobile phase.
c) Increase the salt concentration of the mobile phase.
d) Decrease the pH of the mobile phase.

A
  • *b) Increase the ionic strength of the mobile phase.
    c) Increase the salt concentration of the mobile phase.
    d) Decrease the pH of the mobile phase.​**
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21
Q

How can you get protiens off affinity and ion-exchange columns?

A

By varying:

  • salt concentration in the buffer
  • the ionic strength of the buffer
  • the pH of the buffer.
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22
Q

Why do we carry out micrococcal nuclease digestion at 37ºC and in the presence of CaCl2?

A

37ºC is the optimal temperature for the action of the micrococcal nuclease enzyme, and CaCl2 provides the Ca2+ ions the enzyme requires

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23
Q

What effect does a competitive inhibitor have on Vmax and KM?

A

Vmax – no change

KM – increased

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24
Q

What is the Beer-Lambert Law equation, rearranged to find concentration?

A
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25
Name the techniques that can be used to determine the catalytic activity of an enzyme.
* Spectrophotometry * Isotopic labelling * Titration * Quantitative chemical analysis
26
What is the final concentration of an ortho-nitrophenol-β-galactoside (ONPG) solution if 0.2 ml of a 0.5 M solution is made up to a final volume of 3 ml?
**33.3 mM**
27
**Which one of these statements describing the separation of proteins by SDS-PAGE is correct?** ## Footnote a) The largest proteins migrate the furthest. b) Proteins migrate towards the positive electrode. c) Proteins migrate towards the positive or negative electrode, depending on their native charge. d) Proteins retain their structure and function. e) Proteins can be visualized by staining with the fluorescent dye ethidium bromide.
**b) Proteins migrate towards the positive electrode.**
28
**Put these steps in an enzyme assay in order:** 1. Measure absorbance values over time. 2. Mix the contents of the cuvette thoroughly. 3. Add the enzyme. 4. Add the buffer.
1. **Add the buffer.** 2. **Add the enzyme.** 3. **Mix the contents of the cuvette thorougly.** 4. **Measure absorbance values over time.**
29
Define assay
An analytic procedure for quantitatively measuring (or qualitatively assessing) the presence, amount, or functional activity of a target entity
30
What produces the observed colour when white light is passed through solutions containing coloured compounds?
Certain wavelengths are selectively absorbed and the resultant colour is due to transmitted light
31
What is AEBSF?
A water soluble serine protease inhibitor that inhibits the degradation of some proteins
32
What would be the concentration of a solution with 5.85 g NaCl dissolved in 1 litre of solution? (The molecular mass of NaCl is 58.5 g.)
**0.1 M (0.1 mole/litre)**
33
Define absorbance
The measure of the specific capacity of a substance to absorb light of a specified wavelength
34
What is the advantage of a Lineweaver-Burk plot over a Michaelis-Menten plot?
It is a linear plot so it is possible to determine Vmax and KM manually
35
Why are ion-exchange resins ideal for separating small molecules (e.g. amino acids) but not large ones (e.g. proteins)?
Larger molecules cannot penetrate the tightly linked structure of the resin
36
Why is salt precipitation a good first step for purification of a protein?
It is a cheap, bulk-friendly method
37
Why does the absorption spectrum of ONP have a peak at 410nm, and what property does this represent?
ONP absorbs light the most at this wavelength, meaning it reflects yellow light so appears yellow
38
Why does DNA migrate twoards the positively charged anode in an electric field?
Because it is a polyanion
39
Define elution
The removal of an adsorbed substance by washing with a solvent
40
What does KM effectively measure?
The affinity of an enzyme for the substrate
41
Name five factors which can affect the activity of an enzyme.
1. pH 2. Temperature 3. Salt concentration 4. Substrate concentration 5. Presence/absence of a specific molecule
42
Why is a computer required to determine the parameters (Vmax and KM) on a Michaelis-Menten plot?
We cannot accurately determine Vmax and KM from a Michaelis-Menten plot because true saturation is experimentally impossible
43
How does SDS allow proteins to be separated by mass?
It is a detergent that creates denaturing conditions that separate proteins by molecular weight, and coats them in a negative charge
44
What is a polyanion?
A molecule or chemical complex which has negative charges at several sites
45
What does the y-interecept represent on a Lineweaver-Burk plot?
46
What properties of a protein can be exploited to try to separate them?
* Size * Shape * Charge * Hydrophobicity * Affinity
47
Why is turkey blood used when purifying histones?
Turkey red blood cells have nuclei which contain histones, whereas mammalian erythrocytes do not
48
What is the equation for percentage yield?
49
What does beta-galactosidase do to ONPG?
Breaks down the glycosidic bond, resulting in ONP formation
50
What is Triton X-100?
A molecular detergent used to weaken cell membranes to lyse cells so that histones can be extracted
51
Why is micrococcal nuclease digestion carried out when purifying histone proteins?
It targets nucleic acids rather than histones
52
What is the role of EDTA in stop solution?
It chelates Mg2+, which prevents DNase enzymes from working, so DNA and RNA are not degraded
53
Why does the stop solution contain: 1. glycerol 2. bromophenol blue 3. EDTA?
1. to provide enough density to aid the loading into the slots on the gel 2. negatively charged and allows the progress of the reaction to be followed (it is fluorescent) 3. chelates Mg2+ to prevent further endonuclease activity.
54
What are the limitations of a Lineweaver-Burk plot?
* It places undue emphasis on data obtained at low substrate concentrations * Random errors in rate are no longer symmetrical about the mean when reciprocals are taken
55
Why is ONPG colourless in solution?
ONPG doesn't absorb much light in the visible spectrum (400-700nm)
56
Why are measurements of activity extrapolated back to zero?
The concentration of a substrate influences the rate of reaction and the concentration is known at zero. The initial rate can be calculated
57
Which technique is used to separate histone proteins?
Polyacrylamide gel electrophoreseis (PAGE)
58
What are the advantages of spectrophotometric techniques?
* Reactions can be followed continuously * Rapid * Measurable * Measurements can be made at low concentrations
59
When should you disconnect the electrode from the power when running PAGE?
When the dye has moved 2/3 of the way along the polyacrylamide gel
60
Why is DEAE-cellulose used for separating proteins?
* DEAE-C has a pK of 9.0 and a positive charge * MetHb has a pI of 8.73 and a positive charge * GFP has a pI of 5.8 and a negative charge * GFP will therefore bind to the DEAE and the MetHb will not – it will pass straight through
61
What effect does a non-competitive inhibitor have on Vmax and KM?
Vmax – reduced KM – no change
62
What is the Michaelis-Menten equation?
BS1030: Biochemistry
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