Practical Paper Flashcards
(136 cards)
1
Q
What does a Western blot detect?
A
A specific protein
2
Q
What does a Northern blot detect?
A
Analyse expression of specific RNAs
3
Q
What does a Southern blot detect?
A
Specific DNA sequences
4
Q
What does a Bradford assay measure?
A
Protein concentration
5
Q
What assay would you use to detect a specific protein in a mixture of proteins?
A
Western blot
6
Q
What assay would you use to analyse the expression of specific RNAs?
A
Northern blot
7
Q
What assay would you use to detect specific DNA?
A
Southern blot
8
Q
What assay would you use to measure protein concentration?
A
Bradford Assay
9
Q
What would improve your chances of getting the desired clone either with or without an insert?
A
Blue-White Selection
10
Q
What enables identification of a plasmid with an insert?
A
Blue-White Selection
11
Q
What is cDNA?
A
Complementary DNA
12
Q
What is a cDNA library?
A
Collection of cloned cDNA fragments
13
Q
What is ChIP?
A
Chromatin Immunoprecipitation
14
Q
What does ChIP measure?
A
Interaction between DNA and particular protein
15
Q
What can ChIP determine?
A
If specific proteins are associated with specific genomic regions
16
Q
What is immunofluorescence used for?
A
Protein tagging
17
Q
What does qPCR allow?
A
Quantification of PCR products in real time
18
Q
What is used to amplify a piece of DNA?
A
PCR
19
Q
What can FISH be used to detect and localise?
A
Presence or absence of specific DNA sequences on chromosomes
Specific RNA targets in cells
20
Q
What technique can be used to detect and localise specific RNA targets in cells?
A
FISH (Fluorescence in situ hybridisation)
21
Q
What technique can be used to detect and localise the presence or absence of specific DNA sequences on chromosomes?
A
FISH (Fluorescence in situ hybridisation)
22
Q
What electrophoresis gel is used to separate DNA?
A
Agarose gel
23
Q
What electrophoresis gel is used to separate RNA?
A
Agarose gel electrophoresis
24
Q
What electrophoresis gel is used to separate biological molecules (such as proteins)?
A
SDS-Polyacrylamide gel electrophoresis (SDS-PAGE)
25
What molecules move fastest during agarose gel electrophoresis?
Small molecules
26
How do you plot data with a large dynamic range?
Use log
27
What assay would you use to quantify expressed mRNA levels for a particular gene?
Northern blot
28
What is the Hardy-Weinberg equation?
p^2 + 2pq + q^2 = 1
29
What section of a standard curve will have the most accurate range of standard concentrations?
The most linear section of the curve
30
Why would you plot a logarithmic graph?
To avoid giving any 1 point too much weight
31
What sort of curve is the haemoglobin oxygen saturation curve?
Sigmoidal
32
What does the sigmoidal shape of the haemoglobin oxygen saturation curve show?
Positive cooperativity
Steep portion of curve is moved to physiologically useful range
33
What is often used as a denaturing agent for RNA agarose gel electrophoresis?
Formaldehyde
34
What is the value of V0 when Km = [S] ?
1/2 Vmax
35
When is V0 = 1/2 Vmax ?
When Km = [S]
36
If you plot the graph y=mx+c where y=1/V0 and x=1/[S], what is the y intercept?
1/Vmax
37
If you plot the graph y=mx+c where y=1/V0 and x=1/[S], what is the x intercept?
-1/Km
38
What P value is considered statistically significant?
P < 0.05
39
What sort of inheritance is this?
Autosomal dominant
40
What sort of inheritance is this?
Autosomal recessive
41
What sort of inheritance is this?
Mitochondrial
42
What sort of inheritance is this?
X-linked dominant
43
Which primer is complementary to the coding strand?
Reverse primer
44
What primer is the same as the coding strand?
Forward primer
45
Why might there be no bands on a PCR?
DNA quality
DNA quantity
Primer design
Annealing temperature
Buffer composition
Type of thermostable polymerase
46
Why might a PCR have too many bands?
Low stringency
Primer not unique to intended sequence
47
What is used to separate DNA?
Electrophoresis
48
What does real time PCR allow?
Determination of initial number of copies of template DNA with accuracy and high sensitivity over a wide dynamic range
49
Is DNA charged?
Yes, negatively charged due to phosphate backbone
50
Which electrode does DNA migrate towards during electrophoresis?
Positive electrode
51
What does ethidium bromide bind to?
Double stranded DNA
52
What dye is used in a Bradford assay?
Coomassie blue
53
When is coomassie blue dye blue?
When bound to protein
54
When is coomassie blue dye green/brown?
When free in acid solution
55
What is the absorbance peak of NAD+?
260nm only
56
What are the absorbance peaks of NADH?
260nm
340nm
57
What is the absorbance of DNA?
260nm
58
What is the absorbance of amino acids?
280nm
59
What is alkaline lysis used for?
Purifying plasmids
60
What is absorbance equal to?
Absorbance = log (Io/I)
Io = incident light
I = transmitted light
61
What is moles/second equal to?
Katals
62
What is the beer lambert law?
Absorption = molar absorption coefficient x concentration x length of cuvette
63
How does supercoiling effect the movement of DNA in a southern blot?
Supercoiled DNA is more compact so runs further
64
What do restriction endonucleases do?
Make double stranded breaks in DNA
65
Compare the Bradford assay to the Elisa test
Elisa measurements are more sensitive and can detect in a more dilute environment
Eliza is highly specific
66
Compare the ELISA test to western blot
Western blot often used to confirm ELISA as more definite result
WB can get more information such as size and abundance compared to ELISA yes or no
ELISA is quicker and cheaper than WB
67
How do you measure the amount of dye bound to protein?
Using a spectrophotometer
68
How do you measure the amount of LDH (lactate dehydrogenase) in a sample?
Running enzyme assay. NADH absorbs light at 340nm but as it's converted to NAD+ that will decrease as NAD+ absorbs light at 260nm
69
What is the reaction equation that LDH catalyzes?
Pyruvate + NADH -> Lactate + NAD+
70
How many genes code for LDH?
2
71
What complex does cyanide inhibit?
Complex IV
72
What complex does antimycin inhibit?
Complex III
73
What does oligomycin do?
Blocks F0 so H+ can't travel through it and stops ATP production
74
What does cyclosporin A do?
Blocks the permeability transition pore so blocks calcium induced mitochondrial swelling
75
Why could an insulinoma explain low blood sugar?
Can cause excess insulin secretion
76
Suggest a suitable assay that could be used to measure total insulin concentration
ELISA assay with antibody to insulin on plastic, and enzyme-tagged second antibody to insulin (different epitope to solid phase antibody) as solution antibody
77
What is insulin C peptide?
C-peptide is generated by cleavage of insulin during processing in islet beta cells. It is
stored with mature insulin in granules and secreted along with insulin
78
Why would insulin C peptide be measured?
Its secretion along with mature insulin confirms normal processing and secretion of insulin from islet cells
79
If insulin and C peptide are released together, why would C peptide levels decline faster?
C peptide is removed faster as it is processed slightly differently
80
In an experiment to study insulin activity, what would be the significance of insulin receptor phosphorylation?
Phosphorylation indicates insulin-activated receptor leading to insulin stimulating other processes ie glucose uptake, glycogen synthesis
81
To conduct a western blot detection of Glut4 in the plasma membrane of adipocytes, why would you use a buffer not containing detergent?
So that the Glut 4 remained in the plasma membrane and (assuming the plasma
membrane could be separated from rest of the cell), the plasma membrane-associated Glut 4 could be measured
82
If kidney tubular cells were intubated in the cold to inhibit membrane-trafficking reactions but not simple protein-protein binding interactions, where in the cell is most insulin likely to be located?
On the plasma membrane bound to an insulin receptor
83
Kidney tubular cells were incubated at 37 degrees and insulin levels decreased with time. Suggest how the insulin could be degraded
Endocytosis moving insulin into endosomes and finally lysosomes for degradation
Ubiquitnation of insulin-bound receptor could lead to degradation by proteasome
84
How could you estimate the penetrance of an incomplete dominant trait?
(Individuals exhibiting the phenotype) / (individuals who are homo- + hetero- zgyous for gene)
85
What do the results of the disk diffusion test tell about the resistance of the P. aeruginosa bacteria from different individuals to the four antibiotics?
Individual one is only resistant to Meropem
Individual two is resistant to Gentamicin and partially to Carbenicillin
Individual three responds only to Ciproflaxacin
86
What's the difference between bacteriocidal and bacteriostatic?
Bacteriocidal kills bacteria, bacteriostatic prevents growth and reproduction but doesn't necessarily kill existing bacteria
87
How would you know an antibiotic was a bacteriocidal agent?
If the optical density of the bacteria cultures decreases after the antibiotic is added
88
For the analysis of β-lactamase activity, a sample with 25 µg of protein was used in the assay. Given the protein concentration, how would you calculate the volume?
25 µg / protein concentration
89
What are the likely reasons for the Ser to Leu mutation in DNA gyrase to cause resistance to ciprofloxacin?
Lose potential H-bond or polar interaction from Ser
Leu is a larger residue and would result in steric block of the drug binding site
90
Why would you use 3 different dilutions of the same sample for an ELISA test?
To accommodate the fact that undiluted serum will yield luminescent signal values outside the linear range
91
When testing for HIV, what is the difference in the information obtained from an ELISA assay and a Western Blot?
ELISA measures the amount of antibodies the host has raised against the virus, WB measures the amount of HIV proteins in serum
92
Why is the western blot assay a more definitive assay than the ELISA for determining HIV infection?
The western blot assay is a direct assay, measuring for the presence of the virus.
The ELISA is an indirect assay, measuring antibodies reactive towards antigens found in the virus.
The western blot is more sensitive.
93
In a genome-wide association study to identify SNPs with significant association to Alzheimer's, what are two possible explanations for if there are multiple SNPs just below the genome-wide significance threshold?
Random statistical noise
SNPs that in a larger sample might reach significance, as due to small effect size or in a subset of individuals
94
What are the Hardy-Weinberg equations?
p^2+q^2+2pq=1
p+q=1
95
How does the saturation curve for myoglobin differ from haemoglobin and why?
Myoglobin is a monomer so no cooperativity, simple saturation kinetics
96
Where is hydroxyproline found?
Only in collagen
97
Cathepsin K is an enzyme that resides in the lysosome. Why would an assay for cathepsin K be carried out at pH 5.5?
Lysosome is acidic so pH 5.5 likely to be cathepsin K optimum temperature
98
What molecules move furthest down the column in gel filtration chromatography?
Large molecules
99
What complex does rotenone inhibit?
Complex I
100
How can swelling of the mitochondria can be measured by following their absorbance in a
spectrophotometer?
As mitochondria swell, they scatter less light, and thus their apparent absorbance decreases
101
What is specific enzyme activity?
The number of enzyme units per ml divided by the concentration of protein in mg/ml
102
For a Western Blot of cathepsin K, why would a western blot for transferase be included?
Loading control
103
Suggest possible reasons for why the cell lyase blot on a Western Blot for a serum derived from tumour cells contain a second minor band above the main band
Mutation to remove ER targeting signal
Phosphorylation
Glycosylation
Other post translational modifications possible
104
How many moles of solute does 1 litre of a 1 M solution contain?
1 mol
105
How many moles of solute does 1 ml of a 1 M solution contain?
1 mmol
106
How many moles of solute does 1 ml of a 1 mM solution contain?
1 µmol
107
How many moles of solute does 1 ml of a 1 µM solution contain?
1 nmol
108
What is M?
Molar = moles per litre
109
How do you calculate pH?
pH = – log10[H+]
110
Are acids proton donors or acceptors?
Proton donors
111
Are bases proton donors or acceptors?
Proton acceptors
112
What is pK?
The pH at which there are equal concentrations of the acid and its conjugate
base, in other words the acid is 50% dissociated
113
Will a strong acid have a low or high pK value?
Low
114
How can you calculate the concentration of NADH through spectrophotometry?
Absorbance = log(incident light/transmitted light) = molar absorption coefficient x length of cuvette x CONCENTRATION
115
How does DNP uncouple?
By acting as a protonophore
116
What gel would you use for the analysis of PCR products?
Agarose
117
What gel would you use for separation of RNA for Northern Blotting?
Formaldehyde Agarose gel
118
What gel would you use for analysis of proteins in a cell lysate?
SDS-PAGE
119
What gel would you use in a gel shift experiment to analyse complex formation?
Native-PAGE
120
What gel would you use for analysis of protein dimerization?
Native-PAGE
121
How can theoretical ratios for the P:O ratio be calculated?
122
How is the respiratory control ratio (RCR) calculated?
By dividing the state 3 rate of respiration by
the state 4 rate
123
How is the P:O ratio calculated experimentally?
By dividing the amount of ADP (nmol ADP) added, by the amount of oxygen (nmol O) consumed in state 3
124
How does oligomycin impact ATP synthase?
Inactivates it by blocking proton flow through the synthase
125
What is a zymogen?
An inactive precursor of an enzyme
126
Identify X, Y, Z
X- Phosphoenolpyruvate
Y- Pyruvate
Z- Lactate
127
Why would an empty plasmid be transfected in a western blot?
Empty vector control
128
What are the stages of mitosis?
Prophase
Metaphase
Anaphase
Telophase
Cytokinesis
129
How does histone acetylation affect gene expression?
Normally increases it
130
What amino acid is most positively charged?
Arginine
131
What amino acids have positively charged side chains?
Arginine
Lysine
Histidine
132
What amino acids have negatively charged side chains?
Aspartic acid
Glutamic acid
133
What amino acid can't form hydrogen bonds in proteins?
Proline
134
What is the smallest amino acid?
Glycine
135
What's the difference between serum and plasma?
Plasma = serum + clotting factors
136
What happens at the different temperature stages of PCR?
94 degrees - DNA melted
58 degrees - Primers annealed
72 degrees - DNA synthesised
