Bio lab exam 2 Flashcards
1
Q
what is an enzyme?
A
an organic molecule (usually a protein) that speeds up a chemical reaction
2
Q
What does an enzyme act as?
A
a catalyst
3
Q
what would happen if a chemical reaction didn’t have an ezyme?
A
it would happen very slowly
4
Q
what is a catalyst?
A
a molecule that speeds up a chemical reaction but is not permanently altered by the reaction
5
Q
how does an enzyme catalyze a reaction?
A
the enzyme binds to one or more of the reactant molecules
6
Q
what are the substrates of an enzyme?
A
the reactant molecules that the enzyme binds to
7
Q
what is the active site of an enzyme?
A
the part of an enzymes shape that comes in contact with the substrate
8
Q
what can effect an enzymes activity?
A
temperature, pH, substrate concentration
9
Q
how does temperature effect an enzymes activity?
A
enzymes denature at a high temperature, but at warm temperatures an enzymes activity is higher
10
Q
when a substrates concentration is higher what happens to its activity?
A
it gets higher
11
Q
what happens when an enzyme reaches saturation?
A
any additional increase in substrate concentration will not increase enzyme activity
11
Q
what does it mean when an enzyme is saturated?
A
all available enzyme active sites are occupied and working at maximum efficency
11
Q
what is Vmax?
A
when an enzyme is at maximum activity
12
Q
what usually ends in “ase”?
A
enzyme
13
Q
A
14
Q
Would an enzymes activity be higher at 0°C or 25°C?
A
25°C, because the random movement would be higher, but the temperature wouldn’t be high enough to denature the protein
15
Q
What enzyme did you measure the activity for in lab six?
A
Glucose oxidase
16
Q
In lab six, you measured the absorbency of the pink dye, what does the rate of increase in the absorbency mean?
A
Glucose oxidase enzyme activity
17
Q
What is the formula for a serial dilution?
A
DF = V1+ V2 / V1
18
Q
How do you find the dilution factor?
A
Divide all of the concentrations by the next concentration they should all equal the same number
19
Q
How do you find V2?
A
The final volume
20
Q
When making a serial dilution what are the steps?
A
- Transfer V2 of water to each of the cuvettes
- Transfer V1 of the stock solution to the first Cuvette
- Transfer V1 from the first Cuvette to the second Cuvette
- Repeat
- Discard V1 from the last Cuvette
21
Q
What is chromatography?
A
A technique used to separate molecules that are in a mixture
22
Q
What type of chromatography did we use in lab seven?
A
Thin layer chromatography ((TLC))
23
What did we use thin layer chromatography for in lab seven?
To separate photosynthetic pigment molecules from an extract of spinach leaves
24
When using thin layer chromatography, the pigments separate from each other according to what?
Charges or lack of charges
25
What are the two phases in a TLC set up?
The stationary phase and the mobile phase
26
What is the stationary phase?
The coating of silica on the TLC plate, which is a rectangular strip of plastic
27
What charge does silica have?
Polar
28
What is the location on the plate where the line of extract is applied called? (TLC)
The origin
29
What is the mobile phase?
A mixture of two nonpolar organic solvents: petroleum ether and acetone
30
What is the developing chamber? (TLC)
A sealed glass jar that contains the solvent mixture
31
What does the developing chamber prevent? (TLC)
Evaporation
32
What molecules are attracted to the silica coating? (TLC)
Charged pigment molecules
33
Why are charged pigment molecules attracted to the silica coating?
Because the silica coating is polar
34
What molecules are attracted to the mobile phase?
Uncharged molecules
35
Why are uncharged molecules attracted to the mobile phase?
Because the mobile phase is nonpolar
36
What molecules will migrate slowly? (TLC)
Charged pigments
37
What molecules will migrate faster? (TLC)
Uncharged pigments
38
What is the solvent front? (TLC)
The leading edge of the solvent
39
When is the TLC plate removed from the developing chamber?
When the solvent front is near the opposite end of the TLC plate
40
What are some common problems that can occur in TLC?
At the start of TLC spinach extract dissolves into solvent, at the end of TLC pigment bands on the TLC plate appear wavy or curved instead of straight, at the end of TLC, all pigment bands are faint, at the end of TLC pigment bands are very broad and overlap each other
41
What could cause the spinach extract dissolving into the solvent at the start of TLC?
There could be either too much solvent in the developing chamber or the spinach extract was applied to low on the TLC plate
42
How can you prevent spinach extract dissolving into the solvent at the start of TLC?
Apply the extract at a location that will be above the level of the solvent in the chamber
43
How can you prevent pigment bands on the TLC plate, appearing wavy or curved instead of straight?
Wear gloves when handling the TLC plate, apply the extract in as straight of a horizontal line as possible, use a light touch when applying the extract, after inserting the TLC plate into the developing chamber close the lid of the chamber so that it is tightly sealed
44
What could be the cause of all pigment bands, being faint at the end of TLC?
Two little spinach extract was originally applied to the TLC plate
45
How can you prevent all pigment bands, being faint at the end of TLC?
Make sure the line of extract at the origin is densely colored (apply several layers of extract)
46
How can you prevent pigment bands, being very broad and overlapping each other at the end of TLC?
When applying the spinach extract allow each layer to air for a few seconds before applying the next layer
47
What is RF? (TLC)
The migration of a pigment band along the TLC plate relative to the migration of the mobile phase
48
How do you calculate RF?
Distance from origin to pigment band divided by distance from origin to solvent front
49
What unit is RF?
RF has no units
50
What does an absorption spectrum measure?
Measures the relative amount of light, a molecule absorbs within a range of light wavelengths
51
What is an absorption maximum?
A major peak in the spectrum
52
What is the unit of wavelength?
Nanometers (nm)
53
In TLC what is the gray pigment called?
Pheophytin
54
In TLC what is the red pigment called?
Anthocyanin
55
In TLC what is the orange pigment called?
Carotene
56
In TLC what is the yellow pigment called?
Xanthophyll
57
In TLC what is the blue green pigment called?
Chlorophyll a
58
In TLC what is the yellow green pigment called?
Chlorophyll b
59
What does GFP stand for?
Green fluorescent protein
60
What is the structure of a functional fluorescent GFP molecule?
A protein quaternary structure
61
What is a GFP’s quaternary structure consist of?
Two GFP proteins bonded together as a dimer
62
Each GFP protein in the dimer is called a what?
Monomer
63
What holds together the two GFP monomers?
Interactions between beta pleated sheets on the outside of their cylindrical shapes
64
Functional BFP is a dimer consisting of what?
Two BFP proteins
65
What is size exclusion chromatography?
A techniques often used to isolate specific molecules from a molecule mixture
66
What is the point of using a size exclusion column?
To purify GFP and BFP from protein mixtures
67
What is the point of size exclusion chromatography?
To separate proteins by size, therefore, allowing GFP and BFP, to be purified from their protein mixtures
68
What is the column matrix?
Small spherical polysaccharide based suffix beads that are suspended in an aqueous buffer
69
What size protein will likely elude from the column first? (size exclusion chromatography)
Large
70
What is electrophoresis?
the process of placing molecules in a porous matrix, such as a gel, and applying an electrical current to move them through the matrix.
71
Why is electrophoresis done?
to separate molecules according to their size, shape, and charge
72
What is polyacrylamide?
a porous matrix consisting of cross-linked molecules of acrylamide and bis-acrylamide.
73
higher concentration of cross linked molecules will result in a gel with what sized pores?
small
74
lower concentration of cross linked molecules will result in a gel with what sized pores?
large
75
what is protein size measured by?
molecular weight - Daltons (Da)
76
In lab 8 day 2 you used a premade polyacrylamide gel to separate proteins solely by what?
their size
77
what is a native protein?
a protein that retains its natural shape
78
in electrophoresis, what can interfere with a proteins ability to migrate solely by size?
charges and native shapes
79
what is a protein denaturing solution used for?
to dismantle protein shapes and ensure that all proteins have a net negative charge
80
what is the protein denaturing solution made out of?
denaturing reagents, glycerol, tracking dye
81
What is beta-mercaptoethanol?
a denaturing reagent that breaks disulfide bonds, a type of covalent bond found in many protein tertiary structures
82
what is sodium dodecyl sulfate?
a negatively charged detergent that disrupts weaker bonds contributing to a proteins native shape, it also coats proteins to ensure they all have a negative charge
83
what is glycerol?
a molecule that increases the density of solutions like protein samples so they stay where you load them into the gel
84
what is tracking dye?
a blue negatively charged dye of low molecular weight that will migrate quickly through the gel during electrophoresis
85
what is the point of tracking dye?
the tracking dye migrates faster than the colorless proteins in your samples, so its location will help you determine when to stop the electrophoresis
86
why are protein samples heated to a high temperature?
to eliminate any remaining interactions holding together a proteins native shape
87
what happens if a protein is not treated with a denaturing solution and heated to a high temperature?
proteins will retain their native shape
88
what is a protein marker?
a commercially prepared protein mixture that acts as a standard - the proteins in this mixture have known molecular weights
89
what end of the of the gel is negatively charged?
the cathode end (well end)
90
which end is the cathode end?
the well end (top)
91
what end of the gel is positively charged?
anode end (bottom)
92
what speed will large proteins migrate in electrophoresis?
slow
93
what speed will small proteins migrate in electrophoresis?
fast
94
after getting the value out of your equation what do you have to do to it to get the molecular weight
antilog (2nd log)
95
what is a GMO?
a genetically modified organism - an organism with DNA that has been deliberately altered
96
what is the transgenic organism?
the resulting GMO when genes are transferred from one organism to another
97
what is the polymerase chain reaction (PCR)?
a molecular biology technique used to clone or make identical copies of a specific DNA sequence
98
Where does PCR occur in rather than in a living cell?
a tube
99
PCR copies what instead of all of the DNA in a cell?
a specific target DNA sequence
100
PCR uses what instead of different proteins to carry out most steps of DNA replication?
different temperatures
101
what enzyme copies the DNA sequence of interest in PCR?
DNA polymerase
102
the primers in PCR are what instead of RNA?
single stranded DNA
103
what is template DNA?
a sample of DNA which will be the source of the target sequence
104
what are primers?
short sequences of single stranded DNA that are designed so they will flank the target sequence that is to be copied
105
What does DNA polymerase do?
binds to the primers and extend them to copy the target DNA sequence
106
what are DNA nucleotides?
a supply of DNA monomers that DNA polymerase will link together to make the copies of the target sequence
107
What are the names of the DNA nucleotides?
deoxyadenosine triphosphate (dATP), deoxycytosine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxythymidine triphosphate (dTTP)
108
what does the buffer do?
provides the ideal PH and salt concentration for PCR, the buffer also contains magnesium ions (Mg^2+)
109
what are the 3 steps to the PCR cycle?
denaturing, annealing, extension
110
what is the denaturing step?
the reaction is heated to a high temperature to break apart the base pairs holding together the nucleotide strands of the double-stranded template DNA
111
what temperature does the denaturing step occur at?
94 degrees celsius
112
what is the annealing step?
the reaction is cooled to a lower temperature so the primers will form base pairs with each single stranded template DNA
113
what temperature does the annealing step occur at?
59 degrees celsius
114
what is the extension step?
the step where the target sequence is replicated. the temperature is raised to the ideal temperature for DNA polymerase to bind to the annealed primers, "read" the single stranded template DNA, and copy the target sequence by making a complimentary strand, adding free DNA nucleotides to "extend" the primer
115
what temperature does the extension step occur at?
72 degrees celsius
116
how many cycles does a PCR reaction take to complete?
30-40
117
what is agarose?
a polysaccharide purified from seaweed
118
what charge do nucleic acids have?
negative charge
119
