Bio lab exam 2 Flashcards
what is an enzyme?
an organic molecule (usually a protein) that speeds up a chemical reaction
What does an enzyme act as?
a catalyst
what would happen if a chemical reaction didn’t have an ezyme?
it would happen very slowly
what is a catalyst?
a molecule that speeds up a chemical reaction but is not permanently altered by the reaction
how does an enzyme catalyze a reaction?
the enzyme binds to one or more of the reactant molecules
what are the substrates of an enzyme?
the reactant molecules that the enzyme binds to
what is the active site of an enzyme?
the part of an enzymes shape that comes in contact with the substrate
what can effect an enzymes activity?
temperature, pH, substrate concentration
how does temperature effect an enzymes activity?
enzymes denature at a high temperature, but at warm temperatures an enzymes activity is higher
when a substrates concentration is higher what happens to its activity?
it gets higher
what happens when an enzyme reaches saturation?
any additional increase in substrate concentration will not increase enzyme activity
what does it mean when an enzyme is saturated?
all available enzyme active sites are occupied and working at maximum efficency
what is Vmax?
when an enzyme is at maximum activity
what usually ends in “ase”?
enzyme
Would an enzymes activity be higher at 0°C or 25°C?
25°C, because the random movement would be higher, but the temperature wouldn’t be high enough to denature the protein
What enzyme did you measure the activity for in lab six?
Glucose oxidase
In lab six, you measured the absorbency of the pink dye, what does the rate of increase in the absorbency mean?
Glucose oxidase enzyme activity
What is the formula for a serial dilution?
DF = V1+ V2 / V1
How do you find the dilution factor?
Divide all of the concentrations by the next concentration they should all equal the same number
How do you find V2?
The final volume
When making a serial dilution what are the steps?
- 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
What is chromatography?
A technique used to separate molecules that are in a mixture
What type of chromatography did we use in lab seven?
Thin layer chromatography ((TLC))
What did we use thin layer chromatography for in lab seven?
To separate photosynthetic pigment molecules from an extract of spinach leaves
When using thin layer chromatography, the pigments separate from each other according to what?
Charges or lack of charges
What are the two phases in a TLC set up?
The stationary phase and the mobile phase
What is the stationary phase?
The coating of silica on the TLC plate, which is a rectangular strip of plastic
What charge does silica have?
Polar
What is the location on the plate where the line of extract is applied called? (TLC)
The origin
What is the mobile phase?
A mixture of two nonpolar organic solvents: petroleum ether and acetone
What is the developing chamber? (TLC)
A sealed glass jar that contains the solvent mixture
What does the developing chamber prevent? (TLC)
Evaporation
What molecules are attracted to the silica coating? (TLC)
Charged pigment molecules
Why are charged pigment molecules attracted to the silica coating?
Because the silica coating is polar
What molecules are attracted to the mobile phase?
Uncharged molecules
Why are uncharged molecules attracted to the mobile phase?
Because the mobile phase is nonpolar
What molecules will migrate slowly? (TLC)
Charged pigments
What molecules will migrate faster? (TLC)
Uncharged pigments
What is the solvent front? (TLC)
The leading edge of the solvent
When is the TLC plate removed from the developing chamber?
When the solvent front is near the opposite end of the TLC plate
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
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
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
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
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
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)
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
What is RF? (TLC)
The migration of a pigment band along the TLC plate relative to the migration of the mobile phase
How do you calculate RF?
Distance from origin to pigment band divided by distance from origin to solvent front
What unit is RF?
RF has no units
What does an absorption spectrum measure?
Measures the relative amount of light, a molecule absorbs within a range of light wavelengths
What is an absorption maximum?
A major peak in the spectrum
What is the unit of wavelength?
Nanometers (nm)
In TLC what is the gray pigment called?
Pheophytin
In TLC what is the red pigment called?
Anthocyanin
In TLC what is the orange pigment called?
Carotene
In TLC what is the yellow pigment called?
Xanthophyll
In TLC what is the blue green pigment called?
Chlorophyll a
In TLC what is the yellow green pigment called?
Chlorophyll b
What does GFP stand for?
Green fluorescent protein
What is the structure of a functional fluorescent GFP molecule?
A protein quaternary structure
What is a GFP’s quaternary structure consist of?
Two GFP proteins bonded together as a dimer
Each GFP protein in the dimer is called a what?
Monomer
What holds together the two GFP monomers?
Interactions between beta pleated sheets on the outside of their cylindrical shapes
Functional BFP is a dimer consisting of what?
Two BFP proteins
What is size exclusion chromatography?
A techniques often used to isolate specific molecules from a molecule mixture
What is the point of using a size exclusion column?
To purify GFP and BFP from protein mixtures
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
What is the column matrix?
Small spherical polysaccharide based suffix beads that are suspended in an aqueous buffer
What size protein will likely elude from the column first? (size exclusion chromatography)
Large
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.
Why is electrophoresis done?
to separate molecules according to their size, shape, and charge
What is polyacrylamide?
a porous matrix consisting of cross-linked molecules of acrylamide and bis-acrylamide.
higher concentration of cross linked molecules will result in a gel with what sized pores?
small
lower concentration of cross linked molecules will result in a gel with what sized pores?
large
what is protein size measured by?
molecular weight - Daltons (Da)
In lab 8 day 2 you used a premade polyacrylamide gel to separate proteins solely by what?
their size
what is a native protein?
a protein that retains its natural shape
in electrophoresis, what can interfere with a proteins ability to migrate solely by size?
charges and native shapes
what is a protein denaturing solution used for?
to dismantle protein shapes and ensure that all proteins have a net negative charge
what is the protein denaturing solution made out of?
denaturing reagents, glycerol, tracking dye
What is beta-mercaptoethanol?
a denaturing reagent that breaks disulfide bonds, a type of covalent bond found in many protein tertiary structures
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
what is glycerol?
a molecule that increases the density of solutions like protein samples so they stay where you load them into the gel
what is tracking dye?
a blue negatively charged dye of low molecular weight that will migrate quickly through the gel during electrophoresis
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
why are protein samples heated to a high temperature?
to eliminate any remaining interactions holding together a proteins native shape
what happens if a protein is not treated with a denaturing solution and heated to a high temperature?
proteins will retain their native shape
what is a protein marker?
a commercially prepared protein mixture that acts as a standard - the proteins in this mixture have known molecular weights
what end of the of the gel is negatively charged?
the cathode end (well end)
which end is the cathode end?
the well end (top)
what end of the gel is positively charged?
anode end (bottom)
what speed will large proteins migrate in electrophoresis?
slow
what speed will small proteins migrate in electrophoresis?
fast
after getting the value out of your equation what do you have to do to it to get the molecular weight
antilog (2nd log)
what is a GMO?
a genetically modified organism - an organism with DNA that has been deliberately altered
what is the transgenic organism?
the resulting GMO when genes are transferred from one organism to another
what is the polymerase chain reaction (PCR)?
a molecular biology technique used to clone or make identical copies of a specific DNA sequence
Where does PCR occur in rather than in a living cell?
a tube
PCR copies what instead of all of the DNA in a cell?
a specific target DNA sequence
PCR uses what instead of different proteins to carry out most steps of DNA replication?
different temperatures
what enzyme copies the DNA sequence of interest in PCR?
DNA polymerase
the primers in PCR are what instead of RNA?
single stranded DNA
what is template DNA?
a sample of DNA which will be the source of the target sequence
what are primers?
short sequences of single stranded DNA that are designed so they will flank the target sequence that is to be copied
What does DNA polymerase do?
binds to the primers and extend them to copy the target DNA sequence
what are DNA nucleotides?
a supply of DNA monomers that DNA polymerase will link together to make the copies of the target sequence
What are the names of the DNA nucleotides?
deoxyadenosine triphosphate (dATP), deoxycytosine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxythymidine triphosphate (dTTP)
what does the buffer do?
provides the ideal PH and salt concentration for PCR, the buffer also contains magnesium ions (Mg^2+)
what are the 3 steps to the PCR cycle?
denaturing, annealing, extension
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
what temperature does the denaturing step occur at?
94 degrees celsius
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
what temperature does the annealing step occur at?
59 degrees celsius
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
what temperature does the extension step occur at?
72 degrees celsius
how many cycles does a PCR reaction take to complete?
30-40
what is agarose?
a polysaccharide purified from seaweed
what charge do nucleic acids have?
negative charge
