Lab Test 1

Question 1

Describe what a standard curve is and explain what it is used for

Answer 1

A standard curve is a graph which describes the relationship between signal intensity and
concentration of the analyte. A standard curve is used to determine the amount of analyte in a
sample based on signal intensity of analyte standards of differing concentrations in order to
accurately measure the concentration of the analyte.

Question 2

What is on each axis of a standard curve and their respective units of measurement?

Answer 2

the y-axis that
corresponds to the assay measurement
of the unknown substance and follows
a line to intersect the standard curve.
The corresponding value on the x-axis
is the concentration of substance in the
unknown sample (micrograms/mL)

Question 3

What protein standard did you use to design your standard curve in this lab?

Answer 3

Bio-Rad RC DC protein assay

Question 4

Why were the protein standards and unknown fish species protein samples blue in this lab?

Answer 4

Bromophenol Blue, used to track the proteins in the column as they descend towards the cathode

Question 5

Why were the unknown muscle protein samples diluted? What is the detection limit in a
standard curve?

Answer 5

Often the samples that must be measured are in a solution that is too concentrated and are not within
the range of the standards of the standard curve. In such cases, a dilution is necessary.

The dilution factor is the
total number of unit volumes in which your material will be dissolved.
Figure 1. Example of a Standard curve. The graph is created using
standards of known quantity. The resulting curve is used to detect the
amount of analyte (i.e. protein) in the sample. In this graph the
relationship is linear and is described by the equation for the line: y = mx
+ b

For example a 1:5 dilution entails combing 1 unit volume of diluent (the material to be diluted) + 4 unit
volumes of the solvent medium (hence 1+ 4 = 5 = dilution factor).
The dilution factor can be calculated as: total volume of diluted sample / volume of undiluted
sample used in the dilution. A common way of describing a dilution is as a: n times (or X) dilution or a
n-fold dilution.
Example:
5 ml of serum is diluted to a final volume of 100 ml with saline. What is the serum dilution or the dilution
factor?
Set up the problem as 5 ml serum + X ml saline = 100 ml of the final solution.
X = 100 - 5
X = 95 ml of saline
⮚ The serum dilution is the amount of serum in the amount of total solution; hence, this is a
5/100 serum dilution which would equal a 1/20 dilution. The dilution factor is 20.

Question 6

Calculating unit conversions (ie. mg to (micrograms)g, cm to mm)

Answer 6

mg = 1/1000 gram
microgram = 1/10000000 gram
cm = 1/100 m
mm= 1/1000 m

Question 7

explain the purpose of adding Laemmli buffer to each protein sample

Answer 7

Laemmli buffer, which contains SDS, was added to fish samples to linearize the proteins by breaking up secondary and tertiary protein structure, ensuring that proteins will only move by size.
Also, coats proteins to be electrically charged so it will migrate down the gel
Laemmli sample buffer is used to increase the solubility of the proteins in the fish samples. The glycerol in
the buffer increases the density of the samples, allowing them to sink into the wells

Question 8

explain why the proteins were heated to almost 100 degrees
Celcius

Answer 8

heat is also responsible for the actual denaturation of the protein by allowing the SDS to bind in the hydrophobic regions to complete the denaturation. Heat disrupts the 2, 3, and 4 protein structures to form linear amino acid chains that can move down the gel according tot he molecular weight .

Question 9

explain the purpose of centrifuging

Answer 9

separate heterogeneous mixtures into their various components – liquids in liquids, solids in liquids, and liquids in gases, based on the different densities of the components. One of the most common uses is to separate red blood cells and other blood components from whole blood.

Question 10

explain the purpose of vortexing

Answer 10

The overall goal of the vortex mixer is to mix various samples of liquids rapidly. The function of the device is achieved through a motor that drives a rubber cup in a circular motion to create a vortex or a spiral flow within the sample.

Question 11

explain the purpose of blanking the spectrophotometer

Answer 11

A blank always needs to run each time a spectrophotometry experiment is conducted. A blank will account for absorbance caused by scratches in the cuvette, dust, and other solutes in the buffer that absorb at the selected wavelength.

Question 12

describe what would be in the blank control cuvette for the spectrophotometer

Answer 12

The ‘blank’ allows you to set the spectrophotometer to zero before you measure your ‘unknown’ solution. The ‘blank’ solution will contain everything that the ‘unknown’ solution (the one you want to measure) except for the think you wish to measure.

Question 13

What is the general purpose of SDS-PAGE electrophoresis?

Answer 13

Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is commonly used to obtain high resolution separation of complex mixtures of proteins. The method initially denatures the proteins that will undergo electrophoresis.

Question 14

How did SDS-PAGE electrophoresis help distinguish protein profiles of your different fish
species?

Answer 14

https://www.youtube.com/watch?v=MILiO1XnuqQ&si=fm2238XYIoLk2gvY

Question 15

Why were actin and myosin protein used as standards in the gel?

Answer 15

The actin and myosin have a known kilodalton so they’re used to compare the protein sizes to the
known size of the actin and myosin and precision plus protein kaleidoscope.

Question 16

Why was a protein ladder standard included in the gel?

Answer 16

Protein ladders help in the analysis of experimental proteins by creating reference bands within the gel through protein gel electrophoresis. They consist of a mixture of proteins with known molecular weight and other characteristics.

Question 17

What is the purpose of the following constituents of Laemmli buffer: Tris-HCl, SDS, Glycerol,
Bromophenol Blue, and DTT?

Answer 17

SDS
SDS is a well-known detergent that’s often used to denature proteins. It does so by disrupting non-covalent bonds to destabilize the secondary, tertiary, and quaternary structural assemblies. Denaturing the protein analytes in SDS-PAGE is crucial so that their separation is solely a function of molecular weight.
It is also used to impart a net negative charge onto all of the protein analytes so that they all migrate in the same direction during an SDS-PAGE experiment.
TRIS-HCL
Tris functions to maintain a pH of 6.8 to keep the Laemmli buffer chemically stable. Tris also inhibits a number of enzymes and so can prevent proteases from degrading your analytes.
Preparing the Laemmli buffer at pH 6.8 also helps achieve maximum resolution for your SDS-PAGE Experiment.
GLYCEROL
It’s all well and good doing fancy biochemical alterations to your analytes, but remember that your SDS-PAGE gel is submerged in running buffer. Pipetting in your analytes at this stage would see them just diffuse into the gel tank. Sad times. A good dollop of glycerol can prevent this.
BB
A dye, usually bromophenol blue is added to the sample buffer and enables us to see our samples as we load them onto the SDS-PAGE gel. It’s not strictly necessary but you need good eyesight to forgo it.
Similar to the Cl– mentioned already, the dye molecules will migrate through the resolving gel much quicker than your protein analytes. All the dye molecules actually migrate together in a line called the “dye front.”
As long as the dye front is still on the gel, you can be confident that even the lightest of your analytes is also still on the gel.
DTT
Remember when I said that SDS will break apart analyte quaternary structure? I was lying.
Some quaternary structures are held together by disulfide bonds, which are covalent. Because they are covalent, SDS won’t touch them and a specific reagent is needed to break them apart. Enter the reducing agent.

Question 18

Why is knowing the direction of electrical current flow in a gel so important? From which
electrode to which electrode should current flow?

Answer 18

The SDS-coated,
negatively charged proteins migrate through the gel away from the negatively charged
anode toward the cathode, with the larger proteins moving more slowly than the smaller
proteins (figure 2). This technique was developed by U.K. Laemmli, in 1970 and is still the
predominant method used in vertical gel electrophoresis of proteins.
As soon as the electric current is applied, the SDS-coated proteins begin their race toward
the positive electrode. The smaller proteins can move through the gel more quickly than the
larger ones, so over time, the proteins will be separated according to their sizes. o they start moving through the matrix of the gel towards the positive pole. When the power is turned on and current is passing through the gel, the gel is said to be

Question 19

Before loading a protein sample into a gel well, which two main steps must be done to the
proteins?

Answer 19

the proteins are treated with the detergent sodium dodecyl
sulfate (SDS) and heated.

Question 20

What is the unit of measurement for protein mass?

Answer 20

Daltons

Question 21

What is a cladogram?

Answer 21

. A fish family tree, or cladogram, can be constructed based on protein bands that
the fish have in common. Cladistic analysis assumes that when two organisms share a
common characteristic, they also share a common ancestor with that same characteristic

A cladogram is a diagram that shows relationships between species.

Question 22

-Reading the separation of proteins and determining their molecular weight to a known
protein standard ladder in a gel
- Designing a cladogram based on a comparison of protein bands on a gel

Answer 22

x

Question 23

-Drawing a standard curve (Absorbance vs. analyte concentration) and understand how it
can be used to determine quantity of an analyte in unknown samples. Remember to
label axes, use as much of grid as possible and give a descriptive caption at the bottom.
-Determining the correct dilution to produce a sample that falls within the range of
values on your standard curve. Understand what a dilution factor is

Answer 23

The dilution factor in chemistry is how much we dilute something by, and it’s usually written as a ratio. For example, diluting by 1:10 factor means diluting 1 mL of sample by 9 mL of diluent.

https://youtu.be/-59EcZXQsqM?si=QTyKuD0Kkni-pvL9

Question 24

Know the structure and function of enzymes. How are they affected by changes in pH and
temperature?

Answer 24

The function of the enzyme depends upon the binding of the substrate to the active site of the enzyme. They catalyse chemical reactions in the body.

pH: Each enzyme has an optimum pH range. Changing the pH outside of this range will slow enzyme activity.
In high temperature, the hydrogen bonds and non-polar hydrophobic interactions of enzyme get disrupted.

Question 25

Know the function of catalase enzyme and where it is found.

Answer 25

catalyzes rx and is found in mammals

Question 26

What reaction does catalase catalyze? What is the substrate? The products?

Answer 26

Catalase enzymes break down hydrogen peroxide (H2O2) -the substrate- to water and oxygen molecules, which protects cells from oxidative damage by reactive oxygen species.

Question 27

Understand the relationship between quantity of product produced over time for a reaction
catalyzed by an enzyme.

Answer 27

There is usually a hyperbolic relationship between the rate of reaction and the concentration of substrate.

(A) At low concentration of substrate, there is a steep increase in the rate of reaction with increasing substrate concentration. The catalytic site of the enzyme is empty, waiting for substrate to bind, for much of the time, and the rate at which product can be formed is limited by the concentration of substrate which is available.

(B) As the concentration of substrate increases, the enzyme becomes saturated with substrate. As soon as the catalytic site is empty, more substrate is available to bind and undergo reaction. The rate of formation of product now depends on the activity of the enzyme itself, and adding more substrate will not affect the rate of the reaction to any significant effect.

Question 28

Know the purpose of doing control runs (e.g. no substrate or boiled enzyme runs)

Answer 28

-Confirms that without
a substrate for the
enzyme to bind to no
reaction will occur
because the active
site of the enzyme will
remain empty
-Confirms that when
the enzyme is
denatured it cannot
perform reactions
anymore and all its
functions are lost
-Peroxide doesn’t randomly form from oxygen on its own, it needs catalase

Question 29

What is meant by the initial reaction rate (V0)? Why did most of the initial reaction rates decline
over time?

Answer 29

V0= The initial rate of reaction is the gradient of the straight line portion of the plot, shown by the dotted red line. The initial rate of reaction is when concentrations of enzyme and substrate are known, so this allows fair comparison if you then change initial concentrations of enzymes or substrate.
-The decrease in reaction rate over time means that average reaction rates do not accurately represent the actual rate of reaction at all time points, due to decrease in substrate concentration.

Question 30

Understand the relationship between reaction rate and substrate concentration. Why does it
plateau at a certain substrate concentration?

Answer 30

Initially, an increase in substrate concentration leads to an increase in the rate of an enzyme-catalyzed reaction. As the enzyme molecules become saturated with substrate, this increase in reaction rate levels off. The rate of an enzyme-catalyzed reaction increases with an increase in the concentration of an enzyme

Question 31

What does Vmax and Km from an enzyme saturation curve mean about the relationship between
enzyme, substrate and product of a reaction?

Answer 31

The Michaelis-Menten equation for this system is: Here, Vmax represents the maximum velocity achieved by the system, at maximum (saturating) substrate concentrations. KM (the Michaelis constant; sometimes represented as KS instead) is the substrate concentration at which the reaction velocity is 50% of the Vmax.

Question 32

Know the difference between competitive and non-competitive inhibitors. How they affect the
rate of reaction by enzymes, and what they are doing at the molecular level. Understand how a
change in Vmax or Km can help you identify which type of inhibitor is affecting an enzyme

Answer 32

competitive: directly obscure access to the access site
non-competitive: changes the shape of the enzyme which deterrs substrate from entering the active site.
Affecting the amount of substrate that can access the active site slows down the reaction rate.

change in vmax: non-competitive
change in km: competitive

Question 33

Convert daltons to g/mol

Answer 33

10kD = 10,000g/mol

Question 34

How to draw a cladogram using protein

Answer 34

How to Construct a Cladogram:
EXAMPLE: Muscle proteins from 5 species (A, B, C, D, E) of organisms were extracted & analysed by SDS-PAGE. Each organism showed a number of protein bands (A=8) some of which were also found in other species. The number of proteins that were common to 2 species were noted (A & B =2) as in table A.

Table A: Number of Analysed Proteins Common Between Species.

Species A
8
Species B
2, 10
Species C
2,10,13
Species D
2,5,5,7
Species E
2,3,4,2,12
The last number is always the total count
Everything above is what each sample has in common with one another (ABCDE)

First draw a line to form the trunk of your cladogram. Find the fish with the least bands in common. In the example above it is species A, which has only 2 bands in common with any of the other fish. Then draw a side branch off the line near the bottom of the trunk and label that branch with the fish’s name, in this case, species A. This fish is the outlier, i.e., it is the least similar to any of the others. The node (where the side branch meets the trunk) represents an ancestor that is common to all the fish in this analysis.

Now, find the two fish with the most bands in common (in this example it is species B and C, which have 10 bands in common). Draw a side branch off the trunk near the top and label the two ends with the fishes’ names, in this case, species B and species C (it doesn’t matter which branch gets which label). The node represents a common ancestor of species B and species C that had all the same characteristics (proteins).

Now, identify those fish species with the next most bands in common. In this example, species D has five bands in common with species B and species C, which indicates species D is the same cladistic distance from B and C (i.e. species D is not more closely related to either B or C). Draw a branch further down the trunk. This node represents an ancestor that is common to species B, C, and D that had these 5 characteristic proteins.

The last fish to add to the cladogram in this example is species E, which shares four bands with species C, three bands with species B, and only two bands with species A and D. This fish may seem trickier to place than the others because it shares more characteristics with species B and C than it does with D, but D shares more characteristics with B and C than E does. So, to place this fish you might ask: Does species E share the five proteins that the common ancestor of species B, C, and D had? Answer (no).
Does species E share more proteins with B, C, and D than A? Answer (yes). Therefore, species E gets its own branch in between the D and A branches to indicate that it has more shared characteristics with B, C, and D than A, but fewer shared characteristics with B and C than D. This is the finished cladogram that corresponds to the Table A example on the top of p.11.
In the table below, both the row and column headings are the types of fish. Using the table on page 10 (or your excel file), separately compare the number of bands (X’s) in common with every other fish sample from your gel and put those numbers into the table below, such that each fish is individually compared with every other fish. Your table will be the basis for drawing your cladogram.