BIOC23 FINAL EXAM STUDY

GUIDE

BIOC23 Zoom Drop In Office Hours NO LABS THIS WEEK!

Time: Apr 12, 2023, 1:00 PM -2:30 PM Alkaline Phosphatase report due
[Link] April 5
Meeting ID: 885 3819 5701
Passcode: 565939 Please fill in the course evaluation!
 FINAL EXAM

• Saturday April 15th from 5pm to 7pm in SW309.

• Covers the entire term

• Mix of multiple choice and short answer questions. Will require understanding and application of the techniques discussed.

• Some basic calculations will be included. (You may bring a non-programmable calculator). You will not be tested on the details of specific

labs (do NOT memorize your labs), but rather understand the approaches and be able to apply what you did in lab to new context.

• Use this review session as a guide for the depth of knowledge (level of detail!) and types of application questions to expect.

• In addition to the questions posted here, you should understand the basic calculations we have been practicing. Look back at the quizzes and

make sure you understand unit conversions, dilutions, c1v1 = c2v2, using standard curves, and concentration/amount calculations. You should

also understand from enzyme kinetics: Michaelsi-Menten plot vs Linweaver-Burk plot, how to calculate Vo, Vmax, and Km, what these values

mean, and how different types of inhibitors affect them.

• I am planning to post your final quiz marks and then open the lecture questions for unlimited practice attempts.
 ANTIBODY-BASED METHODS PRACTICE QUESTIONS

• What are antibodies, where do they come from, and why are they useful in research? How specific are they?
What is the difference between monoclonal and polyclonal?

• Describe how indirect detection works (use terms variable region and constant region). What are 3 different examples
of possible detection methods that could be used? How would the process/imaging of detection differ for each?

• You should know the general principle/purpose for the following approaches. Which of the following use
antibodies for a purification approach? Which use antibodies for detection?
Immunoprecipitation ELISA
ChIP-Seq MALDI-TOF
Flow cytometry / FACS Affinity chromatography
Western blotting Immunofluorescence

Northern blotting Immunohistochemistry

Bradford assay
 ANTIBODY-BASED METHODS PRACTICE QUESTIONS

• What are antibodies, where do they come from, and why are they useful in research? How specific are
they? What is the difference between monoclonal and polyclonal?

(goat,
donkey,
camel)

One B cell differentiates into a plasma producing

cell and produces a single version of an antibody
Monoclonal = A single antibody sequence that binds
a single epitope (antigens can have many epitopes)
Modified from Garcia-Calvo et al. 2020
 ANTIBODY-BASED METHODS PRACTICE QUESTIONS

• What are antibodies, where do they come from, and why are they useful in research? How specific are
they? What is the difference between monoclonal and polyclonal?
• Describe how indirect detection works (use terms variable region and constant region). What are 3 different
examples of possible detection methods that could be used? How would the process/imaging of detection
differ for each? Recall that
constant
regions are
species-
Label for specific (Why
detection was this
Secondary important?)
antibody
variable Can also have
radiolabeled
constant antibodies,
Primary though this
antibody isn’t as
variable commonly
antigen used. (Why?)
 ANTIBODY-BASED METHODS PRACTICE QUESTIONS

• What are antibodies, where do they come from, and why are they useful in research? How specific are they?
What is the difference between monoclonal and polyclonal?

• Describe how indirect detection works (use terms variable region and constant region). What are 3 different examples
of possible detection methods that could be used? How would the process/imaging of detection differ for each?

• You should know the general principle/purpose for the following approaches. Which of the following use
antibodies for a purification approach? Which use antibodies for detection?
Immunoprecipitation ELISA
ChIP-Seq MALDI-TOF
Flow cytometry / FACS Affinity chromatography
Western blotting Immunofluorescence

Northern blotting Immunohistochemistry

Bradford assay
 GELS AND BLOTS PRACTICE QUESTIONS

If you were using gel electrophoresis to separate proteins, why would results differ if you analyzed the
same sample by 2DGE (non-reducing conditions) vs SDS-PAGE under reducing conditions vs SDS-
PAGE under non-reducing conditions? Use the below example proteins to help you consider your
answer. What would you see if these were the only 3 proteins in the sample analyzed?

S S
30 S S
60
KDa
KDa

Protein X Protein Y Protein Z

80 KDa 80 KDa 90 KDa
pI = 7.2 pI = 3.4 pI = 5.4

FYI: The SDS-PAGE portion of 2DGE can be done under reducing or non-reducing conditions. The reduction is typically done after the isoelectric focusing (IEF) step.
 GELS AND BLOTS PRACTICE QUESTIONS

If you were using gel electrophoresis to separate proteins, why would results differ if you analyzed the
same sample by IEF-2DGE (non-reducing conditions) vs SDS-PAGE under reducing conditions vs
SDS-PAGE under non-reducing conditions? Use the below example proteins to help you consider
your answer. What would you see if these were the only 3 proteins in the sample analyzed?

IEF-2DGE SDS-PAGE (reducing) SDS-PAGE (non-red.

S S
30 S S - - -
60
KDa
KDa

Protein X Protein Y Protein Z

80 KDa 80 KDa 90 KDa
pI = 7.2 pI = 3.4 pI = 5.4 + + +
pH3 pH10
 WESTERN BLOTTING PRACTICE QUESTIONS

• What is the purpose of each of the following in the western blot protocol? (& you should
know the general steps in a western blot)
• SDS
• Transfer
In SDS-PAGE, do proteins migrate towards the cathode or
• Gel electrophoresis
anode?
• HRP
Which proteins migrate faster and why?
• Membrane
• BSA or milk If you were studying a very large protein (200 Kda), how
• Primary antibody might this impact the % acrylamide you want to use to
make your gel? Why?
• Luminol
What could go wrong? Why might a band be missing?
Why might a band be present at the wrong size?
 WESTERN BLOTTING PRACTICE QUESTIONS

• What is the purpose of each of the following in the western blot protocol? (& you should
know the general steps in a western blot)
• SDS
In SDS-PAGE, do proteins migrate towards the cathode (-) or
• Transfer anode (+) ?
• Gel electrophoresis
Which proteins migrate faster and why? Smaller, easier fit
• HRP through pores
• Membrane
If you were studying a very large protein (200 Kda), how
• BSA or milk might this impact the % acrylamide you want to use to make
• Primary antibody your gel? Why? Need lower % or protein won’t
separate/migrate well
• Luminol
What could go wrong? Why might a band be missing? Why
might a band be present at the wrong size?
 GELS AND BLOTS PRACTICE QUESTIONS
You want to understand which proteins interact with a particular protein: okRTK (a receptor tyrosine kinase), which is activated
by the Ok ligand. You transfect cells with DNA to express a GST-tagged okRTK or a GST-tagged kinase dead okRTK or the
GST tag alone. You then treat cells with the Ok ligand for 15 minutes before lysing cells and isolating GST-associated protein
complexes (What are 2 different methods to do this?). Samples were run on a gel and stained with Coomassie Blue and also
investigated by western blotting with an anti-phosphorylated tyrosine antibody.
Coomassie Gel Western blot (anti-pTyr)
D) If you next wanted
Transfected: GST-okRTK GST-okRTKkd GST tag GST-okRTK GST-okRTKkd GST tag
A) How would the to know which
gel look if the Ok ligand: - + - + - + - + - + - + proteins are
samples were phosphorylated
whole cell lysate directly by okRTK,
instead of purified which protein bands
complexes? from this gel would
you choose to
B) Why do the
investigate and why?
bands on the blot
look different than E) How could you
the gel? identify those
C) Which band do proteins?
you think is
F) How could you
okRTK and what
determine if okRTK
do you think this
phosphorylates them?
band is? How
(how would that result
could you confirm
differ from this
this?
experiment?)
 GELS AND BLOTS PRACTICE QUESTIONS
You want to understand which proteins interact with a particular protein: okRTK (a receptor tyrosine kinase), which is activated
by the Ok ligand. You transfect cells with DNA to express a GST-tagged okRTK or a GST-tagged kinase dead okRTK or the
GST tag alone. You then treat cells with the Ok ligand for 15 minutes before lysing cells and isolating GST-associated protein
complexes (What are 2 different methods to do this? - Binding to immobilized glutathione or antibody). Samples were run on a
gel and stained with Coomassie Blue and also investigated by western blotting with an anti-phosphorylated tyrosine antibody.
Coomassie Gel Western blot (anti-pTyr)
D) If you next wanted
Transfected: GST-okRTK GST-okRTKkd GST tag GST-okRTK GST-okRTKkd GST tag
A) How would the to know which
gel look if the Ok ligand: - + - + - + - + - + - + proteins are
samples were phosphorylated
whole cell lysate -ve controls directly by okRTK,
instead of purified which protein bands
complexes? from this gel would
< GST- < GST-okRTK auto- you choose to
B) Why do the
bands on the blot okRTK phosphorylation investigate and why?
look different than E) How could you
the gel? identify those
C) Which band do proteins? Cut out of
you think is gel and mass spec
okRTK and what GST > F) How could you
do you think this
determine if okRTK
band is? How
phosphorylates them?
could you confirm
Kinase assay with
this?
purified proteins and
radiolabeled ATP
 CHROMATOGRAPHY PRACTICE QUESTIONS

What is chromatography? What is partition chromatography vs adsorption chromatography? How

would the options/process of elution be different for each?

Explain the general process of equilibration, binding, and elution for ion exchange chromatography.

You want to purify a target protein that is a weak acid with a pI of 5.5

Will you choose a strong cation or strong anion exchanger? What pH will
you equilibrate/bind at? What charge will the stationary phase have? What
charge will your target protein have?

You decide to use a stepwise elution with a change in pH. What pH will you
elute at?
 CHROMATOGRAPHY PRACTICE QUESTIONS
What is chromatography? Separation of molecules based on differential affinity for stationary and mobile phase
What is partition chromatography (molecules partition between 2 liquid phases) vs adsorption
chromatography (bind to the stationary phase)? How would the options/process of elution be different for
each? Adsorption would require changing buffer conditions to disrupt binding

Explain the general process of equilibration, binding, and elution for ion exchange chromatography.

Equilibrate with
counter ions and then Elution by increasing salt
displace them with concentration (ionic strength)
binding of target or changing pH
molecules (initial
ionic strength and pH Can be gradient or stepwise.
conditions important
considerations to
allow this)
 CHROMATOGRAPHY PRACTICE QUESTIONS

Functional groups attached to the

resin determine what type of
You want to purify a target
exchanger it is. Want reversible
binding (want to easily be able to protein that is a weak acid
change conditions and elute). with a pI of 5.5
Strong exchangers maintain
charge at pH extremes, but weak Will you choose a strong
exchangers do not. cation or strong anion
exchanger? What pH will
you equilibrate/bind at?
What charge will the
Target protein –vely charged at pH 6.5, can bind to (+ve) anion exchanger stationary phase have? What
charge will your target
protein have?

You decide to use a stepwise

elution with a change in pH.
What pH will you elute at?

Target protein +vely charged at pH 4.5, can elute

 CHROMATOGRAPHY PRACTICE QUESTIONS

• You decide to try anion exchange chromatography (DEAE column) to purify the splop 1
enzyme from an ammonia sulphate cut of 55%. (which you confirm contains splop enzyme
activity based on conversion of the blop substrate to the glop product that absorbs at 420 nm)
(What equipment/reagents would you need for this absorbance assay?)
• What additional step might you perform after the ammonia sulphate precipitation but
before using the sample for other steps/applications? How does this technique work?
2
Ion Exchange Chromatography:
• You equilibrate your column with column buffer (0.1M NaCl, pH 7.5). You then apply your
sample to the column, and collect the flow through (FT). Next, you elute with increasing
concentrations of NaCl and collect fractions(#1-5). Finally, you assay each fraction to
determine the presence of enzyme activity.
• Shown are three possible (separate!) outcomes of this experiment. Explain what each
3
outcome tells you about the properties of splop (pI or anything else that could explain
your results) under the conditions you ran the column (pH 7.5, variable NaCl)

Modified from Brunt 2022

 CHROMATOGRAPHY PRACTICE QUESTIONS
• You decide to try anion exchange chromatography (DEAE column) to purify the splop enzyme from an ammonia sulphate
cut of 55%. (which you confirm contains splop enzyme activity based on conversion of the blop substrate to the glop product
that absorbs at 420 nm) (What equipment/reagents would you need for this absorbance assay? – spec set to 420nm, blob
substrate, appropriate buffer, and a blank)
• What additional step might you perform after the ammonia sulphate precipitation but before using the sample for
other steps/applications? How does this technique work? Dialysis. Works by placing sample inside a sealed
semipermeable membrane and allowing time for diffusion of small molecules (like salts) into fluid reservoir outside of
the membrane. Most proteins are above the molecular weight cutoff of the membrane and therefore stay within the
membrane.

Modified from Brunt 2022

 CHROMATOGRAPHY PRACTICE QUESTIONS

DEAE weak anion exchanger is +vely charged at pH 7.5

And remember: pH > pI = -ve charge, pH < pI = +ve charge 1

Possible interpretations:

1. Splop is negatively charged at pH 7.5 because it sticks to the column. This means that human
Splop's pI must be less than 7.5.
2
2. Splop is positively charged or neutral at pH 7.5 because it flows through the column. This
means that human Splop's pI must be greater than or equal to 7.5.

3. Splop does not elute off the column at the salt concentrations you used for elution (very
negatively charged; pI << 7.5) or Splop is unstable at pH 7.5. Thus, once loaded on the column, 3
it denatures and loses activity. Or perhaps there is something wrong with your setup of your
absorbance assay (wrong wavelength? Wrong substrate? Forgot to pipette a component?)

18

Modified from Brunt 2022

 CHROMATOGRAPHY PRACTICE QUESTIONS

Why do molecules move

quickly in gel filtration
chromatography while
others are very slow?

How does the fractional

range of the matrix impact
results? Would more
crosslinking during
production decrease or
increase the fractional
range?

19
 CHROMATOGRAPHY PRACTICE QUESTIONS

Why do molecules move

quickly in gel filtration
chromatography while others
are very slow?

How does the fractional range

of the matrix impact results?
Can’t separate components outside
the fractional range as they would all
be entirely excluded or entirely
included in pores. Would more
crosslinking during production
decrease or increase the
fractional range? Makes smaller
pores, better for smaller proteins

20
 CHROMATOGRAPHY PRACTICE QUESTIONS

Why do molecules move in thin layer chromatography? Why are some molecules slowed down more than
others?
If the molecules you want to separate are all clumped at the origin line, would you next try making your
solvent more polar or less polar? Why? What would happen if you change it too much?

How would you

determine the Rf value
for this compound?

21
 CHROMATOGRAPHY PRACTICE QUESTIONS

Why do molecules move in thin layer chromatography? Why are some molecules slowed down more than
others?
If the molecules you want to separate are all clumped at the origin line, would you next try making your
solvent more polar or less polar? Why? What would happen if you change it too much?

Molecules are slowed

Less polar down by how much they
mobile interact with stationary
phase phase vs mobile phase.
Less polar molecules
move faster

Polar Could also run in

stationary “reverse phase” where
phase mobile phase is more
polar than stationary
phase.
22
 CHROMATOGRAPHY PRACTICE QUESTIONS

Why do molecules move in thin layer chromatography? Why are some molecules slowed down more than
others?
If the molecules you want to separate are all clumped at the origin line, would you next try making your
solvent more polar or less polar? Why? What would happen if you change it too much?

Too polar wouldn’t

B Rf = A /
interact with B
stationary phase at
More polar all and would How would you
migrate near determine the Rf value
solvent ront
for this compound?

A
23
 CHROMATOGRAPHY PRACTICE QUESTIONS

Below are the components of a mixture and the reagents you may use to devise a separation strategy that will result in the
separation of all seven compounds into separate samples.

Create a flow chart of your strategy and clearly indicate at which step you have purified each of the seven coloured components

Remember you are a researcher, you want to take as few steps as possible • Sephadex G, 75 equilibrated in 0.05 M NaAc, pH 6
(This medium has a fractional range of 3000–
The 7 components of the mixture are: 80,000) 
• DEAE-Sephadex 75 equilibrated in 0.05 M NaAc, pH
compound colour MW (Da) Ionic Character at pH 6
or pI 6 (This medium has a fractional range of 3000 to
80,000 and a weak +ve charge at pH 6).
Blue dextran blue >500,000 Very strong anion
• CM-cellulose (weak -ve charge at pH 6)
Yellow dextran yellow 10,000 Strongly anionic
• DEAE-cellulose (weak +ve charge at pH 6)
Green actin green 44,000 pI of 4.8
Cytochrome c orange 12,400 Strong cation, pI of 10 • 0.05M sodium acetate, pH 6

Vitamin B12 red 1357 Weak cation • 0.1 M sodium acetat), pH 6

DNP-glycine yellow 241 Weak anion , pI of 3 • 0.5 M sodium acetate, pH 6
myoglobin red 15,600 Very weak cation
• 1.0 M sodium acetate pH 6
• 2.0 M sodium acetate, pH 6
• 1M HCl 24

Modified from Brunt 2022

• 1M NaOH
 TRANSGENES/TAGS PRACTICE QUESTIONS

Match the tag/protein to the most likely matrix for affinity purification (matrix would have which of these options immobilized?)

Myc tag Nickel

GST tag An antibody

HA tag
Protein A
FLAG tag
Streptavidin
Polyhistidine tag
Biotin tag Glutathione

IgG
If you wanted to design your protocol so that you could elute your tagged protein from a column while maintaining
enzyme function, what options would you have?

Understand how the following relate to producing a recombinant protein: cloning, ORF, fusion protein, in frame,
promoter, selectable marker, multiple cloning site, vector, E. coli, lac repressor, IPTG

How does tagging a protein with biotin differ from tagging a protein with GST? Are there different types of information
that could be gained from this type of tagging?
 DIFFERENTIAL CENTRIFUGATION PRACTICE QUESTIONS

You want to separate a cell lysate into a fraction of whole cells (C), nuclei (N), mitochondria (M), and small organelles/proteins
(P). Draw a flowchart to show how differential centrifugation could be used to do this. Add letters to indicate where the
different components are at each step.

Now you want to isolate a particular protein from your mitochondrial fraction using salting out. What does this technique
entail and what are the effects/considerations related to temperature, pH, and hydrophobicity of the target protein?
 DIFFERENTIAL CENTRIFUGATION PRACTICE QUESTIONS

You want to separate a cell lysate into a fraction of whole cells (C), nuclei (N), mitochondria (M), and small organelles/proteins
(P). Draw a flowchart to show how differential centrifugation could be used to do this. Add letters to indicate where the
different components are at each step.

Should have shown a process of going from a slower centrifugation speed to a faster and faster one, pelleting
different components at each step before collecting supernatant (with everything else) to spin again. Slowest
speed (ex. ~600 rpm) would pellet whole cells, ~2000 rpm would pellet nuclei, ~13 000 rpm should pellet
mitochondria, and everything else is left in the supernatant (could be separated by ultracentrifugation at very
high speeds)

Now you want to isolate a particular protein from your mitochondrial fraction using salting out. What does this technique
entail and what are the effects/considerations related to temperature, pH, and hydrophobicity of the target protein?
 SALTING OUT

Salt ions attract water

away from protein

Protein molecules
interact with each other
and aggregate

When enough salt is added, protein precipitates (without denaturing = enzymatic activity can survive!)
Proteins with greater hydrophobicity generally precipitate first and cold temperature aids the precipitation
For a target protein with a pI of 6, what pH should you choose?
How could you use this technique to collect multiple fractions of proteins? What property are you
separating proteins based on?
 MASS SPEC PRACTICE QUESTIONS

• If each peak in the chromatogram is

a peptide, what technique was likely
used to obtain this data?

• Why is it necessary to perform

separation of peptides prior to mass
spec analysis?

• Which is more specific for

identification of proteins, the
chromatogram retention time or the
mass spectra?

• How can a protein sequence be

determined by tandem mass spec?
What are the steps involved?
 ISOTOPE LABELING PRACTICE QUESTIONS

• What is radioactivity and what advantages and disadvantages are there to using it in
the lab?
• Which particle type is the largest? Which is the most ionizing? Which is the most
penetrating?
• How do radioisotopes produce ionizing radiation? What is the “half-life” of a
radioisotope and how is it calculated?
• How would detection methods differ for heavy isotope labeling vs radioisotope labeling?
• What are the major considerations if planning an experiment using radioactivity?
• Give an example of a research experiment that could not be done by fluorescence or
chemiluminesence instead of radioactivity?
• Explain how radioisotopes can be useful in imaging/diagnosis of live patients?
 TECHNIQUES OVERVIEW PRACTICE QUESTIONS

• Which techniques can be used directly in a step contributing to purifying a protein?

• Which are often used for analysis rather than purification? (some can be both)
• Which techniques separate/detect components/molecules on the basis of size/mass?

Affinity chromatography Ion exchange chromatography

SDS-PAGE Thin layer chromatography (You should have a
IEF-2D gel electrophoresis Spectrophotometry general understanding of
Western blotting Homogenization purpose/uses and
LC-MS approach for each of
Co-immunoprecipitation
these)
Bradford assay Dialysis
Differential centrifugation Ammonium sulfate
Electrospray ionization precipitation
Ponceau Red stain Gel filtration chromatography
SILAC MALDI-TOF
 TECHNIQUES OVERVIEW PRACTICE QUESTIONS
• Which techniques can be used directly in a step contributing to purifying a protein?
• Which are often used for analysis rather than purification? (some can be both)
• Which techniques separate or detect components/molecules on the basis of size/mass?
(there is some wiggle room for interpretation here (exam questions will either be more specific/unambiguous or will
be open ended for you to explain your reasoning. – important thing is you understand the uses for the techniques)

Affinity chromatography Ion exchange chromatography

SDS-PAGE ** Thin layer chromatography (You should have a
IEF-2D gel electrophoresis ** Spectrophotometry general understanding of
Western blotting Homogenization purpose/uses and
Co-immunoprecipitation LC-MS ** approach for each of
Bradford assay Dialysis ** these)
Differential centrifugation ** Ammonium sulfate precipitation
Electrospray ionization Gel filtration chromatography **
Ponceau Red stain MALDI-TOF **
SILAC *
 GOOD LUCK!

AND THANK YOU FOR BEING

PART OF MY FIRST SEMESTER AT
UTSC!

(And please fill in the course evaluation)