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Biochem Lab Exam 2 Review

Experiment 4- Purification of rGFP using Ni+2 Agarose

1) Underlying principle that allows for the purification of rGFP (how to purify
rGFP)?
-Exploiting the physical difference between your molecule of interest and all other
molecules.
- Chromatography separates proteins via differential interactions with both a mobile
and stationary phase.

2) Why does freezing and then thawing the bacterial pellet result in lysis?
- freezing and thawing to make crude extract
- From freezing, ice crystals form, that break open neighboring cell walls.
- Thawing helps make it more efficient because from damaged bacteria, the
cytoplasm is released in the breaking buffer. This lysozyme will lyse neighboring
bacteria and begin a chain reaction on lysing

3) Purpose of the wash step when developing the column?
- the washes pull out any contaminants and basically any proteins that arent His6-
tagged (rGFP) that dont bind to the column.

4) How are we monitoring the presence of rGFP?
-by using handheld UV light to see presencenot amount! That would be using a
fluorescent microplate reader. So the microplate reader show proteing amount and
protein activity.

If we were purifying a histidine tagged phosphatase, how would we monitor
its presence during the purification procedure?
- Monitor presence by reaction with PPP (colorimetric assay)

5) What is the underlying principle that allows for the elution of rGFP from the
Ni+2 agarose column?
- Imidazole, which is in the elution buffer, is very similar to hisitdine and competes
for the binding sites in the column. So basically, the his6 tags get released out of the
column when imidazole comes in.

6) What are the five major types of column chromatography and the underlying
scientific principles that make them useful for separating proteins?
1. Gel-filtration chromatography- separates based on molecular weight
2. Ion exchange chromatography- based on charge
a. Anion-exchange: binds negative charge
b. Cation-exchange: binds positive charge
3. Hydrophobic interaction chromatography- based on hydrophobic properties
4. Affinity chromatography- based on specific molecular binding properties.
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- His6 tag with Ni+2 binding.
5. Partition chromatography- polarity or water solubility

Experiment 5 - Determining Total Protein Amount in rGFP Fractions

1) Based upon the design and principle of spectrophotometry, what improper
laboratory techniques could result in incorrect absorbance reading?
-Scratches, fingerprints and other residues on cuvettes can mess up readings as well
as improper orientation of the cuvette into the reader.

2) Given the conditions of an enzyme assay, be able to describe what components
of that assay should be placed in the reference cuvette to properly blank the
machine.

3) Be able to understand and correctly use the following terminology:
Wavelength- difference between two crests of a waveor any two points on a
wave.
Wavelength scan- measures the absorbance of a sample as you vary the
wavelength. Example: 260 nm/ 280 nm
Maximum wavelength- highest wavelength that displays the most absorbance.
Absorbance- amount of light absorbed by a substance at a certain wavelength.
Logarithmic measure.
Transmittance- ratio of light once it has passed through a sample to the intensity of
the light when it first entered the sample.

4) What are the differences between a spectrophotometer and a
spectrofluorometer?
- Spectrophotometer measures absorbance. Reads the amount of light the sample
absorbs at a specific wavelength.
- Spectrofluorometer measures fluorescence. Reads the amount of light the sample
generates at a specific wavelength when a different wavelength is shone on it.

5) Given then necessary data, construct and use a standard curve to identify the
amount and concentration of an unknown protein sample.
Using known protein concentration and its absorbance (ex: BSA) make standard
curve and then extrapolate for unknown.

6) Define specific activity.
In a sample, it is the ratio of activity over total amount of protein.
- RFU/ mg (of protein) make sure to convert to mg!






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7) What are the two common procedures for quantifying protein
concentration that we used in this weeks lab?
8) What are the underlying principles of each of those procedures?
9) What are the advantages and disadvantages of each procedure?

Absorbance @ 260 nm/ 280 nm Bradford Assay
-Using spectrophotometer
-Measures absorbance max (280 nm)
caused by aa trp, tyr, and phe
- nucleic acids also absorb light at 280
so measure at 260 and use formula
-Measures absorbance of a sample with
varying wavelengths.
-standard curve to determine protein
amounts
-dye ( coomassie blue) binds primary
amines and carboxyls..Lys Arg
-the more the dye binds, the darker the
band and more absorbance

Advantages: Does not destroy your
sample, very rapid
rapid, reproducible, sensitive
Disadvantages: nucleic acids, turbidity
of solution, plus scratches on cuvette will
give false results. So you need purity!
Detergents interfere

Experiment 6- SDS/PAGE/ Commassie Blue Analysis of rGFP Fractions

1) What is the main purpose behind a SDS-PAGE gel?
To estimate the amount of a certain protein in a mixture of proteins.
- size via ladder
- purity

2) Be able to draw and correctly label a basic SDS-PAGE gel
Buffer, stacking gel, running gel, buffer
Negative electrode positive electrode
Wells, glass plates, spacer

3) What is the underlying principle behind SDS-PAGE gel?
First, denature proteins with detergent (SDS)this binds to proteins and makes
them negative.
Protein will be separated on the basis of size!...will be drawn to the positive
electrode (bottom of apparatus)
Smaller proteins move down faster, larger ones slower and on the top
Ladder helps determine molecular weight
So basically, you can tell size and how much of protein + purity is present

4) What is the purpose of SDS in the system?
Detergent, so it denatures proteins and makes them negative so they can be
separated based on size! (Smaller ones move down)
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5) What is the purpose of the B-mercaptoethanol in the sample-loading buffer?
- helps unfold proteins by breaking disulfide bonds
- even though SDS unfolds proteins, it DOES NOT disrupt disulfide cross-links
between polypeptide chains
- usually added with SDS
- if NOT added, there will be one heavier band vs. two lighter bands

6) What is the purpose of glycerol in the sample-loading buffer?
-Basically increases the density of the sampleloading aid
-non-ionic, not reactive
-adds viscosity

7) What is the purpose of bis-acrylamide?
Polyacrylamide gels are formed by polymerization of acrylamide with the cross-
linking agent bisacrylamide. Pore size of gel can be adjusted by varying amounts of
acrylamide and bisacrylamide. Lower amounts, larger pore size. Larger the pore
size, the faster the proteins will migrate.

Gives high porosity.
Cross linking agent (helps the acrylamide polymerize)
In stacking gel- causes the samples to be concentrated into a narrow zone at the top
of the resolving gel
APS-TEMED reactioncross linkage between acrylamide
Allows separation of the SDS-coated proteins by size

8) What would happen if the chemicals listed above were left out of the system?
- glycerol: sample might not sink because the samples are not dense enough.
Keeps in the loading zone until electrophoresis starts.
- Bis-acrylamide: there will be low porosity, we will not be able to load
samples into gel. Acrylamide will not polymerize and entire thing will be
ruined. The acrylamide will polymerize into long strands and not a porous
gel.
- SDS PAGE: proteins will not denature or run through the gel because of mass
- B-mercaptoethanol: disulfide bonds will not break and any proteins that has
a bridge will oligomerize

9) Given an appropriate figure of a SDS-PAGE gel, be able to determine: mw,
%purity, yield of protein
-MW: migration distance= log mw.
-using ladder
- more mw= less migration distance
- if you are given data of MW and migrations distance (y,x). Draw trend line,
and then extrapolate data
- %purity: how much protein of interest per total amount of protein in mg= specific
activity.
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- determine intensity of each band
- look at the boldness of color of each and estimate %
- yield: amount of protein of interest
ex: scaled, multiple by purity from actual experiment

10) Be able to construct and use a standard curve to determine the MW of a
protein.
-on y-axis log, so m=numbers are unevenly spaced out. Plot MW from ladder.
Negative slope because MW and migration distance are inversely related.
-measure bands from ladder in cm
-plot and draw standard curve
-extrapolate E3

11) Design an experiment to determine if a protein is monomeric or
oligomeric. If oligomerix, does the protein consist of only one type of subunit?
If oligomeric, are they held together by disulfide bonds?
Use B-mercaptoethanolget lighter band..without, darker

Experiment 8- SDS-PAGE/Western Blot

1) What is the main difference between staining and immunological detection?
-staining does not involve antibodies and immunological detection does
- immunological detection you use chromophore/chemi-luminescent (attached to 2
ab) and for staining, you use dye
- detection is more specific (one protein)
- staining detects all proteins

2) What are the three types of membranes discussed in class? Why is one chosen
over another?
1) Nitrocellulose: inexpensive, low tensile strength..breaks easily. Supported
nitrocellulose is a type that doesnt break as easily
2) Nylon: strong synthetic polyamide sheet, canreprobe and use multiple
reagents
3) PVDF (Polyvinylidene difluoride): high chemical resistance=high tensile
strength. High binding and retentive capacity. Expensive. Ideal for protein
sequencing and for using chemiluminescent detection system.

3) What is the purpose of incubating the membrane with BSA, gelatin, or a
mixture of dry milk?
These all are blocking reagents- saturate all the sites on the nitrocellulose that
are not already bound to the protein.
-if unoccupied sites are NOT blocked, 1 and 2 ab will bind non-specifically to
sited making it impossible to localize the protein of intrest.
-makes sure that our ab only bind where they are supposed to


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4) What is the difference between a primary and secondary antibody?
Primary antibodies are raised against a specific antigen: ex:human anti-flu
ab
Secondary is an antibody that binds to primary antibodies. Typically labeled
with probes( chromophores) that make them useful for detection. Ex: if we
want to expose anti flu ab to goat, we would develop goat anti human flu