BCHE5080. BIOCHEMISTRY: HW 2 - Answers for Protein
Purification 100% Correct.
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Pre Activity Assignment
Critical Thinking Questions
1. Which technique(s) could you utilize to separate two proteins that (a) differ greatly by size, (b) differ
by pl, and (c) that have similar physical characteristics (e.g. size and pl), but have very different
functional characteristics? In each case briefly describe how the separation would be accomplished.
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2. On the computer go to the link https://www.youtube.com/watch?v=IWZN_G_pC8U and watch the
animation. In this example both protein samples contain 3 bands. Does that mean that we have 3
different proteins in each fraction or are there other possibilities? Discuss the different options (if any).
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3. Imagine protein with subunits. This 100 kDa protein has two subunits (70 kDa and 30 kDa) joined by
two disulfide bonds. Draw the gel electrophoresis pattern for SDS-PAGE with a lane that includes 2mercaptoethanol (2-ME) and one lane without 2-ME. Be sure to include a molecular weight (MW)
marker lane. Note: 2-ME is one of two commonly used reducing agents. The other is DTT
(dithiothreitol). Explain your drawing. Are subunits always kept together with disulfide bonds?
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4. Both SDS-PAGE and gel filtration chromatography are used to separate proteins based on size. On
the computer go to the link https://www.youtube.com/watch?v=oV5VB5kO3tQ and watch the
animation. Imagine you have a mixture of proteins:
(a) If your experimental goal is to determine as accurately as possible the molecular weight of
proteins in the mixture, would gel filtration or SDS-PAGE be preferable? Why?
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(b) If your experimental goal is to collect each intact protein for further analysis, would gel filtration
or SDS-PAGE be preferable? Why?
5. Watch the animation on ion exchange chromatography on the GE healthcare site:
http://proteins.gelifesciences.com/knowledge-library/video-hub/ or
https://www.youtube.com/user/gelifesciences.
A graph is generated during the purification procedure, the so-called elution profile. Redraw the graph
and label all the axes. Explain how such a graph is obtained in the lab. Refer to the labels of the x- and
y-axes to help determine what data were collected. What is being detected in the broad gray peak?
What is being detected in the colored peaks? How would a scientist wishing to isolate a particular
protein use the information given in the plot?
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6. What techniques can be used to determine the amount of a specific protein in a sample? What
characteristic(s) of the protein does each technique take advantage of?
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7 An isoelectric focusing experiment with a protein sample shows that there are 3 protein present with a pI
of 3 (protein A), 7 (protein B) and 9 (protein C). Since the focusing experiment can only be done with
very small amounts of protein an ion exchange column purification step is designed to separate the
three proteins. Describe how you would design this experiment: What column will you use (cation or
anion exchange)? What will be the pH of the buffer used in the procedure? Draw the expected elution
profile for your column.
8. A biochemist discovers and purifies a new enzyme, generating the purification table below. This is
another example of a more realistic set of steps when trying to purify an enzyme for the first time.
This includes a lot of trial-and-error. A good examination of the purification table will help to remove
unnecessary steps for future purifications.
activity in units
(µmol•min-1)
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Procedure or step
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total protein
(mg)
specific activity
(units•mg-1)
1. Crude extract
4,000,000
20,000
200
2. Precipitation (salt)
3,000,000
5,000
600
3. Precipitation (pH)
1,000,000
4,000
250
4. Ion-exchange chromatography
800,000
200
4000
5. Affinity chromatography
750,000
50
15000
6. Size-exclusion chromatography
675,000
45
15000
(a) From the information given in the table, calculate the specific activity of the enzyme after each
purification procedure. Why do we calculate this?
(b) Which of the purification procedures used for this enzyme is most effective (i.e., gives the greatest
relative increase in purity)?
(c) Which of the purification procedures is least effective?
(d) Is there any indication based on the results shown in the table that the enzyme after step 6 is now
pure? What else could be done to estimate the purity of the enzyme preparation?
(e) Would you advice the biochemist to perform the purification the same way the next time he performs
this or would you recommend any changes in the procedure. If any, what changes would you
recommend?
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9. Refer to the purification table for methyl-coenzyme M reductase. Describe and discuss the different
trends in the different columns of the table. For example, the total amount of protein is going down. Is
that a good thing or a bad thing?
10. Shown in this document is a very polished purification procedure for methyl-coenzyme M reductase.
The first time this enzyme was purified, however, the information provided would not have been
enough to decide it was pure at this stage. Discuss, why that is and what other purification steps and or
procedures would have been performed to conclude that the enzyme was pure after the Q-sepharose
step.
After you did this, the professor will provide you with data for an additional purification step. Discuss
this new data. Does it confirm your proposal?
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Purification Table
Procedure or step
activity in units
(µmol•min-1)
activity
(%)
total protein
(mg)
specific activity
(units•mg-1)
7970
7030
4830
4040
5140
4980
100
88
61
51
65
62
3789
3981
521
452
230
225
2.1
1.8
9.3
8.9
22.3
22.1
After sonication
cell extract
70% (soluble fraction)
100% (resuspended pellet)
Q-sepharose
Superdex-200
Purification profile for Superdex-200
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