PETER O.
CALONIA, JR ACTIVITY 8
ADVANCE BIOCHEMISTRY
SAQ 8-1
1. Given a mixture of the following proteins and their corresponding pI:
a. Human serum albumin 4.9
b. α1 -Lipoprotein 5.5
c. Hemoglobin A 7.1
d. Thymohistone 10.6
Sketch the migration pattern of the proteins after isoelectric focusing.
Label the bands and the terminals.
ANSWER
The proteins will migrate to the pH value in the gel that corresponds to
their isoelectric point (pI). The lower the pI, the further the protein will
migrate toward the acidic (low pH) end of the gradient. Conversely, the
higher the pI, the protein will migrate toward the basic (high pH) end.
Sketch and Labeling:
1. X-axis (pH gradient). This will range from acidic on the left (pH 1) to
basic on the right (pH 12).
2. Protein migration:
o Human serum albumin (pI = 4.9) will migrate to a position near
pH 4.9 on the gel, closer to the acidic end.
o α1-Lipoprotein (pI = 5.5) will migrate slightly to the right of
albumin, near pH 5.5.
o Hemoglobin A (pI = 7.1) will migrate closer to the neutral/basic
region, near pH 7.1.
o Thymohistone (pI = 10.6) will migrate towards the far right, near
pH 10.6.
Terminal Labeling
Left terminal. Acidic (low pH), where proteins with lower pI will focus.
Right terminal. Basic (high pH), where proteins with higher pI will
focus.
Sketch Description
A gel with a gradient of pH, from left (acidic, pH 1) to right (basic, pH
12).
Bands corresponding to each protein will be plotted:
o Human serum albumin at pI 4.9 (leftmost).
o α1-Lipoprotein at pI 5.5 (a bit to the right of albumin).
o Hemoglobin A at pI 7.1 (closer to the neutral zone).
o Thymohistone at pI 10.6 (rightmost).
PETER O. CALONIA, JR ACTIVITY 8
ADVANCE BIOCHEMISTRY
SAQ 8-2
2.A mixture of a homodimer, heterodimer and a monomer protein is
analyzed by SDS PAGE, with the following gel result:
kD M 1 2 3 4 5 6
80 --- --- --- --- ---
60 --- ---
40 --- ---
20 --- ---
Lanes 1-3, non-reducing conditions, Lanes 4-6, reducing conditions,
M = molecular weight marker. Interpret the gel results above. Determine the
molecular size of the following:
(a) subunits of the homodimer
(b) subunits of the heterodimer
(c) monomer
ANSWER
Molecular Size of Subunits
Homodimer. Each subunit is 40 kDa.
Heterodimer. The subunits are 60 kDa and 40 kDa.
Monomer. The monomer has a molecular weight of 20 kDa.
SAQ 8-3
3. Which of these techniques can separate proteins based on molecular size?
a) PAGE
b) Affinity Chromatography
c) Isoelectric Focusing
d) ELISA
e) centrifugal dialysis
ANSWER
a) PAGE
e) Centrifugal Dialysis
PETER O. CALONIA, JR ACTIVITY 8
ADVANCE BIOCHEMISTRY
SAQ 8-4
[Link] current method for sequencing proteins is more conveniently done by
sequencing the cDNA rather than the protein itself. Why is this so?
Answer
Sequencing cDNA is preferred over direct protein sequencing because
it is easier, faster, and more reliable. DNA is more stable, easier to amplify,
and provides a direct map of the protein's coding sequence without the
complications associated with protein isolation, degradation, and
modification. Moreover, modern sequencing technologies, such as next-
generation sequencing (NGS), allow for high-throughput analysis of cDNA,
making it a more practical and efficient method for determining protein
sequences.
SAQ 8-5
[Link] being labor-intensive, protein rather than DNA sequencing is still
resorted to in certain cases. What is the advantage of protein sequencing
over that of DNA?
ANSWER
While DNA sequencing is efficient and scalable, protein sequencing
offers crucial advantages in studying proteins that involve post-translational
modifications, alternative splicing, and complex conformations. It provides
direct, functional, and structural insights into proteins, their interactions, and
modifications that cannot always be inferred from DNA sequence alone.
Thus, protein sequencing remains invaluable in certain contexts, particularly
when studying functional proteins, novel proteins, and protein modifications
that are key to understanding biological systems.