LABASSIGNMENT
PROTEIN ELECTROPHORESIS
PART I. EXTRACTION OF ERYTHROCYTE GHOST PROTEINS
1) A single lipid has a polar head group and a hydrophobic tail. In aqueous environment, hydrophobic
tails of lipids assemble due to hydrophobic effect and form a lipid bilayer. This shields the
hydrophobic tails from water.
There are two classes of membrane proteins: integral proteins and peripheral proteins.
Integral proteins have at least one transmembrane hydrophobic domain and associate with
membrane through hydrophobic forces.
Peripheral proteins associate with membrane by interacting with lipid head groups and/or integral
proteins. They can interact with lipid head groups through ionic interactions, hydrogen bonding,
amphipathic α-helix and/or hydrophobic loop.
2) Integral proteins must be isolated with detergent, which can disrupt membrane and solubilize the
integral proteins.
Peripheral proteins can be isolated by changing ionic strength of the buffer, which disrupts ionic
and hydrogen bond interactions.
3) TX-100 is non-ionic while SDS is anionic. SDS can disrupt protein-protein interactions and denature
proteins while TX-100 can’t and proteins retain their 3D structures. Tween-20 can be used and is a
non-ionic detergent.
PART II. SDS-PAGE OF EXTRACTED RBC GHOST PROTEINS
1) Labelled image of SDS-page of the erythrocyte membrane proteins
Table of the sample in each lane.
Lane
1
2
3
4
5
6
7
8
9
10
Samples
Molecular weight marker
Total untreated
Supernatant untreated
Pellet untreated
Total TX-100 treated
Supernatant TX-100 treated
Pellet TX-100 treated
Total carbonate treated
Supernatant carbonate treated
pellet carbonate treated
2) Table of Rf value for the molecular weight markers.
Molecular weight/ kDa
Distance travelled/unit
Rf*
180
1.8
0.21
130
2.1
0.24
100
3.0
0.35
70
3.7
0.43
55
4.4
0.51
40
5.4
0.63
35
6.2
0.72
25
7.0
0.81
15
8.0
0.93
*: Total distance travelled by dye front = 8.6 unit. Rf = Distance travelled / Total distance.
Molecular weight standard curve
3) Table of identification of unknown bands
Band
Calculated Rf
Interpolated MW
number
value
/kDa
1
0.14
2
0.16
3
0.18
4
0.30
Greater than the
highest MW standard
Greater than the
highest MW standard
Greater than the
highest MW standard
100
Potential identity
Spectrin α chain
Theoretical
MW /
kDa1
280
Spectrin β chain
275
Ankyrin-1
206
Band 3 anion transport
102
protein
5
0.34
90
Protein 4.1
97
6
0.37
83
Erythrocyte membrane
77
protein band 4.2
7
0.51
55
Actin
41
8
0.58
48
Glyceraldehyde 336
phosphate dehydrogenase
9
0.78
28
Erythrocyte band 7
31
integral membrane
protein
All major bands are preliminarily identified. It’s worth mentioning that band 9 is blurry on the gel.
Bands from 5 to 8 showed a relatively large discrepancy between interpolated MW and theoretical
MW. Band 1, 2, 3, 5, 6, 7, 8 are peripheral membrane proteins while band 4 and 9 are integral
proteins.
4) Table of identified bands in each lane.
Lane
Identified bands
Expected bands
2
Band 1-9
Band 1-9
3
No band
No band
4
Band 1-6, 8.
Band 1-9
Faint band 7, 9.
5
6
7
8
9
10
Band 1-2.
Faint band 8.
Band 1-3, 5, 6, 8.
Faint band 4, 7.
Band 1-6, 8.
Band 1-9.
Band 1-9.
No band.
Band 1-9
Band 1-9
No band
Band 1-9
Band 1-3 and 5-8
Band 4 and 9
Comparison
In untreated sample, membrane proteins
still associate with the membrane debris.
The results showed that all proteins end
up in pellet and no protein in
supernatant as expected. However, band
7 and 9 are not obvious.
In TX-100 treated sample, membrane
structure is disrupted, and membrane
proteins are released. All proteins are
expected to end up in supernatant.
However, the results are quite different.
In carbonate treated sample, peripheral
membrane proteins are expected to end
up in supernatant while integral proteins
in pellet. However, the results are
different.
Explanation: It’s highly likely that carbonate treated and TX-100 treated samples are mixed up
since the expected bands in lane 5-7 correspond to the results in lane 8-10. However, results in lane
5-7 doesn’t correspond to the expected bands of carbonate sample.
It’s worth mentioning that a breakage between loading cells of lane 5 and 6 may have affected the
results. It’s possible than part of the contents in well of lane 5 leaked into lane 6 and the contents
left in lane 5 is not enough to generate obvious bands. This explains why only a few bands are seen
in lane 5. But the contamination (band 4, 9) in lane 6 is not obvious.
In case of lane 7, more than expected bands are seen. It’s possible that TX-100 failed to disrupt all
the membrane debris probably due to the inadequate amount of TX-100.
5) GPA is not very abundant (< 2%) in erythrocyte membrane while Coomassie staining can only give
a detectable band given 10-50 ng of a protein.
6) Coomassie staining is more sensitive to basic amino aids. Moreover, detergents or organic solvents
can interfere with dye-protein interactions. These factors cause some variabilities in staining.
Method like silver staining can be applied to help definitive identification.
PART III. NATIVE AGAROSE GEL ELECTROPHORESIS
1) Labelled agarose gel image
Table of agarose gel samples
Lane
1
2
3
4
5
6
7
Sample
HbA standard
HbS standard
Jane
John
Lisa
Billy
Jimmy
2) A single amino acid substitution (Glu to Val) in β-chain of hemoglobin.
3) HbA is normal and properly folded while HbS aggregates into long, chainlike polymeric structures.
Since HbA is more compact than HbS, HbA moves farther than HbS.
4) Hemoglobin is negative charged at pH 9.2. Since the charge of amino acids change from positive to
negative as pH increase, pI of hemoglobin is below pH 9.2. This corresponds to lab 1 result that pI
of hemoglobin is around pH 7.1.
5) The motility ranks as such: lane 1 = lane 5 > lane 3 = lane 4 = lane 6 > lane 2 = lane 7. The greatest
motility corresponds to Lisa and HbA standard, which indicates that Lisa has normal hemoglobin.
The medium motility corresponds to Jane, John and Billy, which indicates they have both normal
and sickle hemoglobin, or they are the carriers of sickle hemoglobin gene. The lowest motility
corresponds to Jimmy, which means he has only sickle hemoglobin and is a patient of sickle cell
disease.
6) BSA has a pI of around pH 6.5, which is lower than hemoglobin. BSA probably won’t denature at
pH 9.2. BSA also has a compact structure but bears more negative charge than hemoglobin and is
expected to have greater motility on the gel.
Reference
1. The UniProt Consortium UniProt: a worldwide hub of protein knowledge. Nucleic Acids Res. 47:
D506-515 (2019).