Experiment 2 Lab Report

Khai Nguyen
CHEM 443-05 Dr. Surya Manandhar
13 Feb 2015

Results
S

10
Table 1. Migration distances measured from each of the bands in the
standard lane (lane #1) to its corresponding wells bottom. Solvent
front is 105.0 mm. Bio-RADs Precision Plus Protein Dual Color
Standards (cat. #161-0374) is used in both lanes #1 and #6.

250 kDa
150
100
75

Standar
d

50
37

250

2.40

22.0

0.21

150

2.18

28.8

0.27

25
20
15
10

100

2.00

37.1

0.35

75

1.88

43.0

0.41

50

1.70

53.5

0.51

37

1.57

61.3

0.58

25

1.40

72.0

0.69

20

1.30

77.2

0.74

9
10

15
10

1.18
1.00

83.8
90.7

0.80
0.86

SOLVENT FRONT

Figure 1. Gel electrophoresis of various proteins in Bio-RADs MiniPROTEAN 3 Cell with Mini-PROTEAN TGX Precast Gel (7.5%
polyacrylamide, cat. #456-1023) and Precision Plus Protein Dual
Color Standards (cat. #161-0374). Proteins in 2X SDS non-reducing
sample buffer (0.124 M Tris chloride, pH 6.8, 4% (w/v) SDS, 20%
(v/v) glycerol) are: BSA (lane 2), IgG (lane 3), and LDH (lane 4).
Proteins in 2X SDS reducing buffer (non-reducing buffer added with
10% (v/v) 2-mercaptoethanol) are: BSA (lane 7), IgG (lane 8), LDH
(lane 9), and histone H1 (lane 10). Lanes labeled with S contain the
standards.

MW
(kDa)

log
MW

Distance
(mm)

Rf

2.40
2.20
2.00

f(x) = - 1.99x + 2.74

R = 0.99

1.80

Molecular weight (log) 1.60

1.40
1.20
1.00
0.80
0.15

0.35

0.55

0.75

0.95

Rf
Figure 2. Standard curve obtain via linear regression analysis, which
result in equation (1).

From the calibration curve, a relationship between molecular weight

following equation.

(in kDa) and Rf can be expressed in the

log M =1.99 R f +2.74

Equation (1) allows determination of proteins whose molecular weights are shown in Table 2.

(1)

Table 2. Migration distances and Rf values along with molecular weights (MW) estimated from equation (1). As with the standards, the distances are
measured from each band to its corresponding wells bottom. Solvent front distance is 105.0 mm.

Protein

Distance (mm)

BSA
IgG
LDH

53.0
33.5
65.6

Rf

MW (kDa)

Proteina

Distance (mm)

54.4
127
31.4

BSA
IgG Band 1
IgG Band 2
LDH
H1

47.4
54.0
72.5
66.4
68.0

0.50
0.32
0.62

Rf
0.45
0.51
0.69
0.63
0.65

MW (kDa)
69.4
52.1
23.2
30.3
28.3

These bands were obtained using proteins in reducing buffer.

Discussion
BSA is likely composed of a single subunit since it has only one band in electrophoresis in both reducing and nonreducing buffer. However, it may contain intra-chain disulfide bonds. As the reducing buffer breaks these bonds, the
molecule becomes more elongated (or linear). As a result, its migration through the gel is retarded with a higher degree
than that observed in globular molecules. This is because linear molecules have more contact with the gel matrix, resulting
in greater friction. Its electrophoretic mobility, therefore, is lowered.
LDH, on the other hand, does not appear to have any of these intra-chain disulfide bonds since the migration distances in
both non-reducing and reducing buffer are almost the same. Additionally, LDH only contains a single subunit since only
one band is produced in both reducing and non-reducing buffer.
Electrophoresis of IgG in non-reducing buffer produces only one band. However, two bands are observed in reducing
buffer. Moreover, the sum of MW for these two bands are approximately half of MW of a single band in non-reducing
lane. Hence, IgG is likely to contain two pairs of identical subunits, each of MW 26.05 kDa (heavy chain) and 11.6 kDa
(light chain).
Histone H1 is likely to be similar in size and/or shape to LDH as its migration distance is close to that of the latter. Further
experiment is required to determine its number of subunits and intra-chain crosslink (if any).

Calculations
Migration band distance measurement: Distance from each band is measured with respect to the bottom of the well of the
corresponding lane. The measurement was done on a printed copy of the gels image.

Rf : For standard #1,

Rf =

22.0
=0.21 . Other
105.0

Rf

values are calculated in a similar fashion.

Standard curve linear regression analysis: Logarithm (base 10) of the standard MWs is plotted against its corresponding
Rf values. Linear regression is performed on a computer spreadsheet program.
MW from standard curve: For BSA in non reducing buffer:
calculated in a similar fashion.

M BSA=101.99 (53.0 /105.0 )+2.74=54.4 kDa . Other MWs are

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