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Name: Date: September 20th 2010

Biochemistry Lab: SDS-PAGE of Proteins.
A) Measure the relative mobility of the protein standards and of the two other proteins
A/B/C provided. Record your data in the form of a suitable table.

Using equation 1:
Relative Mobility = Distance moved by Protein/Distance Moved by Very Small Molecule
Distance moved by very small molecule = 5.5cm
TABLE 1: RELATIVE MOBILITY OF PROTEIN STANDARDS AND PROTEIN A
AND B

Protein Standard
Band Distance moved by Protein Relative
(cm) Mobility
1 2.1 0.382
2 2.6 0.473
3 3.2 0.582
4 4.0 0.727
5 5.1 0.927
Dye Front 5.5 1.000
Protein A
Band Distance moved by Protein Relative
(cm) Mobility
1 3.4 0.618
2 5.1 0.927
Protein B
Band Distance moved by Protein Relative
(cm) Mobility
1 2.6 0.473
2 3.4 0.618
3 3.8 0.691
4 4.9 0.891
Protein C
Band Distance moved by Protein Relative
(cm) Mobility
1 1.4 0.255
2 3.1 0.564
3 5.1 0.927
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B) Using the data generated from the standards, plot a graph of Log Mol Wt versus
relative mobility.

Table 2: Table showing the Log of Molecular Weight (MW) of protein standards and
their Relative Mobility (Rf).

Molecular Weight 6500 14200 20000 24000 29000

(MW) of standards
(Daltons)
Log (Molecular 3.813 4.152 4.301 4.380 4.462
Weight)
Rf Standard 0.927 0.727 0.582 0.473 0.382

Please see attached for Graph showing the Log (Molecular Weight) versus Relative
Mobility for the Protein Standard

C) Deduce the identity of your proteins using your graph and the relative mobility of A/B/C
together with your absorption spectra.

Relative Y Intercept Approximate

Mobility Molecular Weight
(x-axis) (Da)
Protein B1 0.473 4.40 25,118
Protein B2 0.618 4.23 16,982
Protein B3 0.691 4.15 14,125
Protein B4 0.891 3.94 8,709
Protein C1 0.255 4.60 39,810
Protein C2 0.564 4.30 19,952
Protein C3 0.927 3.92 8,317
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D) Compare your SDS-PAGE results to that of the published data provided; account for your
results including any differences.

Wavelength of Max. Absorbance (nm)

Protein B (non-reduced) 410
Protein B (reduced) 425
Protein C (non-reduced) 410
Protein C (reduced) 415
Hemoglobin (non-reduced) 407
Hemoglobin (reduced) 429
Myoglobin (non-reduced) 409
Myoglobin (reduced) 433
Cytochrome C (non-reduced) 409
Cytochrome C (reduced) 414

Based on the results the molecular weight of myoglobin was more than that of the
published data (17000) and the cyctochrome c value was less than that of the published
data (12000). These values were mainly as a result of :

1) Human error in measuring distances moved by the dye front and proteins.

2) Improper loading of protein solution

3) Breaking off of side chain

4) Allowing the gel to run for an incorrect time period

E) What changes, if any, would you expect if sickle rather than normal hemoglobin was
employed.

A molecule of Hb S contains two normal -globin chains and two mutant -

globin chains (S), in which valine replaces glutamate at the sixth position. Due
to this alteration, during electrophoresis, Hb S migrates more slowly to the
anode in alkaline pH than normal hemoglobin (Hb A). This reduced mobility is as
a result of the absence o the negatively charged glutamate residues I the two
chains, making the Hb S less negative than Hb A.

If Hb S was employed no change would be observed because in SDS-PAGE gels,

distance migrated will be a function of the size of the polypeptide, and not the
charge.
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F) Compare the advantages and draw back for SDS-PAGE and exclusion chromatography.

Both SDS-PAGE and exclusion chromatography separate proteins based on their primary
structure or size, but not amino acid sequence. Therefore, if two different proteins that
were both the same size, they would travel together through the gel in a mixed band. As a
result, we would not be able to use SDS-PAGE or exclusion chromatography to separate
these two proteins of the same molecular weight from each other. On the other hand, both
these methods provide good sensitivity providing good separation between large and small
molecules.