Isolation & Characterisation of Proteins. The purpose of conducting this experiment is to study protein separation using two different methods, which are the SDS-polyacrylamide gel and ion exchange chromatography

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Name: Ng Yen Pheng

Student ID: 22353046

Day and date: Tuesday, 3 April 2012

Title: Isolation & Characterisation of Proteins

Aim:

The purpose of conducting this experiment is to study protein separation using two different methods, which are the SDS-polyacrylamide gel and ion exchange chromatography. This practical also aims to study what types of ion exchangers which are more suitable to separate different types of proteins. This experiment also aims to compare the separation of proteins by SDS-PAGE and ion exchange chromatography.

Results:

Part A: SDS-PAGE

Figure 1: Image of SDS-Polyacrylamide gel electrophoresis.

Table 1: Distance migrated (cm) by different length of protein fragments denatured by SDS

[( - ) in the table indicates that the band is missing]

Figure 2: Graph of log (Molecular weight) against distance migrated for marker.

Table 2: Distance migrated and size of fragments of 3% haemoglobin being denatured by SDS with different dilution factor in the presence of 1% of BSA.

Calculation:

To calculate molecular weight for samples

Using the equation in the graph above, y = -52.836 x + 139.1 where x = log (MW)

For sample A, when the protein fragment has migrated 43 mm

y = 43

43 =

x =

   = 1.819

Molecular weight = 101.819

     = 65.892

Another band in sample A migrated 90 mm

y = 90

90 = -52.836 x + 139.1

x =

   = 0.929

Molecular weight = 100.929

     = 8.497

For sample B, when the protein fragment has migrated 44mm

y = 44

44 = -52.836 x + 139.1

x =

  = 1.780

Molecular weight = 101.780

     = 63.083

Another band in sample B migrated 91 mm

91 = -52.836 x + 139.1

x =

   = 0.910

Molecular weight = 100.910

     = 8.135

For sample C, when the protein fragment has migrated 7 mm,

45 = -52.836 x + 139.1

x =

x = 1.781

Molecular weight = 101.781

     = 60.392

Another band in sample C migrated 92 mm

92 = -52.836 x + 139.1

x =

x = 0.891

Molecular weight = 100.891

     = 7.788

Part B: Ion-Exchange Chromatography

Determination of haemoglobin

i) DEAE column

Table 3: Absorbance, mass and concentration of diluted haemoglobin sample tubes in DEAE column

Calculation:

To calculate concentration of 1% eluent fraction, using the formula A = εcl, provided that ε (1%) = 3.5, where A = absorbance, ε = coefficient extinction, c = concentration, l = path length

Concentration of heamoglobin in eluent fraction (1%) in tube 1, g/mL

By using equation A = εcl,

0.002 = 3.5 x c x 1

c =

  = 5.714 x 10-4 g/100mL

Concentration of heamoglobin in eluent fraction (3%) in tube 1, g/mL

5.714 x 10-4 g/100mL x 3

= 1.714 x 10-3 g/100ml

100ml consists of 1.714 x 10-3 g

1ml consists of   = 1.714 x 10-5 g/ml

Mass of haemoglobin eluent fraction (3%) in tube 1, µg

Concentration of haemoglobin = 1.714 x 10-5 g/ml

Thus, mass of haemoglobin, µg = 1.714 x 10-5 g/ml x 106

                                                   = 17.143µg

Same method of calculation was applied to tube 2 – 10 included diluted samples in order to calculate mass and concentration of haemoglobin eluent fraction (3%).

Total mass of haemoglobin recovered in DEAE column from tube 1 to 10, µg

= 17.14 + 128.57 + 771.43 + 1037.14 + 471.43 + 462.86 + 437.14 + 368.57 + 171.43 + 77.14 = 3942.85 µg

Percentage recovery of haemoglobin in DEAE column, %

= [  ] × 100%

=  × 100%

= 98.28 %

Figure 3: Elution profile of mass of haemoglobin against test tubes haemoglobin (3%) in DEAE column

ii) CM column

Table 4: Absorbance, mass and concentration of diluted haemoglobin sample tubes in DEAE column.

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Calculation:

To calculate concentration of 1% eluent fraction, using the formula A = εcl, provided that ε (1%) = 3.5, where A = absorbance, ε = coefficient extinction, c = concentration, l = path length

Concentration of heamoglobin in eluent fraction (1%) in tube 11, g/100mL

By using equation A = εcl,

0.012 = 3.5 x c x 1.0

c =

  = 3.429 x 10-3 g/100mL

Concentration of heamoglobin in eluent fraction (3%) in tube 11, g/100mL

3.429 x 10-3 g/100mL x 3

= 0.0103 g/100ml

Mass of haemoglobin in each eluent fraction (3%) in tube 1, µg/mL

Concentration ...

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