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Affinity
Chromatography
of Lactose
Dehydrogenase

Title: Affinity Chromatography of Lactose Dehydrogenase

Aim:
Method: As seen in BC21D Lab Manual 2009 pp 42-48. The following changes
were made to the method shown:
-

The Bradford assay was done using BSA following the method given

on page 28 of the BC21D lab manual.

A 1:10 dilution was done for the BSA sample and also for the
fractions and supernatant.

Sample Calculations

Volume of Pyruvate
From the calibration curve; y = 0.716+ 0.023
For supernatant

x=

x = 0.326 ml
Assuming that the relationship between mls pyruvate and moles pyruvate
formed/min is linear
0.3 mls = 0.5 mol/min

0.326ml =

= 0.54469 mol/min
1 mol = 1000mmol
0.54469 mol = 544.69mmol
Activity

Activity =

= 10893.86
Since a 1:10 dilution was done activity = 10800 * 20 = 217877.10
Bradford estimation of protein concentration
Using concentration 0.05;

C1V1=C2V2

1.0mg/ml*V1=0.05mg/ml*0.1ml
V1 =0.005ml5l
Therefore amount of water required = 100l - 5l = 95l

Using calibration curve from the Bradford assay:

For supernatant :y = 0.764x +0.031

x=

x=
x = 0.102 mg/ml
Since a 1:10 dilution was done the concentration = 10 * 0.102mg/ml
Therefore protein concentration in fraction 1 = 1.02 mg/ml

Table
Samp

Volume/

Protei

Activity

Specific

Purificati

le

ml

mmol/min/

Activity

on Factor Activity

(mg/

ml

mmol/min/

mmol/m

mg

in

ml)
S

0.2

4.59

217877.1

47467.78

F1

2.0

1.02

11638.73

11410.52

0.222222

F2

2.0

0.76

24906.89

32772.22

0.165577

F3

2.0

1.02

11871.51

11638.73

0.222222

F4

2.0

0.67

10242.09

15286.70

0.145969

F5

2.0

1.22

4189.944

3434.38

0.265795

F6

2.0

0.56

5121.043

9144.72

0.122004

Total

43575.4
2
23277.4
7
49813.7
8
23743.0
2
20484.1
7
8379.88
8
10242.0
9

Recovery
of Activity/
100%
100
53.4188
114.3162
54.48718
47.00855
19.23077
23.50427