Problem Set 3 Part 1

Colin Wakeham

Fraction

dAbs/dt

Units

Units/mL

Total Units

%
Recovery

[Protein]

Specific
Activity

Fold
Purification

Lysed

0.25

0.004019293

0.803858521

32.15434084

234.939759

188.8583272

Spun

0.23

0.003697749

0.739549839

28.10289389

87.4

114.4578313

84.64727076

0.448205128

FT

0.063

0.001012862

0.112540193

5.627009646

17.5

81.3253012

9.152365087

0.048461538

Wash

0.012

0.000192926

0.032154341

0.482315113

1.5

5.421686747

0.174330764

0.000923077

Pool

0.61

0.00980707

9.80707395

19.6141479

61

43.9759036

431.274939

2.283589744

Absorbtion at 595nM

Absorption vs Amt of Protein

1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

y = 0.0166x
R = 0.9477

20

40

60
Protein (ug)

80

100

120

Lysed Absorption vs Time

0.5
0.45

Absorbtion

0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
0

50

100

150

200

150

200

Time (s)

Spun Absorption vs Time

0.5
0.45

Absorption

0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
0

50

100

Time (s)

FT Absorption vs Time
0.12

Absorption

0.1
0.08
0.06
0.04
0.02
0
0

50

100

150

200

150

200

150

200

Time (s)

Wash Absorption vs Time

0.025

Absorption

0.02
0.015
0.01
0.005
0
0

50

100

Time (s)

Pool Absorption vs Time

1.2

Absorption

1
0.8
0.6
0.4
0.2
0
0

50

100

Time (s)

Lysed:
First 5 data points used
dAbs/dt = (0.26 0.01) / (1min) = 0.25
Units = (dAbs/dt) / [(6220) x (1)] x (106 uM/M) x (10-4 L) = 0.00402
Units/mL = units / 0.005mL = 0.804
Total Units = units / mL * 40mL = 32.154
% Recovery = total units Lysed / total units Lysed * 100 = 100
[Protein] = abs / 0.0166 * dilution = 234.040 (using the standard curve: abs = 0.0166[protein])
Specific Activity = units/mL * [protein] = 188.858
Fold Purification = specific activity / specific activity of Lysed = 1
Spun:
Data points 2-5 were used
dAbs/dt = (0.21 0.04) / (0.75min) = 0.23
Units = (dAbs/dt) / [(6220) x (1)] x (106 uM/M) x (10-4 L) = 0.00370
Units/mL = units / 0.005mL = 0.740
Total Units = units / mL * 38mL = 28.103
% Recovery = total units Spun / total units Lysed * 100 = 87.4
[Protein] = abs / 0.0166 * dilution = 114.458
Specific Activity = units/mL * [protein] = 84.647
Fold Purification = specific activity / specific activity of Lysed = 0.448
FT:
Data points 2-7 were used
dAbs/dt = (0.075 0.023) / ( 0.83min) = 0.063
Units = (dAbs/dt) / [(6220) x (1)] x (106 uM/M) x (10-4 L) = 0.00101
Units/mL = units / 0.009mL = 0.113
Total Units = units / mL * 50mL = 5.627
% Recovery = total units FT / total units Lysed * 100 = 17.5
[Protein] = abs / 0.0166 * dilution = 81.325
Specific Activity = units/mL * [protein] = 9.152
Fold Purification = specific activity / specific activity of Lysed = 0.0485
Wash:
Data points 2-4 were used
dAbs/dt = (0.008 0.002) / (0.5min) = 0.012
Units = (dAbs/dt) / [(6220) x (1)] x (106 uM/M) x (10-4 L) = 0.000193
Units/mL = units / 0.005mL = 0.0322
Total Units = units / mL * 15mL = 0.482
% Recovery = total units Wash / total units Lysed * 100 = 1.5
[Protein] = abs / 0.0166 * dilution = 5.422
Specific Activity = units/mL * [protein] = 0.174
Fold Purification = specific activity / specific activity of Lysed = 0.000923
Pool:
Data points 2-5 were used
dAbs/dt = (0.61 0.15) / (0.75min) = 0.61
Units = (dAbs/dt) / [(6220) x (1)] x (106 uM/M) x (10-4 L) = 0.00981
Units/mL = units / 0.005mL = 1.691
Total Units = units / mL * 2 mL = 3.923
% Recovery = total units Pool / total units Lysed * 100 = 12.2
[Protein] = abs / 0.0166 * dilution = 43.976
Specific Activity = units/mL * [protein] = 86.255
Fold Purification = specific activity / specific activity of Lysed = 0.457