Purification Table Calculations

Corrected Activity
Activity Concentration Total
Volume
diluted Undiluted
of Protein
Activity
(ml)
(units/ml) (units/ml)
(mg/ml)
(units)

Total
Protein
(mg)

Fraction

Volume
(ml)

homogenate

148.0

148

1S

42.5

87.3

0.01797

215.661

109.919

18827.2

2S

57.5

118.0

0.00713

128.370

57.236

15147.6 6753.82

2P

15.3

31.4

0.03537

2.122

-20.163

66.6427 -633.11

3S

60.0

123.1

0.02710

1.626

5.854

200.163 720.585

3P

15.3

31.1

0.01184

142.06247

151.220

4418.14 4702.93

3P-D

16.0

32.6

0.0407930 489.51647

117.073

15958.2 3816.59

Pooled

2.3

8.3

0.0071317

21.395

1.626

177.578 13.4959

Conc

0.9

3.3

0.0138354

41.506

-5.772

135.31

pooled = volume of combined fractions

9595.9

-18.818

Specific
Activity
(units/mg)

Yield (%)

A340/min

Dilution
Factor

Dye Binding

Dilution

Absorbance
to Protein

1.96

100.0

0.126

200

0.427

200

549.593496

2.24

80.5

0.05

300

0.265

200

286.178862

-0.11

0.4

0.248

0.027

200

-100.81301

0.28

1.1

0.19

0.107

200

29.2682927

0.94

23.5

0.083

200

0.554

200

756.097561

4.18

84.8

0.286

200

0.449

200

585.365854

13.16

0.9

0.05

50

0.109

50

32.5203252

-7.19

0.7

0.097

50

0.018

50

-115.44715

A595 = 0.0137x
109918.70

109.919

57235.77

57.236

-20162.60
5853.66

-20.163 ***
5.854

151219.51

151.220

117073.17

117.073

1626.02
-5772.36

1.626 ***
-5.772

x = A595/0.0137

LDH Activtities
0.700

0.600

0.500
Absorbance in A340

LDH Activities
Absorbance at 340nm vs time
1:400 dilution
Time
Absorbance
0
0.623
5
0.617
10
0.609
15
0.602
20
0.597
25
0.590
30
0.584
35
0.578
40
0.571
45
0.565
50
0.557
55
0.552
60
0.547
65
0.540
70
0.533
75
0.526
80
0.520
85
0.514
90
0.507
95
0.501
100
0.494
105
0.488
110
0.482
115
0.476
120
0.470
125
0.463
130
0.457
135
0.450
140
0.445
145
0.438
150
0.432

0.400

0.300

0.200

0.100

0.000
0

20

40

LDH Activtities - Absorbance at 340nm vs time

y = -0.0013x + 0.6221
R = 0.9999

40

60

80
Time (Seconds)

100

120

140

160

160

dilution

fraction

0.076

1:400

0.0025 4.33604336

0.124

1:200

0.005 7.07459706

0.233

1:00

0.01 13.2933961

0.073

1:300

0.00333333 4.16488375

0.0025

0.076

0.005

0.124

0.01

0.233

A340/min v
0.25

0.2

A340/min

A340/min

0.15

0.1

0.05

0.00333333

0.073
0
0

A340/min versus Enzyme Concentration

y = 22.092x + 0.0114
R = 0.9858

0.002

0.004

0.006
0.008
enzyme concentration

0.01

0.012

Activity vs. Fraction Number

Fraction #
1
2
3
4
5
6
7

A340/min
Dilution
Activtity
0.002
1 0.00028527
0.01
1 0.00142633
0.01
1 0.00142633
0.053
100 0.75595493
0.054
400 3.08087291
0.024
100 0.34231921
0
1
0

Fraction #
1
2
3
4
5
6
7

Activtity
0.0003
0.0014
0.0014
0.7560
3.0809
0.3423
0.0000

Activtity
3.5000
3.0000

Units/ml protein

2.5000
2.0000
1.5000
1.0000
0.5000
0.0000
0

4
Fraction #

Activtity

Standard Curve

Dye-Binding Stan
1.4

1.2

Absorbance A595

mgrams protein Absorbance

0
0
10
0.229
20
0.375
30
0.511
40
0.543
50
0.709
60
0.885
70
1.003
80
1.02
90
1.146

0.8

0.6

0.4

0.2

0
0

10

20

30

40

Binding Standard Curve

y = 0.0123x + 0.089
R = 0.9799

40

50
mgrams of protein

60

70

80

90

100