Essay on Exp 4

Submitted By cpandrews
Words: 629
Pages: 3

Experiment 4

Post-Lab

Milliliters of 20,000 x g supernatant: 32.5
Grams (NH4)2SO4 used for 40% cut: 7.475
Milliliters of 40% supernatant: 34
Grams of (NH4)2SO4 used for 65% cut: 5.644
Milliliters of 65% supernatant: 35
Milliliters of 65% pellet after resuspending: 3

Fraction Isolated
Sample Assayed μL
Dilution Made
Absorbance Change/min
20,000 x g supernatant
1000
none
.846
(.973-.127)
40% (NH4)2SO4 supernatant
1000
none
.355
(.499-.144)
65% (NH4)2SO4 pellet
1000
none
.662
(1.224-.562)
65% (NH4)2SO4 supernatant
1000
none
.181
(.296-.115)

Bradford Assay

LDH Fraction
Quantity Assayed μL
Dilution Used
Absorbance
20,000 x g supernatant
10
1:20
.352
40% (NH4)2SO4 supernatant
10
1:20
.104
65% (NH4)2SO4 pellet
10
1:20
.275
65% (NH4)2SO4 supernatant
10
1:20
.081

1. Calculation of activity and reaction activity.
Activity= change in absorbance/change minutes x 10^6μM/M x .001L= μmole/min 6200M-1cm-1
For example the 20,000 x g supernatant: .846A/Min x 10^6 μM/M x .001L =.136μmole/min 6200M-1cm-1
The same calculation was carried out for each absorbance; the results are in the table.

Relative Activity=units/volume assayed
For example the 20,000 x g supernatant: .136/1mL=.136

The same calculation was carried out for each absorbance: the results are in the table.
2. Total Units
Total Units= relative activity x total volume fraction
For the first absorbance .136/mL x 32.5mL=4.42 total units
The same calculation was done for each absorbance using the total volume for the fraction. The results are in the table.

3. Protein Concentration
Found using the standard Bradford curve from the previous lab. The chosen protein concentrations are in the following table.

4. Specific Activity
Relative Activity/quantity protein
Ex: for absorbance number 1 .136/16=.0085
The same concept was used for each calculation, in the table below.

Fraction
Units
Units/mL
Total Units
Protein (mg/mL)
Specific Activity
20,000 x g supernatant
.136
.136
4.42
16
.0085
40% (NH4)2SO4 supernatant
.057
.057
1.94
5.9
.0097
65% (NH4)2SO4 pellet .106
.106
.318
13
.0082
65% (NH4)2SO4 supernatant
.029
.029
1.02
5
.0058

Questions
1. The change in absorbance/change in minute was .622. The relative activity would then be .30/3mL times 1/6 which equals .0167.
4. A percent saturated solution of ammonium sulfate is the max amount of solute that can be…