t=0 sec

t=15 sec

t=30 sec

t=45 sec

Crude
extract
PBS
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
Pre-lab questions:
1. Table 1: Absorbance values at 450 nm for lysozyme assay

t=60 sec

2. Table 2. Absorbance values at 595 nm for Bradford assay

Tube Number

Sample Name

Blank

2
3
4
5
6
7
8
9
10
11
12

Protein
Concentration
0.00 mg/ml

Added dye or
protein
0.50 L dH2O; 1.5
mL dye

OD at 595
0.00

crude enzyme
crude enzyme
pooled enzyme
fraction

3. Table 3: Serial dilution procedure

Tube
1
2
3
4

Initial BSA conc.

200 l of 2 mg/ml
200 l of 1 mg/ml
200 l of 0.5 mg/ml
200 l of 0.25 mg/ml

dH2O
200 l
200 l
200 l
200 l

Final BSA conc.

1 mg/ml
0.5 mg/ml
0.25 mg/m
0.125 mg/ml

4. In the lysozyme assay, increased activity actually makes for a lower absorbance reading,
due to the fact that lysozyme breaks down cell walls, providing for less scattering of
light.

5. Absorbance is due to molecules in the solution blocking light. The solution itself prevents
some light rays to pass through. Light scattering occurs when particles like bacteria in a
solution refract light and the light beams dont reach the sensor on the other side.
6. Specific activity is a measure of enzyme purity as it is units of activity/amount protein. It
should express the retaining of the enzyme and loss of junk protein, and therefore the
SA should go up.