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Protein Purification Prelab

The document provides tables of absorbance readings from lysozyme and Bradford assays performed on crude and fractionated enzyme samples at various time points. It also includes a serial dilution procedure for BSA standards and answers to pre-lab questions about the relationship between enzyme activity and absorbance as well as the meaning of specific activity.

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63 views3 pages

Protein Purification Prelab

The document provides tables of absorbance readings from lysozyme and Bradford assays performed on crude and fractionated enzyme samples at various time points. It also includes a serial dilution procedure for BSA standards and answers to pre-lab questions about the relationship between enzyme activity and absorbance as well as the meaning of specific activity.

Uploaded by

shdwnightwolf
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd
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t=0 sec

t=15 sec

t=30 sec

t=45 sec

Crude
extract
PBS
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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.

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