Isolation and Purification of Egg White Proteins

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Title                              : Isolation and Purification of Egg White Proteins

Date                              : 10 May 2011

Session                         : Morning (9 a.m. - 12 p.m.)

Demonstrator’s Name : Yan Qi

INTRODUCTION

OBJECTIVE

The aim of this experiment was to isolate and purify ovalbumin, conalbumin, ovoglobulin present in the egg of the domestic fowl for the first week. In week two, the ion exchanger, carboxymethylcellulose column was used to purify the lysozyme at alkaline pH. Besides that, the effect of pH and heat on the state of tyrosine was investigated. In week three, the determination of protein content of conalbumin, ovalbumin, lysozyme A, B C and D was done by a using a standard curve obtained from the Biuret test on a standard protein solution containing bovine serum albumin. Next, the iron binding capacity of conalbumin will be determined by titrating ‘iron-free’ conalbumin with Fe3+. In week four, the enzymic activity was measured by its action on Micrococcus lysodeikticus using a spectrophotometer. Furthermore, the point of saturation of the conalbumin with iron was determined.

RESULTS

Week 1

Isolation of Ovalbumin

Isolation of Conalbumin

Calculations:

Mass of ammonium sulphate required to produce 8% (w/v) solution = 8% x 98 = 7.840 g

Week 2

Volume of egg white used = 26.5mL

Volume of sodium bicarbonate buffer added = 26.5 x 4= 106 mL

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Total volume = 132.5 mL

Isolation of Lysozyme A

Isolation of Lysozyme B

Isolation of Lysozyme C

Isolation of Lysozyme D

State of Tyrosine in Ovalbumin

*1mL of Ovalbumin sample from week 1 was added with 9mL of water

Table 1:

Spectrum Diagram Obtained

Figure 1

Figure 2:

Figure 3:

Week 3

Concentration of standard protein containing Bovine Serum Albumin (BSA) = 10mg/mL

Table 2: Different amount of protein contains in different sample

Table 3: Respective data of dilution, absorbance, suitability of ...

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