2: Preparation of Ovalbumin and Lysozyme
- Page ID
- 169758
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)The most abundant protein in avian egg white is ovalbumin, occupying about 55% of the total proteins in newly deposited eggs. Ovalbumin is made of a single polypeptide with a molecular weight of 44,500 Da. It is thought to be a storage protein serving as an amino acid source for developing embryo. Lysozyme (Mr 14,600 ) is an enzyme also abundant in egg white as well as human tears that catalyzes the hydrolytic cleavage of polysaccharides in the protective cell walls of some families of bacteria. Lysozyme, because it can lyse (or degrade) bacterial cell walls, serves as a bactericidal agent.
In Exp 2-4, we will prepare these two proteins from chicken egg white applying the general strategy for protein purification. In Exp 5, we will carry out protein gel elecrophoresis to check the purity of these two proteins.
Getting Started with Protein Purification
Protein purification is usually a multi-step process exploiting a wide range of biochemical and biophysical characteristics of the target protein, such as its source, relative concentration, solubility, charge, and hydrophobicity. The ideal purification strives to obtain the maximum recovery of the desired protein, with minimal loss of activity, combined with the maximum removal of other contaminating proteins.
A typical protocol for purification of a soluble cellular protein involves the disruption of the cellular membrane, followed by a differential centrifugation in a density gradient to isolate the protein from subcellular particles and high molecular weight aggregates. Further purification may utilize selective precipitation by addition of inorganic salts (salting out) or addition of miscible organic solvent to the solution containing the protein. Final purification will include a combination of techniques that separate based on molecular charge, molecular size, and affinity.
- 2.1: Protein Fractionation
- This page describes protein purification via fractional precipitation using ammonium sulfate's salting-out technique, where increased salt concentration induces protein aggregation and precipitation. The method involves gradually adding ammonium sulfate to egg white, followed by centrifugation to collect protein aggregates. The crude albumin is resuspended in NaCl for further experiments, with specific steps and precautions outlined for effective recovery.
- 2.2: Protein Quantification
- This page discusses methods for accurately determining protein concentrations, focusing on the use of a UV spectrophotometer at 280 nm and the Bradford Assay. The Bradford Assay requires adding Coomassie Brilliant Blue dye to protein solutions, with quantification based on absorbance changes.
- 2.3: Gel Filtration Chromatography
- This page describes gel filtration chromatography, a technique that separates molecules by size using porous gel. Smaller molecules enter the gel's pores while larger ones do not, producing distinct peaks corresponding to molecular weight. The process involves packing a column with Sephadex G-50, adding a protein sample, and measuring absorbance at 280 nm for analysis.
- 2.4: Ion-exchange Chromatography
- This page discusses how charged groups on protein surfaces influence their solubility and solvent interactions, introducing the isoelectric point (pI). It describes ion exchange chromatography using DEAE-cellulose for protein separation based on charge, emphasizing salt concentration variations during elution.
Video of Experimental Procedures: 2.1 & 2.2
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