Protein Purification Lab Report

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Purification of GFP from E.coli via Hydrophobic Interaction Chromatography
Priscilla Mariel M. Cadiz
Biology Department, De La Salle University, 2401 Taft Ave, Manila, Philippines
*Email: cadizpriscillamariel@yahoo.com

Protein purification is an important techniquie in order to study the function and structure of proteins. Protein purification involves the process of removing contaminants and isolating desired proteins. Chromatography is one of the methods used for purifying and isolating proteins. Green Fluorescent Protein (GFP) is the desired protein to be isolated from the bacteria Escherichia coli which allows the bacteria to fluoresce. GFP contains abundant hydrophobic sites and because of this, Hydrophobic Interaction Chromatography
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INTRODUCTION

An important pre-requisite in order to know the function and structure of a protein is called protein purification (Berg et al., 2002). It is the process of removing protein contaminants and also to be able to transfer proteins to a stable environment in a form that is required for its’ use (Queiroz et al., 2001). There are different techniques available today for protein purification such as precipitation, which uses a neutral salt for purifying, filtration and chromatography to name a few. One may choose which technique to use depending on the intended use of protein however, it is chromatography that is widely used today because of its’ high resolving power (Saraswat et al., 2013). It can purify any soluble substance with the aid of a right adsorbent material and carrier fluids. It is also used to separate delicate producats due to the unsevere conditions while it is
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Tube 2 under UV light after addition of wash buffer.

Figure 2. Tube 3 under UV light after addition of elution buffer.

Before doing chromatography, lysozyme was first added in order to break open the cell wall and release the components of the cell then it was centrifuged then binding buffer was added to the supernatant. The binding buffer allows the GFP to change its confirmation to expose its’ hydrophobic particles because of the presence of high amount of salt. (Tan & Yiap, 2009). The hydrophobic particles will move to the exterior part of the molecule allowing the GFP to bind to the HIC resin found in the column (Klotz & Urquhart, 1949). After the addition of the binding buffer to the supernatant, the mixture was then transferred to the column containing the equilibration buffer which is used to equilibrate or prime the column for GFP binding (Noor et al., 2014). Because of the binding buffer, the hydrophobic parts of the GFP now binds with the hydrophobic regions of the column. Since the GFP binds with the column, no glow was seen in the 1st tube when exposed to UV light. Only the hydrophilic proteins of the E.coli was drained after the supernatant was added to the column (Noor et al.,

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