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Protein isolation - Lab Report Example

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The existence of a myriad of protein isolation and purification techniques is a direct consequence of these scientific studies (Jordan and Ogren,…
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Protein isolation
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Download file to see previous pages The aim of the experiment is to isolate, purify and identify Rubsico in spinach leaves using Ammonium sulphate, spectrophotometry, and electrophoresis and ion exchange techniques.
Spinach leaves were described, homogenized and rubisco isolated using two grades of ammonium sulphate concentrations (37 % and 50%). Filtrates and supernatants were tested using appropriate techniques to ascertain successful isolation. (Robinson, Streusand, Chatfield and Portis, 1988) The isolate was then purified using DEAE Cellulose fast flow ion exchange chromatography and bound proteins eluted using different shades of salt concentration. Purified proteins were run on a SDS-gel electrophoresis to identify the isolate (Robinson, Streusand, Chatfield and Portis , 1988).
Ammonium sulphate (salt) precipitates proteins by altering hydrogen bond interaction between protein and water molecules. Ammonium sulphate a high affinity for water molecules displaces protein molecules (lowers the solubility) thereby causing precipitation. Different protein molecules precipitate at different concentrations of ammonium sulphate and at the rate at which it is added. The effect of salt concentration on the isolation of rubisco is shown on figure 1. The absence of the rubisco band on sample p2 (protein isolated at 50 % salt concentration) shows a significant amount of the protein was isolated at 37% concentrations of ammonium sulphate. A protein molecular weight ladder shown in figure 2 was used to establish a calibration curve that used to identify the protein isolate.
Figure 1 The effect of salt concentration on the isolation of proteins; the top arrows represent the samples ( from left to right: MW ladder, leaf extract filtrate, Supernatant of first precipitate (S37%), Pellet of first precipitate (S37%) at low salt, Pellet of first precipitate (S37%) at medium salt, Pellet of ...Download file to see next pagesRead More
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