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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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Extract of sample "Protein isolation"

Protein Purification Protein Purification Introduction Majority of scientific studies today require the isolation, purification and study of micro-molecules in biochemical processes. The existence of a myriad of protein isolation and purification techniques is a direct consequence of these scientific studies (Jordan and Ogren, 1981). Enzymes are among the most studied proteins due to their crucial role in regulating metabolic processes. Enzymology entails the use of isolation and purification techniques similar to those used in proteomics.

Ribulose 1, 5-bisphosphate carboxylase/oxygenase (rubsico) is one of the most abundant proteins in the universe a reaction on the synthesis of sugar in green plants (Jordan and Ogren, 1981). 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.MethodologySpinach 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).ResultsAmmonium 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: SDS-gel of the different samples tested.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 first precipitate (s 37%) at high salt, Supernatant of second precipitate (S 50%), second pellet at low salt, Second pellet at medium salt, Second pellet at High salt) while side arrows show the molecular weight of different protein bands.

The red circles show the identified Rubisco bands at 55kd and 14kd respectively.Successful isolation and purification of rubisco from spinach leaves was visualized using a SDS-gel as shown in figure 1. The first column on the gel represents the ladder while the rest of the columns represent the different treated samples. A band representing rubisco can be identified on the filtrate and pellet sample obtained from a salt concentration of 37%. Figure 2: SDS-gel of a Protein standard.Figure 2: This is the calibration curve showing protein bands and their different molecular weights.

A characteristic absorbance spectrum of rubisco at wavelength of 280 nm was used to confirm the identity of the isolate. Selected samples were run on a scanning spectrophotometer across wavelengths of 200-400nm and the results are shown in the figure below.Figure 3: Showing OD values of the different samples across 200-400nm wavelength.Figure 3 The OD values of the different samples (samp 1-first pellet, sampl 2- first supernatant, sampl 3 second pellet, samp 4 second pellet medium salt, samp 5- first pellet medium salt, sampl 6- second supernatant) across different wavelength.

The peaks represent maximum absorbance of Rubisco protein.Sample “Samp 3” representing the second isolate at 50% salt concentration shown as purple line as spike at a wavelength close to 280nm, which is the optimal wavelength for rubsico. DiscussionThe aim of this experiment was to isolate and identify rubisco, an enzyme catalysing sugar synthesis in plants. The enzymes identity was confirmed by its molecular weight and OD value at 280 nm. As clearly shown on figure 1 and 2 two bands of molecular weight 55k and 14k represent the two major polypeptide subunits of the enzyme.

These same bands are seen on the filtrate the leaf extract upon which isolation and purification was done. The validity of the molecular weight calibration curve can further be confirmed by comparing the size and position of bands obtained from the run of the standard and the curve. A useful feature of rubisco is in its characteristic absorbance spectrum at wave length 280nm. This can be used a proxy confirmatory test in the identification of the protein. The graphs show one distinct peak at wavelengths close to 280nm, which can e identified as rubisco.

Interestingly the almost all the samples had a distinct peak at a wavelength of around 200nm. However, such observation may be caused by a regular protein present in all of the samples or contamination during assay setup.ConclusionAccording to this experiment, rubisco can be isolated using ammonium sulphate and purified by ion exchange chromatography. The concentration of the isolating reagent as well as elution affects the amount of protein isolate obtained. Rubisco can be firmed by its molecular weight, characteristic wavelength and enzymatic assays.

Works citiedJordan DB, and Ogren WL, “A sensitive assay procedure for simultaneous determination of Ribulose, 1, 5-bisphosphate carboxylase and oxygenase activities.” Plant Physiol 67 (1981): 237-245. Print. Robinson Sp, Streusand VJ, Chatfield JM and Portis Ar “Purification and Assay of Rubisco Activase from Leaves.” Plant Physiol. 88 (1988):1008-1014. Print.

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