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Protein Purification of LDH, Lab Report Example

Pages: 3

Words: 891

Lab Report

Introduction

This lab report is based on the purification of Lactate Dehydrogenase (LDH) and the aim is to purify the proteins. The purification of protein is important when studying the structure, function and particular protein interactions. It is necessary to identify the protein concentration if there are analytical procedures of different variety which are physical. LDH is an enzyme which is found in animals and mostly plants and plays a crucial role in the formation of glycolysis ATP. The reaction with this enzyme creates a reaction of catalyze and NADH to make NAD+ lactate. In order to purify LDH, a sample of tissue is required which contains protein. Majority of the proteins are found inside the cell, so the process of homogenizing is done to separate the protein from the tissue. If there is a solution protein then the process of selective precipitation is used. In the process of selective precipitation the rough method is used for recovering the desired level of protein. There is an interaction chemically and the process depends on the precipitating agent and protein. In this lab, (NH4)2SO4 for LDH precipitation is used.

The process of molecules separation is dialysis in the diffusion rates differences across the embrace which is semi-permeable. A huge number of impurities are eliminated using the process of dialysis and this is done is a solution which is heterogeneous. Once the protein is purified there are many techniques which figure out the concentration and purity of the protein. The first test to run is the properties of enzymes testing. This identifies the chromatography which is found in the protein.  Another method used to identify the purity is gel electrophoresis. Assay Bradford method is another way to find out the concentration and purity of protein.

Purpose of the experiment

The aim of the experiment is to purify the enzyme LDH from cow’s heart and ways to extract using different techniques comprising of protein precipitation, centrifugation, affinity chromatography and dialysis. Many of the procedures of analysis were employed to find out the purity concentration, of LDH.

Goal of Research

The foal of the research is to isolate a single protein away from all the other components of a cell like;

  • Carbohydrates
  • Lipids
  • Nucleic acids
  • Small molecules
  • Other proteins

Purpose

The purpose of the research is to enable a detailed understanding of the fundamental structure and function

  • 1 degree sequence
  • 3 degree sequence
  • Binding
  • Kinetics

Challenges

An analysis of Bradford was done to identify the LDH concentration in a fraction and sample selection that had observed significant activity of enzymes. There are parameters which are specific in the Assay Bradford construction curve standard. The complete protein mass was identified based on the volume measure of each fraction or sample and its concentration of protein corresponding to. The activity of specific LDH is calculated on the fraction or sample total volume. SDS Page was a gel which was run to figure out the LDH purity in the fractions and the samples selected. The routine method used for this analysis is the Assay Bradford method which identities the concentration of protein. A curve standard is constructed with the known concentration of protein using the blue dye Coomassie which binds all the proteins and light is absorbed at 595 mm. The solution absorption on this level shows the interested level of protein concentration.

The results of the Assay Bradford shows the concentration of the data of LDH in each fraction and sample. This also gives the information of the LDH purity. The larger blue samples have specific LDH activity and are purification folded to measure the purity. The specify LDH activity with larger samples and more with the lower value samples. The sample of Chromatography shows the largest concentration of fraction. The analysis shows that LDH was reified from the cow’s heart. There were some impurities which were minor still there. The assay found and the source of protein chosen must be fractionated into the cell. This is done in components and we need to identify which component the interest protein is enriched on. Such scheme of fractionalization are built by error and trial or on experience basis. In the first stage, the homogenate is made which disrupts the membrane of the cell. This mixture is then with the help of centrifugation fractionated yielding a pellet of dense health material at the tube of centrifuged bottom and the light supernatant above is seen. This again is centrifuged at a force which is greater and this supernatant yields yet another pellet. This method is known as differential centrifugation. This yields various decreasing density fraction where each comprises of different hundreds of protein. These are assays subsequently being purified for each activity. Normally there is an enrichment of one fractions for such type of activity and it then serves as the material source to which the techniques of purification are applicable. Numerous of proteins have been purified in the form which is active based on charge, size, affinity and solubility. Normally, the mixtures of protein are subjected to separation series and each is based on a pure protein yield which is different than the other. The preparation is assayed and the concentration of protein is identified in these methods. Purification, the preparation is assayed and the protein concentration is determined.

Ribliography

Berg, J.; Tymoczko, J.; Stryer, L. W H Freeman 2002.

Ziȩtara, M.; Skorkowski, E. Comparative Biochemistry and Physiology Part B: Comparative Biochemistry 1993, 105, 349-356.

Bornhorst, B.; Falke, J. Protein Expression and Purification 2011.

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