Fact finding mission: . What are the different methods used to purify proteins f
ID: 260189 • Letter: F
Question
Fact finding mission: . What are the different methods used to purify proteins from crude extract materials? Whya so many different types of methods required? What is happening in each step of our purification protocol? low does gel electrophoresis work? What characteristics of the protein determine how far it will migrate in a gel? We will run denaturing gels, what does this mean and why is it important? ow do various reagents, such as urea, hydrogen peroxide and change in pH, affect protein activity and stability? .H What is a protein's "specific activity"? . What is the basis for the nitrocefin-based activity assay? How does it work?Explanation / Answer
1. There are four methods of protein purification from crude extracts
(1) Extraction
(2) Precipitation and Differential Solubilisation
(3) Ultracentrifugation
(4)Chromatographic Methods.
2. Depending on the usage purity of the protein is required. For some applications, a crude extract is sufficient. In case of foods and pharmaceuticals, a high level of purity is required. for this this several protein purification methods are typically used, in a series of purification steps.
3.
a. In extraction protein will be dissolved in solutions(by freezing and thawing, sonication, homogenization by high pressure or permeabilization by organic solvents) and seperated frrom tissues using centrifugation (Upper solution is collected)
b.In precipitation proteins will be precipitated with ammonium sulphate and different fractions of precipitate will be collected.
c.Centrifugation is a process that uses centrifugal force to separate mixtures of protein particles of varying masses or densities suspended in a liquid.
d.In chromatographic technique the solution containing the protein is allowed to flow through a column packed with various materials. Different proteins interact differently with the column material, and can thus be separated by the time required to pass the column, or the conditions required to elute the protein from the column, which can be detected at absorbance of 280nm.
3.How does gel electrophoresis work
a. Polyacrylamide gel electrophoresis is performed in the presence of SDS (sodium dodecyl sulfate) which binds to proteins giving them a large net negative charge. Since the charges of all proteins are fairly equal, this method separates them almost entirely based on size. Unwanted proteins are gradually removed from the mixture, the number of bands visualized on the SDS-PAGE gel is reduced, till there is only one band representing the desired protein.
b.Depending on the size and charge (mostly size) protien will be seperated.
c. sodium dodecyl sulfate is a strong detergent agent used to denature native proteins to unfolded, individual polypeptides. When a protein mixture is heated to 100 °C in presence of SDS, the detergent wraps around the polypeptide backbone. In this process, the intrinsic charges of polypeptides becomes negligible when compared to the negative charges contributed by SDS. Polypeptides after treatment become rod-like structures possessing a uniform charge density, that is same net negative charge per unit length. hence,The electrophoretic mobilities of these proteins will be a linear function of the logarithms of their molecular weights.
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