A Study of Nonentein: a Theoretical, Amateur Protein Analysis.
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A Study of Nonentein
Introduction
Since the discovery of the presence of the intracellular protein, nonentein, it has become desirable to learn more about its functions and reactions within the cell; for example, to know more about it which proteins it may interact with, and whether these interactions are both a part of standard cell function and necessary for nonentein to function properly. A more advanced understanding of these aspects will help give an increased insight into the functions of cells and proteins in general and also will give rise to possible future applications for nonentein. Therefore, several procedures are suggested to be undertaken upon nonentein: firstly, Co-immunoprecipitation and Western Blotting in order to discover which proteins nonentein interacts with; secondly, Bimolecular Fluorescence Complementation to determine whether nonentein’s interactions are necessary for normal cell function; finally, protein inhibitors or mutagenesis should be utilised so as to confirm that these interactions are necessary for nonentein’s normal function.
I – Determination of the protein-protein interactions of nonentein
Immunoprecipitation (IP) is a method often used to analyse protein-protein interactions of suspected complexes. The technique immobilises and removes protein complexes from whole cell extracts. The captured protein can then be identified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blotting.
First, the cell is lysed and a whole cell extract is prepared. An antibody specific to the antigen is added to the cell lysis and the mixture is incubated for several hours. During this time, the antibody will interact with the antigen and they will form an immune complex. An agarose gel support – either protein A or protein G – is added to this mixture and – again, over a period of several hours – the immune complex will be bound to the gel. As shown in figure 1, the antibody binds to...
Introduction
Since the discovery of the presence of the intracellular protein, nonentein, it has become desirable to learn more about its functions and reactions within the cell; for example, to know more about it which proteins it may interact with, and whether these interactions are both a part of standard cell function and necessary for nonentein to function properly. A more advanced understanding of these aspects will help give an increased insight into the functions of cells and proteins in general and also will give rise to possible future applications for nonentein. Therefore, several procedures are suggested to be undertaken upon nonentein: firstly, Co-immunoprecipitation and Western Blotting in order to discover which proteins nonentein interacts with; secondly, Bimolecular Fluorescence Complementation to determine whether nonentein’s interactions are necessary for normal cell function; finally, protein inhibitors or mutagenesis should be utilised so as to confirm that these interactions are necessary for nonentein’s normal function.
I – Determination of the protein-protein interactions of nonentein
Immunoprecipitation (IP) is a method often used to analyse protein-protein interactions of suspected complexes. The technique immobilises and removes protein complexes from whole cell extracts. The captured protein can then be identified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blotting.
First, the cell is lysed and a whole cell extract is prepared. An antibody specific to the antigen is added to the cell lysis and the mixture is incubated for several hours. During this time, the antibody will interact with the antigen and they will form an immune complex. An agarose gel support – either protein A or protein G – is added to this mixture and – again, over a period of several hours – the immune complex will be bound to the gel. As shown in figure 1, the antibody binds to...