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Sodium Dodecyl Sulfate-Polyacryl Amaide Gel Electrophoresis - Lab Report Example

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The paper "Sodium Dodecyl Sulfate-Polyacryl Amaide Gel Electrophoresis" presents a method to find the molecular weight of two proteins. SDS was used to denature the sample got from the pig brain. This method is efficient and widely used to separate the proteins…
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Sodium Dodecyl Sulfate-Polyacryl Amaide Gel Electrophoresis
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Extract of sample "Sodium Dodecyl Sulfate-Polyacryl Amaide Gel Electrophoresis"

Genomics and Proteomics al Affiliation Sodium Dodecyl Sulfate-Polyacryl Amaide gel electrophoresis is a method used to separate proteins basically by their difference in size. The difference in size causes the subsequent difference in molecular weight and that way when they are ran on an electrophoretic gel the light ones will be able to move faster and the ones with a large molecular weight will move slowly. This way they are separated and are ready for analysis. In SDS-PAGE the proteins are first denatured so that all the other characteristics are kept inactive as we want them to only retain the amino acid structure. Sodium Dodecyl Sulfate is a detergent soap that is able to dissolve hydrophobic molecules but also has a negative charge impacted by the sulfate molecule. When the cell is incubated with SDS the membranes get dissolved and all the proteins will be solubilized and covered with many negative charges. This process is important as it ensures that all the proteins retain their primary structure and also acquire the negative charges. Now once the proteins are denatured by the SDS and introduced into an electric field they will move to the positive pole due to their charges. However because we want to separate them according to size, we introduce polyacrylamide which is a polymer of acrylamide monomers. This polymer turns into a gel that is made of a labyrinth of tunnels through a meshwork of fibers. Now this is the one that will be able to separate the proteins according to their size. Keywords: Proteins, Separation, SDS-PAGE. Introduction Pig brain extract was used in this experiment. The objective of the experiment was to separate protein molecules according to their molecular weight. Sodium Dodecyl Sulfate was used to denature the proteins. Western blotting is used to transfer the separated proteins onto a membrane and have them detected. A standard sample is used to compare the results we get so that we establish the prospects of our unknown proteins. We then plot a graph of logMW against Rf (relative mobility) where MW is the molecular weight and Rf is the distance a protein moves divided by the one the dye has traveled. We draw a line of best fit and then we establish the value of our unknown protein. 1. Standards (Mr) Distance migrated Log10Mr Mr unknown proteins 0.4 1.924 na 1.0 1.789 na 1.2 1.740 na 1.4 1.490 na na Unknown protein A 0.1 1.940 87.000 kDa Unknown protein B 1.19 1.70 50.119 kDa 2. I have done all the calculations just draw the graph manually as we want the line of best fit and Ms Excel cannot produce it. Log MW is on the y-axis and Rf on the x-axis. Section 2 Question 1 Colum two of the digital image produced by the software shows the standard proteins that were used to establish the values of our unknown protein A and B. The proteins will move according to their molecular weight with the ones with small molecular weight moving the furthest from the origin. The log of molecular weight of the protein is proportional to the Rf of the protein and so when we plot the graph of best fit we are able to establish the molecular weight of our unknown proteins A and B. Question 2 The proteins migrate according to their weight when SDS-PAGE is used because of the following reasons. First Sodium Dodecyl Sulfate is a detergent soap which denatures the proteins. The proteins should be denatured as we want to separate the proteins according to their sizes only. By denaturing them we ensure that other properties of the proteins do not interfere and the molecular weight is the one that will bring the difference in the distance traveled by the proteins. SDS has sulfate which on top of denaturing the proteins covers them with many negative charges and so when they are introduced on a PAGE they will move to the positive node. Polyacrylamide is a polymer which forms a gel that ensures the proteins move exclusively by their difference in molecular weight. The distance traveled can then be used to find the molecular weight of the unknown. Question 3 SDS stands for sodium dodecyl sulfate. It is a detergent soap that is used to denature the proteins in protein separation by size. It is an anionic surfactant mainly used as a cleaning detergent. It is used in lysing of which are then used in protein separation by size. It is preferred because on top of lysing or denaturing the proteins it coats them with many negative charges and so when they are introduced into the electrophoretic chamber they migrate to the positive electrode. It disrupts the non covalent bonds in proteins thus making the proteins to lose their native shape and that way denatures them. The negative charge that they acquire is greater than the original charge of the protein and so it is the one that prevails. According to Gallagher, 2012, SDS binds to the proteins causing a strong electrostatic repulsion that make the proteins to unfold into a rod like shape and that way eliminating shape differences that would have caused unfair grounds in separating the proteins. When these proteins are introduced into the chamber to run the gel, the negative charges makes it to move in the direction of the positive electrode. The gel forms a fiber substance that ensures the proteins are separated by their size. This method is widely used in protein analysis and research because it is reliable and with the use of programs that work to establish the unknown molecular weight and even establish their identity. This has made molecular biology simpler and efficient. This is by far the most widely used technique. One of the main disadvantages of this method is that due to the denaturing process the proteins cannot be used after the gel has been run. This makes it not the best when you have limited sources of your sample. It has also been widely applied in forensic science and it is the one used to find the molecular weight of molecules like DNA and RNA. Conclusion SDS-PAGE has been used in this experiment to find the molecular weight of two unknown proteins A and B. SDS was used to denature the sample that was got from pig brain. After denaturing it and covering it with the negative charges then the proteins can be separated when introduced into the electrophoretic chamber. This method is efficient and widely used to separate the proteins. It’s only main limitation is that it does not work with un denatured protein sample and so in cases where the sample is limited it could prove tricky. Reference Gallagher, S. (2012). Sodium Dodecyl Sulfate-Polyacryl Amide Gel Electrophoresis,SDS-PAGE. Carlifonia: John Wiley Publication. Read More

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