Biochemistry 101

"Po lyacrylamide gels are formed by the reaction of acrylamide and bis-acrylamide ( N,N ’-methylenebisacrylamide) that results in highly cross-linked gel matrix. The gel acts as a sieve through which the proteins move in response to the electric field. Proteins contain an overall positive or negative charge; this enables the movement of a protein molecule towards the isoelectric point at which the molecule has no net charge. By denaturing the proteins and giving them a uniform negative charge, it is possible to separate them based on the size as they migrate towards the positive electrode". One aspect I truly liked about learning this technique was seeing how SDS and PAGE both combined to  use one characteristic, molecular size. The two other physical properties that are dismissed are the charge and structure due to detergents SDS  amphipathic nature. https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/gel-electrophoresis/sds-page

"There are several ways of estimating the protein concentration such as amino acid analysis following acid hydrolysis of the protein; analyzing the changes in the spectral properties of certain dyes in the presence of proteins; and spectrophotometric estimation of the proteins in near or far UV region. Although dye-binding assays and amino acid analysis following acid hydrolysis of the protein can be used for estimating the protein concentration for both pure as well as an unknown mixture of proteins; UV spectroscopic quantitation holds good for the pure proteins. If a protein is pure, UV spectroscopic quantitation is the method of choice because it is easy and less time consuming to perform; furthermore, the protein sample can be recovered back". One of the biggest obstacles as an overall whole, was the calculation part of the experiment, I have attached a video that helps better explains the process of finding the standard curve.  REFERENCES [1] Scopes, R. K. (1974) Measure...