The hypothesis of the experiment was that the molecular weight of N-acetyl-β-D-hexosaminidase would be around 28,000 kDa but to determine the molecular weight the concept of SDS-PAGE had to be understood. The movement of any charged particle through an electric field is determined by its net charge, its molecular radius and the magnitude of the applied field (7). Being that natively folded proteins are not molecular weight dependent, the proteins would be traveling at different speeds in an electric field. In order to separate the proteins based on their molecular weight, the tertiary structure of the protein needs to be destroyed making the protein a linear molecule. Sodium dodecyl sulfate (SDS) plays an important role in this mechanism. Sodium dodecyl sulfate is an amphipathic detergent whose role is to break down the tertiary structure of a protein making it a linear molecule and also masking the native charge of the protein with a negative charge. By using SDS the protein involved in this experiment allowed the different volumes of the protein to migrate down the electrophoresis gel at the same time. SDS is also present throughout the gel to make sure the proteins are linearized and their charges are masked throughout the run. The gel run is conducted through a "discontinuous" system, which means that the buffer in the gel and the tank are different. The system has three different pHs: the stacking gel has a pH of 6.5, the running gel has a pH of 8.5, and the electrode buffer has a pH of 8.3. The different pHs are necessary because glycine can exist in three different states, positive, neutral of negative. For example, when the power is turned on the negatively charged glycine ions in the pH 8.5 electrode buffer are forced to move into the stacking gel where the pH is 6.5. This triggers the glycine ions to switch to its neutral or zwiterionic state this causes the glycine ions to move slowly. On the other hand, the Cl- ions move quickly into the electric field causing an ion front ahead of the glycine ions. The separation of the Cl- ions from the Tris creates a zone with a steep voltage gradient pulling the glycine ions along with it, resulting in two different ion fronts. Once they hit the running gel with a pH of 8.5, the glycine ions accelerate past the proteins leaving them in a band at the interface of the stacking and running gel. Being that the running gel has an increased amount of acrylamide this slows down the movement of the proteins and separation occurs according to size. After the electrophoresis was run, the bands of the protein were made visible using a pre-stained marker. The pre-stained marker although it isn't ideal for accurate estimation of protein size like an unstained marker, it is ideal for confirming the electrophoresis run and the efficiency of transfer to the membrane. The resulting protein bands was used to determine the
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The Rf value for N-acetyl-β-D-hexosaminidase was calculated using the distance of the migration of the protein divided by the total migration of the solvent resulting in a Rf value of 0.3. The linear equation generated from the graph (Figure 2) was used to determine the log (MW), which was determined to be 4.9. The antilog of the log (MW) was used to determine to be 79,432
The Rf value for N-acetyl-β-D-hexosaminidase was calculated using the distance of the migration of the protein divided by the total migration of the solvent resulting in a Rf value of 0.3. The linear equation generated from the graph (Figure 2) was used to determine the log (MW), which was determined to be 4.9. The antilog of the log (MW) was used to determine to be 79,432