SDS-PAGE

Electrophoresis of Proteins from Cauliflower Fractions by SDS-PAGE Laboratory Summary Electrophoresis is a technique where molecules are separated according to their physical properties such as size, charge, and/or shape. Charged proteins are commonly separated in this matter using PAGE (polyacrylamide gel electrophoresis) to identify individual proteins present in samples. In this lab, SDS-PAGE was used. SDS-PAGE separates charged proteins primarily by size because the ionic detergent sodium dodecyl sulfate (SDS) with 2-mercaptoethanol disassociates the subunits, and then completely unfolds these proteins subunits via binding of SDS with a strong negative charge. At the end of this laboratory, I predict that there will be multiple bands showing on each fraction that will be electrophoresized. Protein concentrations of the fractions were determined in the previous lab through protein quantifications of cellular fraction. These fractions were crude homogenate, nuclear, mitochondrial, and lastly cytoplasmic fraction. These concentration values were then used to calculate how many µL are needed for µg of protein. Water and 3X SDS PAGE sample buffer were added into each fraction. In addition, MW standard was also prepared. After the apparatus was assembled, chambers were filled, and the gel completely submerged, the samples were then loaded into separate gel slots. Once all the samples were loaded, the samples were electrophoresized for 30 minutes and stained/destained until the protein bands were clearly visible in the gel. When the gel was analyzed I found that there were multiple bands in each slots especially in the MW standard. This was expected as the larger proteins will have migrated the least, while the smaller proteins the farthest towards the anode during the time that the current was applied.