Biochemistry 101

In this specific lab, we used a purification method,  Gel filtration chromatography, to separate a mixture of two molecules known as Blue Dextran and Fluorescein based on the size of the molecule. A column was prepared using a gel filtration medium called Sephadex G-50. Prior to class Dr.Regmi made an elution buffer of Na2HPO4/ NaH2PO4  pH 7.0 for size-exclusion chromatography. Through the duration of this lab, I  learned that the atoms that are bigger in size normally elute the section first before more modest particles can, due to their exchanges with the fixed stage, the beads. Below is a list of a variety diameters of organelles; this determines the RCF required Below is a video of the Gel-filtration Sephadex G-50 Procedure

In the third lab experiment I was able to isolate the Nuclei and Mitochondria from chicken liver using differential centrifugation. Though this was my first time ever using a centrifuge, I was able to learn and understand the theory behind the process. This technique is very common and was used in the experiment to separate to major organelles, the nucleus and mitochondria,  through a stepwise increase in centrifugation speed. Chicken liver and the cold fractionation buffer was homogenized and filtrated in centrifuge tubes. We used two different settings of RPM. 2500 RPM for the nuclei and 5600 for the mitochondria due to the size of the diameter, the smaller the particle the greater the RCF.  Below is a video of the process of Cell Fractionation. . .