Following graduation, you decide to take a job working at a small startup, Baysh
ID: 1032967 • Letter: F
Question
Following graduation, you decide to take a job working at a small startup, Bayshore Biotech. Here, your manager pairs you with a staff scientist, Biff Clueless. It is Biff's job to help you with your project characterization of the company's new candidate protein therapeutic drug-a Humira or Adalimumab biosimilar drug. For some of the early characterizations of this protein, you are interested in determining how this protein may potentially associate with itself in solution. You decide to perform sedimentation equilibrium to find that out. Since Biff is unfamiliar with this method, you should briefly explain to him how sedimentation equilibrium works and the information you can determine from it. Also, remind him that if you have isolated your treated protein with a non-denaturing buffer (such as Tris-buffered saline at pH 8.0), what type of molecular weight are you determining with this method? (Hint: native or subunit molecular weight?) Fully explain your rationale for your answer. (2.5 pt) 1)Explanation / Answer
Sedimentation equlibrium is an analytical method involving ultracentrifugation of protein solutions to find protein-protein interactions and protein molecular masses. This method involves the sedimentation of proteins by using a centrifugation velocity slower than that required to form a pellet which might destroy protein-protein interactions and the target protein's native structure and a high enough velocity to cause all the particles to accumulate toward the rotor. As the centrifugal force produces a gradient in protein concentration across the centrifuge cell, diffusion acts to oppose this gradient. Over time, an exact balance is reached between sedimentation and diffusion, and the concentration distribution reaches an equilibrium. This equilibrium concentration distribution across the cell is then measured while the sample is spinning, using either absorbance or refractive index. Thus the technique exploits only the molecular mass of the protein in the sedimentation and is independant of the shape. Top of all, in this equilibrium as the protein states are not disturbed, self-assembling oligomers also tend to associate in the centrifugation cell thus clearly appearing in a higher molecular mass in a certain fraction of the target protein thus enabling the identification of the precise nature of the protein isolated as a native structure, dimer, etc. The molecular weight determined by this method is given in kDa.