Sub cellular fractionation

Sub cellular fractionation

• 1.
  Sub-cellular fractionation
• 2.
  Importance of separatingsub-cellular fractionation  To study about a particular subcellular component, we need the sample in large amount.  This technique enable us to separate pure samples of subcellular components in reasonably active states.  Successful approach for large number of proteomic studies is cellular fractionation.  Study of metabolic activity of the components – Mitochondria & Peroxisomes.  A well-established technique for separation of organelles is solely based on two different types of centrifugation, differential velocity and density gradient centrifugation making use of differences in sedimentation coefficients and densities.
• 3.
  Density gradient centrifugation Separates sub-cellular components based on their density  A non-ionic molecule like sucrose, glycerol is used to vary the density of the buffer solution.  The concentration of the molecule is varied and therefore the density of the solution and the concentration is greatest at the bottom of the centrifuge tube and decreased slowly at the top.  A step gradient can also be prepared by using a series of solutions of different concentrations – layered on the top one another with highest concentration at the bottom.  The cell extract is layered at the top of the gradient and the tube is subjected to centrifugation at high speed for several hours.  During this time, the different sub-cellular components move down the tube until they reach the position in the density gradient that corresponds to the density of the component.
• 5.
  Differential velocity centrifugation Separates cellular components based on size/shape and density.  Larger more dense components will pack at the bottom of a centrifuge tube faster and at lower speeds than smaller less dense ones.  Initially, the total cell extract is centrifuged at a low speed for a short time to remove unbroken cells.  Later, the speed and time of centrifugation are progressively increased removing some components. (Packing them at the bottom of the tube as precipitate) and leaving others in the supernatant at each step.  The final supernatant after the step-wise removal of nuclei, mitochondria, vacuole, ribosomes which is called soluble fractions containing soluble proteins and other small molecule components of the cytosol such as tRNAs.
• 7.
  Materials required  Homogenizingbuffer:  0.25 M sucrose  1 mM EDTA  10 mM Tris-HCl (pH 7.4)
• 8.
  Homogenize the cellsin the buffer prepared as mentioned above in homogenizer. Procedure Centrifuge at 1000 g for 10 min Pellet - nuclei Supernatant (Centrifuge at 15000 g for 15 min) Pellet – Mitochondria, Lysosomes & peroxisomes Supernatant (Centrifuge at 100000 g for 60 min) Pellet – Vesicles & Microsomes Supernatant Cytosol