Assignment 1
Bioanalytical Technolog. & Instrument. BIOL-312
Cleio Georgiou

1. What properties determine the sedimentation rate of a particle?
The rate of sedimentation of a particle is determined by the size and the density of the particle and the viscosity of the medium. In addition the gravitational force and the speed of rotation affect the sedimentation.

2. Define the terms homogenate and supernatant

Homogenate is the material that is obtained after homogenization .It is homogenous tissue which is made by grinding the cells and it is used for laboratory purposes. The homogenate most of the times lacks cellular structures and it is just a suspension of cell fragments and constituents.

Supernatant is usually a clear liquid layer that …show more content…
For example for biological work I can use regular glass, nitrocellulose , Corex glass or polyallomer. But depending on the speed the material that I will use, my choices vary. For example if I use glass tubes for a speed above 3.000 RMP, the glass will break. Although there some kind of tubes that can withstand f much higher speeds, such as the Corex glass that is used for speeds up to 18000 RPM, or the preparative centrifuge tubes that are made of polypropylene and can withstand up to 20.000 RPM and the Nitrocellulose tubes that are suitable for speeds up to 10.0000 RPM. In addition we have the Polyallomer tubes that are chemically inert and are slippery and can be used for precipitation …show more content…
We discard the supernatant and we again resuspend the pellet in ice cold buffer following another one centrifugation at 10.000 g. The pellet that we will collect after that will be resuspended in ice cold mitochondrial resuspension buffer (MRB) using a loose fitting glass/ Teflon Potter Elvehjem homogenizer.

Then for the fractionation we add Percoll (a media for density gradient centrifugation cells) in a Polyallomer ultracentrifuge tubes. On top of the percoll we add the suspension of the mitochondria and on top of that MRB solution until we fill we reach about 5 mm below the top of the tube. We centrifuge at 95.000 g for 30 minutes using a Beckman Coultr Optima L-100 XP Ultracentrifuge (AW40 rotor, Beckman, Fullerto). In the end of the centrifugation we will get a dense layer at the bottom containing pure mitochondria, in addition another one layer will appear above the mitochondria containing mitochondrial associated membranes (MAM). We can collect the pure mitochondria layer using Pasteur pipette and resuspend it