Cell fractionation

Cell fractionation is a useful preparative and analytical method for separating cellular compartments. The separation of distinct organelles results from their different physical properties, like size, shape, buoyant density or surface charge density. The basic principle of cell fractionation by centrifugation is represented by the Svedberg-equation, which describe mathematically the sedimentation of a spherical particle in solution:

x: distance from rotor axis... r: radius of particle ω: angular velocity... ρp: density of particle

ρm: density of medium... η: viscosity of medium t: time

The most commonly used solute for cell fractionation is sucrose, since it can easily be prepared in densities that span the range of densities of most organelles. Since sucrose solutions are rather viscous at high concentrations, iodinated non-electrolytes like OptiPrep are often added, which increase the density of the fractionation medium without significantly increasing the viscosity. Another possibility to achieve high density at low viscosity is the addition of colloidal silica like Percoll.

2.6.3.1. Preparation of soluble and particulate fraction from adherent cells

Cells were washed in ice-cold PBS, then hypotonic lysis buffer was added and cells were incubated for 5min on ice. Cells were scraped and homogenized. Depending on the volume of the lysates homogenization was carried out either by using a Dounce homogenizer or by sucking the cell lysates 10 times through a 26G needle. In order to remove intact cells and cell nuclei the homogenized lysate was centrifuged for 3min at 4°C with 700g. The resulting postnuclear supernatant was transferred to a new reaction tube and centrifuged for 30min at 4°C with 30000xg. The supernatant (soluble fraction) was removed and stored on ice. The pellet was carefully washed in hypotonic lysis buffer and resuspended in resuspension buffer (particulate fraction). The protein concentration was determined by Bradford protein assay (2.6.4.2).

Hypotonic lysis buffer

Tris-HCl...10mM pH 7.6 KCl...5mM MgCl2...1.5mM Dithiothreitol (DTT) ...1mM NaF ...10mM Na3VO4...1mM protease inhibitor cocktail...1 tablet/10ml

Triton-X-100 ...1ml Hypotonic lysis buffer...100ml

2.6.3.2. Detergent-free plasma membrane fractionation

In order to fractionate the plasma membrane and to separate the caveolin-rich membrane fraction the method established by Anderson and colleagues (Smart et al. 1995) was slightly modified. Since the caveolin-rich membrane fraction is very small a large amount of starting material is needed. For one experimental condition six 140mm dishes of subconfluent cells were used.

Plasma membrane preparation: Cells were washed two times in buffer A and scraped in 5ml buffer A on ice. Cells were collected by centrifugation for 5min at 1000g at 4°C, the pellet was resuspended in 1ml buffer A and placed in a 2ml Dounce homogenizer and homogenized with 20 strokes. The suspension was transferred into a 1.5ml centrifuge tube and centrifuged at 1000xg for 10min at 4°C. The postnuclear supernatant (PNS) was removed and stored on ice. The pellet was resuspended in 1ml buffer A, homogenized again, and centrifuged for additional 10min at 1000xg. This PNS was combined with the first one and layered on top of 8ml Percoll solution (30% Percoll in buffer A). Cells were centrifuged in a SW41 ultracentrifugation rotor at 84000xg for 30min at 4°C. The plasma membrane fraction bands in the middle of the tube, the cytoplasmic fraction stays located on top. Both fractions were isolated by tube puncture. In order to keep the layering of the gradient it is important not to use any brakes for deceleration of the ultracentrifuge.

Isolation of caveolin-rich membrane fraction: The volume of the plasma membrane fraction was adjusted to 2ml with buffer A. Samples were sonicated 6 times for 6sec with 1-2min on ice in between times. The sonicated samples were mixed with 1.84ml buffer C and 0.164ml buffer A and placed at the bottom of a 12ml ultracentrifuge tube. On top, an 8ml 20%-10% Optiprep gradient was poured. The 10% and 20% Optiprep gradients were produced by mixing buffer A in buffer C. Samples were then centrifuged at 52000xg for 90min at 4°C. The lower 3ml of the gradient represents the non-caveolin-rich membrane fraction. The top 5ml were collected and mixed with 4ml buffer C, placed at the bottom of a new ultracentrifuge tube and overlaid with 2ml of 5% Optiprep (made by diluting buffer C in buffer A). Samples were centrifuged at 52000xg for 90min at 4°C. The caveolin rich membrane fraction appears as an opaque band at the 5% Optiprep interface.

Buffer A

tricine...20mM pH 7.8 EDTA ...1mM Buffer B sucrose ...250mM tricine...120mM pH 7.8 EDTA ...6mM Buffer C Optiprep... 50% sucrose ...250mM tricine...20mM pH 7.8 EDTA ...1mM

Optiprep Density Gradient Medium, Sigma (Cat.No. 1556) Percoll, Sigma (Cat.No. P1644)

2.6.3.3. Preparation of the Triton-X insoluble cytoskeletal fraction

To isolate the cytoskeletal fraction of adherent cells, a 140mm dish of sub-confluent fibroblasts was washed once with ice-cold PBS followed by addition of 1.5ml cytoskeletal extraction buffer. Dishes were incubated for 15min on ice, cells were scraped off and the lysate was collected in a 2ml centrifugation tube. Lysates were centrifuged at 4°C with 15000g for 15min. The supernatant representing the soluble fraction was taken off and stored on -80°C. The pellet was washed two times with 2ml cytoskeletal extraction buffer and finally resuspended in 400µl RIPA buffer. The RIPA lysate was sonicated once for 5-10sec and centrifuged at 4°C with 15000g for 5min. The cytoskeletal rich supernatant was taken off and either directly processed or frozen down on -80°C.

Cytoskeletal extraction buffer

NaCl...50mM sucrose ...150mM PIPES...10mM pH 6.8 Triton-X-100 ... 0.5% NaF ... 10µM Na3VO4...2mM protease inhibitor cocktail...1 tablet/10ml RIPA buffer

Tris-HCl...50mM pH 7.6 EDTA ...1mM Na-deoxycholate ... 1% SDS ... 0.1% Triton-X-100 ... 1% NaF ...10mM Na3VO4...1mM protease inhibitor cocktail...1 tablet/10ml