Preparation of Heavy and Light Membrane Fractions From MDCK Cells
version 2 as of 29-VI-95
Start with 16 dishes of almost confluent cells. Scrape off cells with a rubber policeman (four dishes fit nicely into a 50 ml Falcon tube).
Spin down cells (2 min. at setting 2 in the clinical centrifuge).
Resuspend all the cells in one 50ml volume of PBS and wash twice with room temp. PBS. Wash once with 30ml hypotonic buffer (don’t waste the PIN by putting it in this wash, also don’t waste any time once you put the cells in the hypotonic buffer).
Resuspend in 15mls cold hypotonic buffer and leave on ice for 5 minutes (some cells have to be left for longer periods of time, but MDCK swell pretty quickly--you can always monitor this using the tissue culture microscope).
Pre-cool the Dounce homogenizer (15ml). OK - once your cells are swollen you are ready to start.
Rupture cells by 3 sets of 10 strokes in the Dounce with pestle B. Keep the homogenizer in the ice during homogenization and give the cells 1-2 minutes between sets to keep them cold. The cold helps depolymerize the cytoskeleton.
Add 5 mls of compensating buffer to bring the homogenate back to isomotic conditions and homogenize another 10 strokes. Keep your samples cold for the rest of this process! Put the lysate into 2 polypropylene disposable tubes (10ml each).
Pellet nuclei and unbroken cells by centrifugation at 1800 rpm in the Hermle for 10 min. at 4°C.
Pour off supernatants into 35ml flip top polypropylene tubes (one goes in each). Leave these in the cold room throughout your prep.
Resuspend pellets of nuclei and unbroken cells in a total of 14mls of 250mM K Resuspension buffer.To do this, I add 4 mls to each tube, vortex, dump into homogenizer containing the PIN and DTT, then add 3 mls more to each tube, vortex and dump these into the homogenizer.Homogenize 10 strokes and pour into two new polypropylene disposable tubes (7.0 mls each).
Pellet nuclei and unbroken cells by centrifugation at 1800 rpm in the Hermle (800 Xg?)for 10 min. at 4°C.
Pour off supernatants into 35ml the same flip top polypropylene tubes as before (one in each).
Resuspend pellets in a total of 14mls of Resuspension buffer plus 500mM KOAc as above and homogenize 10 strokes.Pour into two new polypropylene disposable tubes (or you can re-use the same ones from last time (7.0 mls each)).
Pellet nuclei and unbroken cells by centrifugation at 1900 rpm in the Hermle for 10 min., at 4°C. Pour of supernatants into the same 35ml flip top polypropylene tubes as before (one in each).
Resuspend pellets in a total of 15mls of Resuspension buffer plus 500 mM KOAc but with no Magnesium, homogenize 10 strokes.Pour into two new polypropylene disposable tubes (7.0 mls each).
Pellet nuclei and unbroken cells by centrifugation at 2000 rpm in the Hermle for 10 min. at 4°C.
Pour off supernatants into the same 35ml flip top polypropylene tubes as before (one in each).The flip top tubes should now each contain about 30mls of post nuclear suprnatant, mix them together so that you end up with 250mM KOAc.
Pellet heavy membranes from the post-nuclear supernatants by centrifugation at 13, 000 rpm in the Hermle (15, 000 Xg) for 10 min. Save supernatant to isolate light membranes.
Resuspend pellets in 14ml volume of Resuspension buffer (as above) plus 500mM KOAc but minus magnesium, homogenize 10 strokes.
Pour into two 10 ml disposable polypropylene tubes and pellet debris at 1900 rpm 10 min. in Hermle.
Pour supernatants into a new tube and pellet heavy membranes at 13,000 rpm in the Hermle, 10 min.
Combine supernatants with the Sup you are saving to isolate light membranes (ER) and adjust the volume to 80 mls total using resuspension buffer plus K minus Mg.
Resuspend pellet (these are your heavy membranes) in 200ml of resuspension buffer (0 K) and freeze in liquid nitrogen.
Layer supernatant from heavy membrane spin on 5 ml cushions in Ti 50.2 polycarbonate bottles (or underlay the cushions--its faster). Each bottle holds 25 mls total so you will need a balance tube. Remember to put 5mls of cushion in the balance tube because 25 mls of water will not weigh as much as your cushions and samples that are full of sucrose).Layer onto cushions carefully with the bottle held at an angle in ice.
Spin at 45, 000 rpm in the Ti 50.2 rotor for two hours at 4°C.
Resuspend pellets in a total of 500ml of resuspension buffer (0 K) and freeze in liquid nitrogen.
Nycodenz gradients can be used to clean up the heavy membrane fraction as follows:
Make solutions of 10, 20, 30, 40% nycodenz in:
Add some of the buffer to the tube before adding nycodenz or it will “ball up” in the bottom of the tube and be hard to dissolve.
For each gradient layer 400ml of each nycodenz solution into a 2ml TLS-55 tube. Let sit overnight in the cold room.
Take 100ml of the heavy membrane fraction (frozen) and mix with 300ml of the above solution without nycodenz or sucrose. Layer this material on top of the gradient.
Spin at 40, 000 rpm(100, 000 Xg) for one hour.(The K factor is a lot lower than the similar spin used in the mito prep).