LAB PROTOCOLS GROUP HUBER
31. January, 2003
HOMOGENIZATION of CELLS

Keep everything strictly on ice

Have protease inhibitors present in all solutions during the homogenization
and the following preparative centrifugation steps

Add protease inhibitors just before use of a certain buffer since they are
instable

Cycloheximide is added to trap the nascent chains in ribosomes on the rough
ER, thus making this compartment heavier and easier to separate from the
lighter compartments (early and late endosomes)

EDTA is added to, firstly, inhibit proteases which are dependent on bivalent
ions and, secondly to deplete Ca++ , in order to weaken intercellular contacts
and prevent aggregation of membranes.
1
IF
INTRODUCTION
(Labeled) Cells are scraped from filters (or plates), homogenized, and centrifuged to
prepare a PNS (Post nuclear supernatant). Gentle homogenization conditions should
be applied to limit possible breakage of endosomes and lysosomes, particularly when
using fluid-phase markers. Clearly, the markers should remain entrapped in vesicles
(latent) after homogenization. In addition, harsh conditions should always be avoided
to limit the breakage of lysosomes and consequent proteolysis by released hydrolases.
Each step of the homogenization process should be monitored using a phase-contrast
microscope to check for cellular breakup and for damage of already released nuclei
which is deleterious to the quality of the preparation. Since epithelial cells grow in
tightly interacting monolayers, sheets of cells which are not homogenized properly
will always be present whereas already released nuclei tend to break upon overhomogenization. The result of the prep will always be a balance between efficiency
(unbroken cells will be lost in the nuclear pellet) and quality of released subcellular
compartments, the latter being more important.
1. SOLUTIONS
1.1 PBScontaining 0.5x protease inhibitors (optional; some cells already break
during the scraping of the cells; 1000x stock of 10 mg/ml Aprotinin,
1 mg/ml Pepstatin and 10 mg/ml Leupeptin and 500x stock of 0.4M
Pefabloc at -20C).
you need 2.5 ml per filter (75 mm, Costar, #3419).
1.2 Homogenization buffer (HB)
250 mM sucrose
17.1 g
3 mM imidazole, pH 7.4
2 ml of 0.3M stock
double distilled H2O
ad 200 ml
Store sterile buffer for 4 to 8 weeks at 4C.
You need 2-2.5 ml (containing 0.5x protease inhibitors) per 75 mm
filter
1.3 HB+
HB containing 1mM EDTA (0.5M stock in the lab)
1x protease inhibitors (see above)
30l of cycloheximide (stock at -20C) per 10 ml
2
IF
1.4 Extraction buffer
20mM HEPES, pH7.0
100mM KCl
1mM DTT
1% Triton X-100
1x protease inhibitors (see above)
2. PROCEDURE
2.1 EpH4 and MDCK cells on filters (also plastic):
Place the lid of the plate up side down on the cold plate (metal plate on ice),
aspirate the last wash (PBS) and place the filter on the lid.
2. Add 2.5 ml of PBS (containing 0.5x protease inhibitors) and scrape the cells in
one continuous sheet using the special scrapers and turning the plate 360 in one
continuous movement while keeping the scraper with slight pressure always in the
fluid (tilt a bit). Remove the sheet of cells from the scraper by fast vertical shaking
within the fluid without touching the filter. Scrape the center of the filter with a
single movement as well.
3. Transfer the scraped cells into a conical 15 ml centrifuge tube using a wide
disposable plastic Transfer pipette (cut the tip) and centrifuge 5 min at 1000 rpm
and 4C.
4. Aspirate PBS- and add 2-2.5 ml of HB (containing 0.5x protease inhibitors) in a
way that the entire pellet floats up.
5. Centrifuge 10 min at 2500 rpm at 4C, aspirate the buffer.
6. Re-suspend the cells gently in HB+ (about 3-5x the volume of the cellular pellet,
approximately 400 l per filter) using a blue tip. (It is important to disrupt sheets
of cells as efficient as possible before homogenization. Check presence of big
aggregates using a lamp).
7. Homogenize the cells by drawing the homogenate three to ten times through a 22G needle attached to a 1-ml syringe. Fill the syringe with buffer and blow out all
air-bubbles trapped in the needle before starting! Air-bubbles will give
unpredictable forces leading to bad homogenization or extensive nuclear
breakage. Suck up the homogenate slowly and blow it out with some forces but
still gently, with the tip of the needle being in contact with the tube. Monitor
homogenization using a phase-contrast microscope. Be careful not to overhomogenize (for MDCK about 3 strokes, for EpH4 about 7 strokes). The ratio
between unbroken cells and free nuclei should be about 10-20%:80% (check in
the microscope). If this is not the case you can do one or more additional strokes.
Nuclei should be intact and free of cellular materials.
3
IF
8. Immediately after checking the percentage of broken cells and unbroken nuclei,
pellet the nuclei at 3000 rpm for 10 min, 4C.
9. Carefully retrieve the supernatant without sucking up any nuclei. PNS is relatively
stable on ice and is ready for preparative gradients.
10. To prepare cellular membranes centrifuge the PNS 30min at 100.000g , 4C.
11. Dissolve membrane pellet in Extraction buffer, keep 30min on ice
12. Centrifuge 30min at 17000g, 4C and perform glycerol density centrifugation of
the supernatant
3. Optional protocol - Nuclear Fraction Purification
9. Pipette away the PNS fraction and adjust the nuclear fraction to the final
concentration of 0.25 M sucrose and 0.35 % Triton X-100. Pellet the nuclei
through a 1ml 10 % sucrose cushion in 1.5 ml Eppendorf tube by 5 min full speed
centrifugation at 4C in a table-top Eppendorf centrifuge.
10. Discard the sucrose cushion and re-suspend the nuclei in PBS containing 0.7 M
NaCl and 0.4 % Tween 20 (final conc.).
11. Sonicate the samples on ice, 2 times, approximately for 10-15 sec, at low intensity
(should not cause foaming of the sample!).
12. Centrifuge 5 min full speed centrifugation at 4C in a table-top Eppendorf
centrifuge.
13. Supernatant contains mainly caryoplasma and pellet nuclear membranes.
Fractions are re-suspended in HB+ buffer and equal protein concentration
adjusted according to the protein concentration measurement.
14. Samples should be snap-shot frozen in liquid nitrogen and later used for e.g.
Western blot analysis.
PREPARATIVE GRADIENTS
SOLUTIONS
10% (w/w) glycerol solution in HB
4
IF
35% (w/w) glycerol solution in HB
PROCEDURE
1. For the preparation of the gradients use the BioComp gradient master. Draw a
mark for the 50% volume of the SW41 tubes using the small metal block lower
level (in the equipment of the gradient master).
2. Fill the tube until a bit above the mark with 10% glycerol solution containing
protease inhibitors and cycloheximide.
3. Using the syringe adjusted with a rubber tubing, load the 35% glycerol solution
(+protease inhibitors/cycloheximide) underneath the light solution until the
interphase lines exactly at the half-volume-mark.
4. Carefully but properly close the tubes with the long, black lids removing all the air
bubbles and the superfluous 10% solution through the whole in the lid. Remove
the overflow fluid out of the lids. Air bubbles will destroy the establishing
gradient during the run in the gradient master.
5. Level the gradient master using the leveling mode (described in the description of
the apparatus) every time before you start the run.
6. Put in the closed tubes balanced (like in centrifuge). Recall program No.1 and
start the run.
7. When the run has completely stopped, take out the tubes, take away the lids
carefully. Have the top of the tubes wrapped in paper to suck away the redundant
fluid trapped at the side of the lids.
8. Place the gradients on ice. When they are cold you can start loading the PNS or
the prepared membranes on top of the gradient. Take care not to disturb the
gradient while you are loading the sample.
9. Mount the tubes in an SW 41 rotor and centrifuge 18 hrs, 40000 rpm, 4C
5
IF
COLLECTION of FRACTIONS
For collection of the fractions use the Auto Densi-Flow. Read the instruction
manual before you start!! Take care not to bend or break the very sensitive tip
(“probe”) and sheath of the instrument. Always leave the instrument in the cold
room. For replacement of the rubber tubing read the instruction manual; take care to
make it long enough to reach the lowest position of the “probe” and to reach all the
Eppendorf tubes you have prepared (the length of a big yellow ice-bucket).
1. Prepare and label 20-24 Eppendorf tubes in an ice-bucket.
2. Level the Auto Densi-Flow using the screws at the bottom of the instrument.
3. Close the screw of the pump assembly and tighten it until the pump starts sucking
water through the tubing from a beaker (in the position ”Deposit”) and turn 1/2
turn further than the point where pumping has started. Remove the water from the
tubing by switching to “Remove”.
4. Fix the centrifuge tube in the instrument with the centering jaws.
5. Switch on the instrument, set the function switch to the position “Down” and let
the “probe” sink down to the surface of the fluid where it stops. Set the pump
speed to 2 and start the fractionation by switching the pump to “Remove”. At this
speed four drops are about 170 µl (giving 24 fractions) and five drops are about
210 µl (19-20 fractions). Lightest fraction is No.1.
6. After each gradient extensively wash the tubing with double distilled water (you
can use high speed for the washing) from the small glass beaker which is supplied
with the instrument. Move the “probe” into the water by pressing the release
bottom in front of the “probe-holder” and carefully moving the “probe” with the
bottom kept pressed. “Remove” and let run dry afterwards.
7. Take off the next gradient as described.
8. After you have finished your last gradient, wash again extensively with water, let
it run dry and open the tightening screw of the pump to keep the rubber tubing in a
good shape as long as possible.
6
IF