Subcellular Fractionation Procedures
and Metabolic Labeling Using
[35S]Sulfate to Isolate Dense Core
Secretory Granules from
Neuroendocrine Cell Lines
Subcellular fractionation techniques have been
developed to allow isolation of a particular subcellular
compartments. Typically subcellular fractionation
is used as a starting point to characterize the composition
of subcellular organelles, but has also been used
frequently to provide membranes for cell-free assays
that reconstitute a particular intracellular event or
Ideally, for both purposes (characterization and cellfree
assays) the subcellular compartment should be
purified to homogeneity. However, this is difficult to
achieve, particularly when the starting material is from
cell lines where amounts are limiting. Therefore, it is
realistic to aim to achieve a highly enriched fraction
(defined here as greater than 90% purity) with a good
yield. In addition, it is important to carefully consider
the purpose of the fractionation protocol as this will
dictate how critical the purity is.
To study the formation of secretory granules in the
PC12 cell line, a protocol was developed to identify
nascent immature granules by optimizing their
separation from the donor compartment, the Golgi
complex (Tooze and Huttner, 1990). A combination
of velocity-controlled differential and equilibrium
density centrifugation achieved a separation of immature
secretory granules and the trans
(TGN). This protocol was also used to obtain a population
highly enriched in immature secretory granules
(ISG) and one highly enriched in mature secretory
granules (MSG) (Tooze et al.
, 1991). MSGs are easier to
obtain in a more purified form as MSGs are denser
than other subcellular compartments. This protocol
has been used to characterise the ISG and MSG (Tooze et al.
, 1991), study low pH-dependent prohormone
processing (Urbé et al.
, 1997), clathrin coat recruitment
to ISGs (Dittié et al.
, 1996), and the role of ADPribosylation
factor (ARF) (Austin et al.
, 2000), sorting
of proteins in the ISG (Dittié et al.
, 1999), cell-free
homotypic fusion (Urbé et al.
, 1998; Wendler et al.
2001), and the recruitment of lipid kinases to ISGs
(Panaretou and Tooze, 2002). This protocol has also
been used by other researchers to investigate sorting
sequences in regulated secretory proteins (Krömer et
al., 1996) and with, for example, the AtT20 cell line
(Eaton et al.
II. MATERIALS AND
PC12 cells are maintained in growth medium consisting
of 10% horse serum, 5% fetal calf serum, and
Dulbecco's modified Eagle's medium (DMEM) with
3.5 g/liter glucose, penicillin/streptomycin, and 4 mM
glutamine (note this is twice the standard concentration)
under 10% CO2
. Reagents for cell culture are
obtained from Sigma Aldrich and GIBCO.
PC12 cells are passaged once a week at a dilution of
1:6. For a standard granule preparation, nine, 245× 245-mm confluent plates of PC12 cells are used. These
plates are prepared from five, 175-cm2
with trypsin, seeded, and grown for about 7 days or
from nine, 175-cm2
flasks harvested with trypsin,
seeded, and grown for about 3 days.
All chemicals are available from Sigma-Aldrich,
except sucrose (ultrapure grade Cat. 15503-02), which
is from GIBCO.
A cell cracker (EMBL Workshop, EMBL Heidelberg,
Germany) with a range of titanium balls. Assemble cell
cracker with chosen ball, precool and wash cell cracker
with 1 ml of homogenization buffer (HB)/protease
inhibitor cocktail (pi) before use. For PC12 cells, use a
Gradients are centrifuged in an SW40 rotor using
Beckman ultraclear centrifuge tubes (Cat. No. 344060).
Velocity gradients are prepared using a BioComp
gradient master (http://www.biocompinstruments.
com), and equilibrium gradients are prepared using a
Labconco Auto Densiflow gradient maker (http://
www.labconco.com). Both velocity and equilibrium
gradients are collected using the Auto Densiflow.
A cell scraper is made from a silicon rubber bung by
cutting the bung with a single-sided razor blade first
horizontally (across the widest part) and then vertically
in half. The tip of a plastic 10-ml pipette is then
inserted into the center of the cut bung.
A. Preparation of a Postnuclear Supernatant
- Tris-buffered saline (TBS): 137 mM NaCl, 4.5 mM KCl,
0.7 mM Na2HPO4, 25 mM Tris-HCl, pH 7.4
- Protease inhibitor (pi) cocktail: 0.5 mM phenylmethylsulfonyl
fluoride and 5-µg/ml leupeptin
- Homogenization buffer (HB): 0.25M sucrose, 10mM HEPES-KOH, pH 7.2, 1 mM EDTA, and 1 mM MgOAc
For preparation of sufficient postnuclear supernatant
(PNS) to load six SW40 gradients, use nine 245
× 245-mm plates. All solutions must be at 4°C.
B. Velocity Gradient Centrifugation
- Place one 245 × 245-mm plate on ice.
- Remove growth medium by decanting and wash
gently twice with 40ml TBS.
- Wash each plate again with 40ml TBS/pi.
- Add 40ml TBS/pi and remove PC12 cells from
the plates by scraping with a cell scraper. Collect the
suspension of cells from each plate into a 50-ml Falcon
tube. Repeat for all nine plates.
- Spin each tube for 7min at 84g. Remove supernatant
and resuspend each pellet in 1 ml of HB/pi.
Pool in a 50-ml Falcon tube containing 24 ml of HB/pi
and divide into six 15-ml Falcon tubes.
- Spin for 7 min at 500g. Remove supernatant and
resuspend each pellet in 700 µl HB/pi and pool all nine
tubes of cell suspension into a 15-ml Falcon tube.
- Using a 1-ml syringe with a 21- or 22-gauge
needle, draw up 1 ml of cells and pass back and forth
through the needle six to seven times. Check that cells
are well dispersed by resuspending 5 µl of cells with
10 µl of trypan blue solution and monitoring by phasecontrast
microscopy. Ensure that the cells are now dispersed
uniformly and not still in clumps. A maximum
of 20% of the cells should be permeable to trypan blue.
Repeat until all the cells have been passed through the
- Using a 1-ml syringe and 21- or 22-gauge needle,
draw up 1 ml of the cell suspension and place syringe
on one port of the cell cracker. Place an empty syringe
on the other port. Pass cells through chamber six to
seven times. Check breakage with trypan blue as in
step 7. Greater than 90% of the cells should be broken.
Nuclei should be intact and spherical but free of subcellular
membranes (see Tooze and Huttner, 1992).
- Remove homogenate and repeat with remaining
cell suspension. Rinse the cell cracker out with 1 ml of
HB/pi. Pool all the homogenate into a 15-ml Falcon
tube. Total volume will be between 9 and 10ml.
- Spin for 10 min at 1700g to pellet nuclei.
Remove postnuclear supernatant, avoiding the nuclear
pellet. Because the interface between the supernatant
and the pellet can be difficult to see, illuminate the tube
from behind when removing the supernatant.
- Respin the PNS for 5 min at 1700g to ensure that
all nuclear material is removed and collect the supernatant.
Adjust volume if necessary to 9.0 ml.
This method achieves separation of organelles by
size rather than density. Thus, larger subcellular
compartments will sediment faster during this centrifugation
than smaller ones. This is the basis for the
separation of ISGs and MSGs from the trans-Golgi network (TGN). In particular, this step is essential for
separation of ISGs from the TGN as these organelles
have the same equilibrium density.
pH 7.2, and 1.2M
C. Preparation of ISGs and MSGs
by Equilibrium Gradient Centrifugation
- Prepare the velocity gradients. Use 5.5ml of
0.3M sucrose and 6ml of 1.2M sucrose per gradient.
Using the Biocomp gradient maker, perform two steps:
step 1 for 10min at a 50°C angle at 30rpm and step 2
for 1 min at a 80°C angle at 12rpm. If preparing the
gradients manually, use a 15-ml gradient maker, gently
mixing the heavy into the light. Using a narrow
pipette, pump solution from the light chamber into the
bottom of the tube and displace the solution upward
with the heavier solutions.
- Load 1.5 ml of PNS on top of each gradient. Spin
at 25,000rpm in a SW40 rotor at 4°C. Using maximum
acceleration, allow centrifuge to reach 25,000rpm and
then spin for exactly 15 min with the brake applied at
the end of the run.
- Collect thirteen 1-ml fractions from the top.
Fraction 1 will contain most of the soluble proteins
from the PNS. Fractions 2-4 will contain the ISG and
constitutive secretory vesicles (CSVs), fractions 5-7
will contain MSGs, and fractions 8-11 will contain
TGN membranes. Note: There will be other subcellular
membranes in these fractions so the ISGs, MSGs, and
TGN fractions are only slightly enriched. Pool fractions
1-4 for preparation of ISGs and fractions 5-7 for
preparation of MSGs.
This method achieves separation of organelles by
density, allowing vesicles of the same size but different
densities to be separated. ISG, which have an
average diameter of 80nm, can be separated Golgiderived
CSVs, which are reported to have a diameter
of 50-200nm (Salamero et al.
sucrose, 10 mM
pH 7.2; 1.0M
sucrose, 10 mM
pH 7.2; 1.2M
HEPES-KOH, pH 7.2;
HEPES-KOH, pH 7.2; and 1.6M
sucrose, 10 mM
HEPES-KOH, pH 7.2
D. Other Procedures
- Prepare equilibrium gradients. Pipette 1 ml of
1.6M and then layer 2ml of 1.4, 1.2, 1.0, and then
1.0 ml of 0.8 M sucrose into a SW40 tube, either by hand
or using the Auto Densiflow, with probe moving
- Adjust pooled fractions from the velocity gradient
to a final volume of 4 ml per gradient with 10mM HEPES-KOH, pH 7.2. Load pooled fractions from
velocity gradient onto prepared equilibrium gradients.
- Spin gradients in a SW40 rotor at 25,000rpm
overnight or for at least 5.5 h at 4°C.
- Collect twelve 1-ml fractions from the top of the
gradient. If preparing ISGs, the ISGs will be found in
fractions 7-9. If preparing MSGs, the MSGs will be
found in fractions 10-12. These respective fractions can
be pooled and aliquoted for storage for up to 6 months
in liquid nitrogen.
The centrifugation procedure can be checked by
assaying for TGN, secretory granule, or other
compartment-specific markers in each fraction from
the velocity and equilibrium gradients. This can be
done by Western blotting using antibodies to the
marker proteins or by metabolic labelling. For the
technique described earlier using PC12 cells, the most
accurate method is posttranslational labelling of proteins
S]sulfate on tyrosine residues (for a
review, see Moore, 2003).
Protein tyrosine sulfation is a posttranslational
modification found in some secretory proteins,
including secretogranin II (SgII) and an unidentified
constitutively secreted heparan sulphated proteoglycan
(HSPG). The enzyme tyrosylprotein sulfotransferase
responsible for sulfation is a resident TGN
protein (Lee and Huttner, 1985). Thus, by incubating or
pulsing the cells for a short period of time by the addition
S]sulfate, proteins that contain the sulfation
motif can be labelled in the TGN and can be used to
identify TGN membranes. In addition, exit of sulphated
proteins from the TGN into CSVs, which contain HSPG,
and ISGs and MSGs, which contain SgII, can be followed
accurately during the chase. Identification of
CSV and ISGs by sulfate label of the specific markers,
followed by subcellular fractionation, can be used to
confirm that the cultured cells are correctly sorting regulated
and constitutively secreted proteins into separate
vesicles (Tooze and Huttner, 1990). Metabolic
labelling with sulfate can also be applied more generally
(Tooze, 1999). It is advisable to use a scaled down version of the procedure described earlier to avoid
having to use large amounts of radioactivity.
- Labelling medium (sulfate-free DMEM): DMEM is prepared
by substituting MgCl2·6H2O for MgSO4·7H2O and reducing the normal concentration of cysteine
and methionine to 1% of the original concentration;
0.1% dialysed horse serum; 0.05% dialysed fetal calf
serum; and 2mM glutamine. It is important not to
include any sulphated antibiotics.
- [35S]Sulfate (40-100mCi/ml): Amersham Biosciences
(Cat. No. SJS.1).
- Chase medium: growth medium supplemented with
1.6 mM NaS04.
To check the ability of PC12 cells to sort regulated
proteins using the gradients described earlier, two 150-
dishes of PC12 cells should be prepared for each
condition. Ideally the experiment should confirm the
position of the TGN on the gradients, identified by a
5-min pulse of sulfate; the CSVs and ISGs, identified
by a 5-min pulse followed by a 15-min chase; and the
MSGs, identified by a 5-min pulse and 90-min chase.
Alternatively, MSGs can be labelled using one-tenth
the amount of [35
S]sulfate for 6 h and chased overnight.
- Wash each 150-mm 2 dish once with labelling
medium at 37°C. Replace with 20 ml labelling medium.
Incubate for 20min at 37°C to deplete endogenous
- Remove medium and replace with fresh medium
at 37~ containing lmCi/ml [35S]sulfate, or 0.1mCi/
ml for long-term MSG labelling. Incubate at 37°C for
precisely 5 min. Dishes can be rocked gently to reduce
the amount of labelling medium required. Five to 7 ml
is sufficient when dishes are being rocked.
- Remove labelling medium. To identify the TGN,
transfer the dishes immediately to 4~ and add 10ml
of TBS at 4°C. To identify ISGs, add 20ml chase
medium at 37°C and return cells to incubator for 15 min.
To label MSGs, add 20ml chase medium at 37°C and
return cells to incubator for 90min. Alternatively, use
the longer label-and-chase protocol for MSGs.
- At the end of the chase period, transfer remaining
dishes to 4°C remove the chase medium, and add
10 ml TBS at 4°C.
- Wash and harvest dishes as described previously.
Pool the two dishes from each condition. Prepare a
PNS as described earlier and load the PNS from both
labelled 150-mm 2 dishes on one velocity gradient.
- Collect thirteen 1-ml fractions from each gradient.
Remove 30µl from each fraction and analyse the [35S]sulfate-labelled proteins by SDS-PAGE. For
optimal resolution of SgII and HSPG, use a 7.5% gel.
- Save the remaining material for equilibrium gradient
centrifugation. Pool fractions 1-4 from the ISG
gradient, and pool fractions 5-7 from the MSG gradient.
Load each pool onto on an equilibrium gradient
and continue as described in Section III.C.
- The PC12 cells are not passaged correctly and
thus do not achieve maximum confluency, resulting in
a very low yield of cells. The cells must be plated as
single cell suspensions. This is best achieved by
tituration using a flamed narrowed disposable glass
- The cells are not completely homogenized,
resulting in fewer broken cells as judged by trypan
blue. This will result in a reduced yield.
- The homogenate is very viscous. This is most
likely a result of nuclei lysis during homogenization.
A clean postnuclear supernatant cannot be obtained
from this homogenate, and the experiment should be
stopped as the subcellular organelles will not be
separated properly on the gradients.
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