(1) Adherent epithelial cells are plated at 90-100%
confluent density at 18 hours before utilization for biochemistry.
(2) On the first day of purification, cells are
quickly rinsed four times with ice cold 10 mM HEPES, pH 9.0, and incubated in
the same buffer for three hours at 4°C without agitation
(3) At the end of three hours, in the cold room, the
buffer is decanted quickly and gently to avoid sloughing off of cells from the
(4) To maintain the highest protein concentration possible
in the initial cell lysate, the remaining buffer is removed as much as possible
by tilting the plate and using a pipetman.
(5) Cell are immediately scraped off the plates and
collected in a 50 ml conical tube chilled on ice.
(6) After all the cells are collected from the plates,
are passed 10 times through a 22 G needle. Efficiency of homogenization is
examined under phase-contrast of trypan blue stained whole cell lysate.
(7) After homogenization,
protease inhibitors (Antipain, 50 μg/ml;
Aprotinin, 2 μg/ml; calpain inhibitor I, 17 μg/ml; calpain inhibitor II, 7 μg/ml;
E-64, 10 μg/ml; leupeptin, 1 μg/ml; Pefabloc SC, 1 mg/ml; pepstatin A, 1 μg/ml;
Roche Molecular Biochemicals) are added immediately with continuous vortex.
(8) Large organelles,
nuclei, and whole cells are removed by
centrifugation at 30,000xg for 10 minutes at 4°C (or 10,000xg for 30 min).
(9) The supernatant is
centrifuged at 100,000 g for 60
minutes at 4°C onto a saturated sucrose cushion.
(10) Membranes are collected at the buffer and sucrose
interface, washed with ice-cold distilled water, and re-centrifuged at 100,000
g onto a saturated sucrose cushion in a new tube.
(11) The washed membranes are collected at the water/sucrose
interface and diluted in 10% CHAPS in distilled water (chilled to 4°C) to a
final concentration of 2% CHAPS.
(12) The membranes are then incubated with primary
antibodies (1-10 ug per 150 mm dish of cells dependent on the copy number per
cell of the protein) for 18 hours at 4°C.
(13) Membranes are centrifuged at 10,000 g for 30 min at
to remove precipitates.
(14) Cleared supernatant is incubated with Protein
A-Sepharose beads (amount of beads needed depends on the binding capacity of
the beads and the amount of primary antibodies added; in general, add two-fold
excess of beads to pull down all antibody-antigen complexes). Rotate the beads for
18 hours at 4°C.
(15) Beads are washed 6 times with ice cold 2% CHAPS in
distilled water. Antibody-antigen
complexes can be eluted with a peptide antigen in 100 mM NaCl, 15 mM HEPES, pH
7.5, for 60 min at 25°C. Add at
least a ten-fold molar excess of peptide should be added for the elution step. If a peptide antigen is not available, partial
elution can be performed using high salt (0.5 M Tris, pH 7.5).
(16) A small fraction of the eluted
immuno-complex can be
used immediately for negative stain electron microscopy. The rest of the sample
is passed through a 25G Sepharose spin column to remove salts and subsequently
concentrated using a speedvac. The concentrated samples can be used immediately
for gel analysis or stored at -80°C.