Biology Reference
In-Depth Information
1.
Rinse HT1080 cells in flask with 2
10 mL PBS to remove medium that could
inhibit trypsin. Drain and add 2 mL of trypsin solution (prewarmed to room tem-
perature) and incubate at room temperature for 3 min. Cells should now be
detached and can be disrupted by gently tapping the flask. Add 8 mL of KHM
buffer and 20
×
g/mL) to the
tissue-culture flask. Transfer cell suspension to a 15-mL Falcon tube on ice.
µ
L soybean trypsin inhibitor (final concentration 100
µ
2.
Pellet cells by centrifugation at 350
×
g
for 3 min at 4
°
C. Aspirate the supernatant
from the cell pellet.
3.
Resuspend cells in 6 mL of ice-cold KHM. Add 6
µ
L digitonin (from 40 mg/mL
g/mL) and mix immediately by inversion and
incubate on ice for 5 min (
see
Note 2
).
4. Adjust the volume to 14 mL with ice-cold KHM and pellet cells by centrifuga-
tion as in
step 2
.
5. Aspirate the supernatant and resuspend cells in 14 mL ice-cold HEPES buffer.
Incubate on ice for 10 min and pellet cells by centrifugation as in
step 2
.
6. Aspirate the supernatant and resuspend cells carefully in 1 mL ice-cold KHM
(use a 1-mL Gilson pipet and pipet gently up and down). Place on ice.
7. Transfer a 10-
stock, i.e., final concentration 40
µ
µ
L aliquot to a separate 1.5-mL microcentrifuge tube and add
10
µ
L of trypan blue.
8.
Count cells in a hemocytometer and check for permeabilization, i.e., whether the
trypan blue permeates into the cell.
9.
Transfer cells to a 1.5-mL microcentrifuge tube and spin for 30 s at 15,000
×
g
.
Aspirate supernatant and resuspend the cells in 100
µ
L KHM using a pipet.
10.
Treat the cells with a calcium-dependent nuclease to remove the endogenous
mRNA. Add 1
µ
L of 0.1
M
CaCl
2
and 1
µ
L of monococcal nuclease and incubate
at room temperature for 12 min.
11.
L of 0.4
M
EGTA to chelate the calcium and inactivate the nuclease.
Isolate the cells by centrifuging for 30 s in a microcentrifuge and resuspend in
100
Add 1
µ
µ
L of KHM.
Use approximately 10
5
cells per 25-
12.
µ
L translation reaction (approx 4
µ
L of the
100
µ
L obtained).
3.2. Transcription In Vitro
The cDNA encoding the protein of interest is ligated into a mammalian
expression vector, such as pBluescript, upstream of a suitable promotor con-
taining an RNA polymerase binding site from which transcription is initiated.
Prior to transcription, the cDNA clone must be linearized by restriction endo-
nuclease digestion to generate a template for mRNA synthesis. This method is
a modification of a method described previously
(9)
.
1.
Prepare a 100-
µ
L reaction mixture containing 44
µ
L H
2
O, 10
µ
L linearized DNA
(5-10
µ
g), 20
µ
L transcription buffer (5X), 10
µ
L 100 m
M
DTT, 1
µ
L RNasin
(20 U), 3
µ
L of each nucleotide.
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