In Situ Electroporation of Radioactive
Nucleotides: Assessment of Ras Activity
or 32P Labeling of Cellular Proteins
Binding to nucleotide(s) can determine a state of
activation of a protein; a number of GTP-binding
proteins such as Ras exist in two distinct, guanine
nucleotide-bound conformations, the active Ras.GTP
state and the inactive Ras.GDP form, so that the fraction
of Ras bound to GTP (percentage Ras.GTP/GTP +
GDP) can determine its state of activation (Lowy and
Willumsen, 1993). Several indirect assays are in existence
for measurement of Ras activity (Scheele et al.
1995; Taylor et al.
, 2001). However, in a number of
instances, a direct measurement of Ras.GTP binding is
necessary (Egawa et al.
, 1999) and is commonly performed
through the addition of [32
the growth medium followed by Ras immunoprecipitation
and guanine nucleotide elution (Downward,
1995). This approach is relatively inefficient due to the
fact that the isotope is incorporated into all phosphatecontaining
cellular components. To circumvent this
P]GTP has been introduced into the cell
and its breakdown into [α32
P]GDP after Ras binding
monitored as described earlier (Downward, 1995).
Because, contrary to free bases or nucleosides, most
nucleotides do not cross the cell membrane, [α32
has to be introduced into intact cells after cell membrane
Protein phosphorylation is a ubiquitous regulator of
a large variety of cellular functions. The in vivo
and detection of phosphoproteins is usually
conducted through the addition of [32
to the culture medium, followed by immunoprecipitation
and electrophoretic separation of
precipitated proteins. Just as in the case of Ras activity
measurement, this method is relatively inefficient,
hence ATP, the common immediate phosphate donor
nucleotide, may be used for in vivo
which must be introduced into intact cells through
This article describes a technique where the introduction
of nucleotides into adherent cells is performed
through in situ
electroporation. Cells are grown on a
glass surface coated with electrically conductive,
optically transparent indium-tin oxide at the time of
pulse delivery, a coating that promotes excellent cell
adhesion and growth. Unlike other techniques of
cell membrane permeabilization, such as streptolysin-
O (SLO) treatment, in situ
electroporation does not
detectably affect cellular metabolism, presumably
because the pores reseal rapidly so that the cellular
interior is restored to its original state; under the
appropriate conditions there is no increase in the
activity of the extracellular signal-regulated kinase
(Erk½) or two stress-activated kinases, JNK/SAPK
or p38hog (Brownell et al.
, 1998). Results show that Ras
activity measurement through electroporation of
P]GTP could be performed using approximately
50-100 times lower amounts of radioactivity; although
P is in the form of [α32
P]GTP exclusively, this
technique offers higher specificity compared to
labelling through the addition of [32
to the culture medium. In addition, labelling of two
viral phosphoproteins, the large tumor antigen of
simian virus 40 and adenovirus EIA, by in situ
P]ATP requires a fraction of the
amount of radioactive phosphorus, while offering
II. MATERIALS AND
Dulbecco's modification of Eagle's medium
(DMEM) is from ICN (Cat. No. 10-331-22). Phosphatefree
DMEM (Cat. No. D-3916) is from Sigma. Fetal calf
serum (Cat. No. 2406000AJ) is from Life Technologies
Inc. Calf serum is from ICN (Cat. No. 29-131-54). The
following reagents are from Sigma: insulin (Cat. No.
1-6643), NaCl (Cat. No. S-7653), HEPES (Cat. No.
H-9136), Lucifer yellow CH dilithium salt (Cat. No. L-
0259), trypsin (Cat. No. T-0646), MgCl2
(Cat. No. M-
8266), Triton X-100 (Cat. No. T-6878), deoxycholate
(Cat. No. D-5760), EDTA (Cat. No. E-5134), EGTA (Cat.
No. E-3889), phenylmethylsulfonyl fluoride (PMSF, P-
7626), aprotinin (Cat. No. A-6279), leupeptin (Cat. No.
L-2023), benzamidine (Cat. No. B-6506), dithiothreitol
(DTT, Cat. No. D-9779), CaCl2
(Cat. No. C-4901), Trisbase
(Cat. No. T-6791), sodium orthovanadate (S-6508),
and LiCl (Cat. No. L-4408). The following reagents are
from BDH: Extran-300 detergent (Cat. No. $6036 39),
SDS (Cat. No. 44244), TLC silica gel plates containing
a fluorescence indicator (Cat. No. M05735-01), isopropanol
(Cat. No. ACS720), concentrated ammonia
solution (Cat. No. ACS033-74), Nonidet P-40 (Cat. No.
56009), and glycerol (Cat. No. B10118). GTP (Cat. No.
106 372) and GDP (Cat. No. 106 208) are from
Boehringer Mannheim. The monoclonal anti-Ras antibody
is from Oncogene Science (Pan-Ras Ab2, Cat. No.
OP22), the monoclonal antibody to simian virus 40
large tumor antigen is from Pharmingen (p108, Cat.
No. 14121A), and the monoclonal antibody to adenovirus
EIA is from Calbiochem (M73, Cat. No. DPll).
Staph. A Sepharose beads (17-0780-03) for immunoprecipitation
are from Pharmacia. X-ray film is from
Kodak (X-OMAT AR, Cat. No. 165 1454). CelTakTM
(Cat. No. 354240) is from BD Biosciences. Tissue
culture petri dishes (6cm diameter) are from Corning
The purity of the material to be electroporated is of
paramount importance. [α32
P]GTP (Cat. No. NEG
006H) and [γ32
P]ATP (Cat. No. NEG 002A) are from
Dupont NEN Research Products (HPLC purified).
The apparatus for electroporation in situ
model EZ-16) is available from Ask Science Products
Inc. (487 Victoria St. Kingston, Ontario Canada). The
inverted, phase-contrast and fluorescence microscope,
equipped with a filter for Lucifer yellow (excitation:
435, emission: 530), is from Olympus (Model IX70).
The technique can be applied to a large variety of
adherent cell types. We have used a number of lines,
such as the Fisher rat fibroblast Flll and its polyoma
or simian virus 40 virus-transformed derivatives, mouse fibroblast NIH 3T3, mouse Balb/c 3T3, mouse
NIH 3T6, mouse C3H10T½ fibroblast derivatives
expressing a ras
-antimessage [e.g., lines R14 and 25B8
(Raptis et al.
, 1997)], Rasleu61
-transformed 10T½, and rat
liver epithelial T51B, as well as a variety of differentiated
adipocytes (Brownell et al.
, 1996). All cells can be
grown in plastic petri dishes in DMEM supplemented
with 5% calf serum in a humidified 5% CO2
with the exception of R14 and 25B8, which are grown
in DMEM supplemented with 10% fetal calf serum.
Cells that do not adhere can be grown and electroporated
on the same conductive slides coated with
-lysine or collagen.
The apparatus for in situ
described in Fig. 1. Cells are grown on conductive and
transparent glass slides, which are placed in a petri
dish to maintain sterility. The cell growth area is
defined by a "window" formed with an electrically
insulating frame made of Teflon. The pulse is transmitted
through a stainless-steel negative electrode,
which is slightly larger than the cell growth area and
is placed on top of the cells, resting on the Teflon
frame. Another stainless-steel block is used as a positive
contact bar. A complete circuit is formed by
placing the electrode set on top of the slide as shown
in Figs. 1A and lB. The frame creates a gap between
the conductive coating and the negative electrode so
that current can only flow through the electroporation
fluid and cells growing in the window. In order to
obtain a uniform electric field strength over the entire
area below the negative electrode, despite the fact that
the conductive coating exhibits a significant amount of
electrical resistance, the bottom surface of the negative
electrode must be inclined relative to the glass surface,
rising in the direction of the positive contact bar, in a
manner proportional to the resistance of the coating
(Raptis and Firth, 1990); glass with a surface resistivity
of 2Ω/sq requires an angle of 1.5°, whereas glass
of 20Ω/sq requires an angle of 4.4°. The procedure
described is for glass with a surface resistivity of
20Ω/sq, which is readily available and relatively
inexpensive, hence it can be discarded after use to limit
exposure to radioactivity (see Comment
the electrode can be made out of inexpensive aluminum
for a single use.
|FIGURE 1 Electroporation electrode assembly. (A) Side view. A Delrin carrier (7) holds the negative electrode
(3) and the positive contact bar (4) so that they form one unit, which can be placed on top of the slide
(1) with its frame (2) and cells (5) in place. Negative (-) and positive (+) signs indicate the electrical connecting
points via which the pulse of electricity is delivered to the electrodes from the pulse generator. The
underside of the negative electrode (3) is machined to a slight angle, which compensates for the surface resistivity
of the conductive slide to provide uniform electroporation of the whole cell growth area. The negative
electrode rests on a Teflon frame (2), which insulates it from the conductive surface. The fluid containing the
material to be electroporated just fills the cavity below the negative electrode. When a capacitor is discharged,
current passes through the electroporation fluid (6) and cells (5) attached to the conductive glass slide (1) to
the positive contact bar (4) and back to the pulse source. Note that the angle of the negative electrode has
been exaggerated to better illustrate the meniscus of the radioactive electroporation solution (6). The slide
and electrode fit in a 6cm petri dish (8) that is locked in place on a stand (9). The top plate supports the petri
dish and is made of transparent acrylic so that the operator can look in the mirror (10) to ensure that the
liquid (6) is properly filling the cavity without air bubbles. (B) Top view. The outline of the conductive slide
(1) with a Teflon frame (2) in place to define the area of cell growth and electroporation are indicated [from
Brownell et al. (1997), reprinted with permission]. Upscaling. (C) Side view. Cells to be electroporated (5) are
grown on a glass slide (1), coated with ITO (1a). The negative electrode (3a and 3b) is a narrow steel bar
mounted across the width of the slide, resting on the Teflon frame (2), which is moved across the surface of
the slide as shown by the arrow, by an insulated carrier. The underside of the negative electrode is curved
in both directions, such as to optimise the uniformity of electrical field. Note that only the area of cells immediately
below the electrode is electroporated by a given pulse. The curvature of the negative electrode has
been exaggerated to better illustrate its contour. Note that due to the narrow shape of the electrode, air bubbles
do not get trapped easily under it, hence a stand with a mirror may not be necessary. (D) Top view. The
outline of the conductive slide with a Teflon frame (2) in place to define the area of cell growth and electroporation
and the counterelectrodes (4) are indicated. The assembly is placed in a 10cm petri dish (8) [from
Raptis et al. (2003) and Tomai et al. (2003), reprinted with permission].
A large number of signal transducers are present in
small amounts in the cell so that a large number of cells may be required to obtain a strong signal. Uniform electroporation
of a cell growth area of 32 × 10 mm u s i n g 20
Ω/sq glass (i.e., an angle of 4.4°) and a Teflon frame with
a thickness of 0.279 mm using the assembly in Figs. 1A
and 1B requires a volume o f ~280 µl. Simple scale-up of
this assembly, e.g., to a cell growth area of 50 × 30mm,
which can be accommodated in a standard, 10 cm petri dish, is faced with the problem of burning the ITO
coating that occurs at the higher voltage and capacitance settings required to electroporate this larger area
because of the resistance generated by the greater distance
the current has to travel. In addition, the volume
required , ~1.7 ml, cannot be held in place by surface
tension, while the cost of purchase and disposal of the isotope can be prohibitive for certain experiments.
These problems can be solved by using an assembly
with a narrow, moveable electrode that electroporates a
"strip" of cells at a time (Figs. 1C and 1D); in this configuration,
only cells immediately below the negative electrode
are electroporated by a given pulse of electricity.
After electroporation of the first strip of cells, the electrode
is translocated laterally, dragging the solution
under it by surface tension so that a new strip of cells is
electroporated using mostly the same solution (Figs. 1C
and 1D). The electric circuit formed during pulse delivery
starts at the negative electrode, passes through the
electroporation fluid, the cells and the conductive slide
surface, to the two positive contact bars, one on each
side of the slide. The two positive contact bars form
parallel circuit paths, both carrying current from the
conductive surface. To compensate for the resistance of
the coating, the bottom surface of the negative electrode
must be inclined toward each of the positive contact
bars; a 25 mm radius on the bottom of the electrode produces
successive strips of even electroporation over the
entire cell growth area. Using this assembly, an area of
32 × 10 mm can be electroporated using less than 50 µl of
solution with a 2.5 mm-wide electrode, whereas an area
of 50 × 30 mm can be electroporated effectively in four,
8 mm-wide strips, with a total of 200 µl of solution.
A. Electroporation of [α32P]GTP for
Measurement of Ras Activity
- Lucifer yellow solution, 5mg/ml: To make 1 ml,
add 5mg Lucifer yellow to 1 ml phosphate-free
- [α32P]GTP: Prepare a solution of 500-2000µCi/
ml in phosphate-free DMEM (see Comment 2).
- Ras extraction buffer: 50mM HEPES, pH 7.4,
150mm NaCl, 5 mM MgCl2, 1% Triton, 0.5% deoxycholate,
0.05% SDS, 1 mM EGTA, 1 mM PMSF, 10 µg/ml
aprotinin, 10µg/ml leupeptin, 10mm benzamidine,
and 1 mM vanadate. The stock solutions can be made
ahead of time. To make 50ml, add 2.5ml of HEPES
stock, 1.5ml of NaCl stock, 0.25ml of MgCl2 stock,
0.5 ml Triton, 0.05 g deoxycholate, 0.25 ml of SDS stock,
and 0.5ml of EGTA stock and then the protease
inhibitors, 0.5ml of PMSF stock solution, 0.05 ml of
aprotinin stock, 0.05ml of leupeptin stock, 0.5ml of
benzamidine stock, and 0.05 ml of vanadate stock and
bring the volume to 50 ml with distilled H2O on the day
of the experiment.
10% sodium dodecyl sulfate stock solution: Dissolve 100 g
in 1 liter H2O. Store at room temperature. Stable for
more than a year.
5 M NaCl stock solution: Dissolve 292.2 g NaCl in 1 liter
distilled H2O. Autoclave and store at room temperature.
Stable for more than a year.
1M MgCl2 stock solution: Dissolve 203.3 g MgCl2·6H2O
in 1 liter H2O. Autoclave and store at room temperature.
Stable for more than a year.
100mM EGTA stock solution: Dissolve 38.04 g in 800ml
distilled H2O. Adjust pH to 8.0 with NaOH and
complete volume to 1 liter with distilled H2O. Stable
for more than a year at room temperature.
100mM PMSF stock solution: Dissolve 17.4mg PMSF in
10ml isopropanol and store in aliquots at -20°C. Stable for several months.
10mg/ml aprotinin stock solution: Dissolve 100 mg in
10ml of 0.01M HEPES, pH 8.0. Aliquot and store at
-20°C. Stable for several months.
10 mg/ml leupeptin stock solution: Dissolve 100mg in
10ml distilled H2O. Aliquot and store at -20°C. Stable for several months.
1M benzamidine stock solution: Dissolve 1.56 g in 10ml
distilled H2O. Aliquot and store at -20°C. Stable for
1M vanadate stock solution: Dissolve 1.84 g in 10ml distilled
H2O. Aliquot and store at -20°C. Stable for
- Guanine nucleotide elution buffer: 2mM EDTA,
2mM DTT, 0.2% SDS, 0.5 mM GTP, and 0.5 mM GDP.
1M dithiothreitol stock solution: Dissolve 3.09g DTT in
20ml of 0.01 sodium acetate (pH 5.2). Store in 1 ml
aliquots at -20°C. Stable for more than a year.
0.5M EDTA, pH 8.0 stock solution: Add 186.1g disodium
ethylenediaminetetraacetate 92 H2O to 800 ml
distilled H2O. To dissolve, adjust the pH to 8.0 with
NaOH pellets while stirring. Bring volume to 1 liter.
Stable for more than a year at room temperature.
10 mM GTP stock solution: Dissolve 52.3 mg in 10 ml distilled
H2O and store at -20°C. Stable for a month.
10mM GDP stock solution: Dissolve 44.3mg in 10ml
distilled H2O and store at -20°C. Stable for a month.
See earlier list for other required stock solutions: To make
500µl of elution buffer, add 10µl of 10% SDS stock
solution, 2µl of EDTA stock solution, 1 µl of DTT
stock solution, 25µl each of GTP and GDP stock
solutions, and bring volume up to 500 µl with H2O.
- Thin-layer chromatography (TLC) running
buffer: 66% isopropanol and 1% concentrated
ammonia. To make 100 ml, mix 66 ml isopropanol, 1 ml
concentrated ammonia solution, and 33ml distilled
H2O in a fume hood. Make fresh the day of the experiment.
Depending on the size of the chromatography
tank, this volume may need to be increased.
The appropriate institutional regulations for isotope
use must be followed for all experiments.
- Choice of slides. Cell growth areas of 32 × 12 mm
are sufficient for most cases. However, for some lines
with low Ras levels, a larger number of cells may be
required to obtain an adequate signal. In this case,
the assembly in Figs. 1C and 1D (cell growth area, 50
x 30mm) may be used. Make sure the glass is clean
and free of fingerprints (see Comment 5).
- Plate the cells. Uniform spreading of the cells
is very important, as the optimal voltage depends in
part on the degree of cell contact with the conductive
surface (see Comment 3). Place the sterile glass slide
inside a 6cm (for 32 × 10mm) or 10cm (for 50 x
30 mm) petri dish. Add a sufficient amount of medium
to cover the slide (approximately 9 ml for a 6 cm dish).
Pipette the cell suspension in the window (Fig. 1) and
place the petris in a tissue-culture incubator.
- Starve the cells from phosphates by placing
them in phosphate-free DMEM and the required
amounts of dialysed serum for 2-3h or overnight,
depending on the experiment.
- Prior to pulse application, remove the growth
medium and wash the cells gently with phosphatefree
- Carefully wipe the Teflon frame with a folded
Kleenex tissue to create a dry area on which a meniscus
can form (see Pitfall 1).
- Add the [α32P]GTP solution. The volume of the
solution under the electrode varies with the electroporation
assembly and cell growth area. For the setup
in Fig. 1A and a cell growth area of 32 × 10mm, the
volume is ~280µl, whereas the assembly in Fig. 1C
requires ~200µl for a cell growth area of 50 × 30mm.
Depending on the exact concentration, this volume
will contain ~200µCi [(α32P]GTP in phosphate-free
- Carefully place the electrode on top of the cells.
Make sure there is a sufficient amount of electroporation
buffer under the positive contact bar to ensure
electrical contact. Make sure there are no air bubbles
between the negative electrode and the cells by
looking in the mirror. If necessary, the electrode can be
sterilized with 70% ethanol before the pulse, and the
procedure carried out in a laminar-flow hood, using
- Apply three to six pulses of the appropriate
strength (40-200V, see Comment 3) from a 10- or 20-µF
capacitor, depending on the apparatus used (Fig. 1A
vs Fig. 1C).
- Remove the electrode set. Because usually only
a small fraction of the material penetrates into the cells,
the [α32P]GTP solution can be carefully aspirated and
- Add phosphate-free medium containing dialysed
serum if permitted by the experimental protocol
and incubate the cells for the desired length of time
(see Comment 4).
- Remove the unincorporated material: wash
the cells twice with phosphate-free medium lacking
- Extract the proteins. Add 1 ml of extraction
buffer to the window area of the slide. Scrape the cells
using a rubber policeman into a 15 ml tube and rock
the tubes on ice for 20min. Centrifuge for 30min
at 1,000rpm in a Beckman J-6 centrifuge to clarify.
Preclear the lysates by adding 100µl packed Staph.
A-Sepharose beads, incubating on ice for 1 h, and
centrifuging for 5 min at 1,000rpm in a Beckman J-6
- Immunoprecipitate Ras. Incubate the precleared
supernatant overnight with pan ras Ab2 antibody
bound to Staph. A Sepharose beads while
rocking on ice.
- Wash the immunoprecipitate four times with
1 ml of extraction buffer lacking the inhibitors. Use a
Hamilton syringe to completely remove all traces of
- Elute GTP and GDP off the beads by adding
10-20µl elution buffer to the beads and incubating at
68°C for 20min.
- Spot the eluate containing the labelled
nucleotides on a silica gel TLC plate containing a fluorescence
indicator. Spot 1 µl each of the stock GTP and
GDP solutions to serve as cold standards, easily visible
under UV light. Develop the plate using a solution of
1% ammonia-66% isopropanol for about 3-4h.
- Dry the TLC plate, expose to Kodak X-OMAT
AR film, and excise the spots for liquid scintillation
counting or submit to phosphorimager analysis (see
|FIGURE 2 Assessment of Ras activity through electroporation of [α32P]GTP. Cells were grown on conductive
glass (cell growth area, 32 × 10mm, Figs. 1A and 1B) and starved from serum and phosphates. A
solution containing [α32P]GTP was added to the cells and introduced with three pulses delivered from a
20µF capacitor at different voltages as indicated. Cells were subsequently placed in a humidified 37°C, CO2 incubator for 3 h. Ras was extracted and precipitated with the pan-ras Ab2 monoclonal antibody, the bound
GTP and GDP eluted and separated by thin-layer chromatography (see text). The plate was exposed for
15h to Kodak XAR-5 film with an intensifying screen. In all panels, arrows point to the positions of cold GTP
and GDP standards, respectively. (A) Electroporation does not induce a rapid breakdown of intracellular
GTP. Lanes 1-5: Mouse 10T½ fibroblasts were electroporated using voltages of 140-200 V as indicated in the
presence of 5µCi [α32P]GTP. Three hours later, nucleotides in a 2µl aliquot of each clarified lysate were separated
as described earlier without Ras immunoprecipitation. Lane M: As a marker, an aliquot of the
[α32P]GTP used in this experiment was run in parallel. (B) Assessment of Ras activity through electroporation
of [α32P]GTP in normal 10T½ and their ras-transformed counterparts. Lanes 1-10:10T½ cells (lanes
1-5) or their rasval12-transformed counterparts, 2H1 (lanes 6-10), were electroporated using voltages of 120-190
V as indicated in the presence of 66 or 33µCi [α32P]GTP, respectively. Proteins were extracted, and the Rasbound
GTP and GDP were separated as described earlier. As a control (lane 11), 2H1 cells growing in a 3cm
petri to were metabolically labelled with 200 µCi [α32P] orthophosphate and processed as described previously.
(C) Assessment of Ras activity in normal 10T½ fibroblasts and their ras-transformed counterparts, 2H1,
using the standard SLO permeabilization assay. This assay was performed as described (Brownell et al.,
1997) and is shown here as a comparison. Lane 1, 10T½; lane 2, rasval12-transformed, 10T½-derived line 2H1; and lane 3, 2H1 precipitated with control rat IgG instead of anti-Ras antibodies.
B. Electroporation of [γ32P]ATP for Labeling
of Cellular Proteins: Labeling of the Simian
Virus 40 Large Tumor Antigen or Adenovirus
- [γ32P]ATP: 600-1000µCi/ml in phosphate-free
- Simian virus large tumor antigen (SVLT) or adenovirus
E1A extraction buffer: 122 mM NaCl, 18 mM Tris-base, pH 9.0, 0.8 mM CaCl2, 0.43 mM MgCl2, 10%
glycerol, 1% NP-40, 10µg/ml aprotinin, 10µg/ml leupeptin,
1 mM PMSF, and 1 mM vanadate. Add 8.0g NaCl, 2.42 g Tris-base, 0.1 g CaCl2, 0.04 g MgCl2, 10 ml
NP-40, and 100ml glycerol to 800ml H2O. Adjust pH
to 9.0 and store at -20°C. On the day of the experiment,
add 0.1 ml aprotinin stock, 0.1 ml leupeptin stock, 1 ml
PMSF stock, and 0.1 ml vanadate stock solutions to
100 ml of this solution.
- Tris-LiCl solution for washing immunoprecipitates:
0.1M Tris-base and 0.5M LiCl. Make a stock
solution of 10X (4 litres). Add 484g of Tris-base and
848 g of LiCl in 4 litres of H2O. Adjust pH to 7.0 using
- SDS gel-loading buffer: 15ml 10% SDS, 1.5ml
mercaptoethanol, 6ml glycerol, 3ml 1.25M Tris, pH
6.8, 0.75 ml 0.3% bromphenol blue, and water to 30ml.
- 10% SDS stock solution
- 1.25M Tris-HCl (pH 6.8) stock solution: Add
151.3g Tris-base to 800ml distilled water. Adjust the
pH to 6.8 using concentrated HCl and the volume to
1 liter with distilled water.
- Plate, starve from phosphates, and wash the
cells with phosphate-free DMEM as in Section III,A.
- Add the [γ32P]ATP solution.
- Apply three to six pulses of the appropriate
strength (40-200 V, see Comment 3) from a 10- or 20-µF
capacitor, depending on the apparatus used (Fig. 1A
vs Fig. 1C). If the setup in Fig. 1 A is employed, then
the solution can be carefully aspirated and reused once
more after the pulse.
- Add phosphate-free medium and incubate the
cells for 2-3 h in a tissue-culture incubator.
- Extract the proteins by adding 1 ml SVLT extraction
buffer to the window, scraping into a 15 ml tube,
and rocking on ice. Clarify by spinning for 30min at
1,000rpm in a Beckman J-6 centrifuge (2,000g).
- Precipitate with the pAbl08 (SVLT) or M73
(E1A) monoclonal antibody and wash three times with
PBS, twice with the Tris-LiCl solution, and once with
H2O. Elute labelled proteins from the beads with SDS
geMoading buffer and resolve by acrylamide gel electrophoresis
|FIGURE 3 Labeling of the simian virus 40 large tumor antigen or adenovirus EIA through in situ electroporation
of [γ32P]ATP. (A) Mouse 10T½ cells (lane 1) or their SVLT-transformed derivatives (line 10SV2b,
lanes 2-8) were grown on 50 × 30mm conductive areas (Figs. 1C and 1D). A solution containing 50µCi
[γ32P]ATP in phosphate-free DMEM was added to the cells, and six capacitor-discharge pulses of 10µE
40-80 V were applied as indicated. Cells were placed in a humidified incubator for 30min. For a comparison
(lanes 9 and 10), the same SVLT-transformed cells were labeled in vivo with the indicated amounts of
[32P]orthophosphate. SVLT was precipitated from detergent extracts with the pAbl08, anti-SVLT antibody
(lanes 1, 3-7, and 10), or normal mouse IgG (lanes 2, 8, and 9) and labeled proteins were resolved by
acrylamide gel electrophoresis. Dried gels were exposed for 1 h to Kodak XAR-5 film with an intensifying
screen. Note the intense and specific labeling of SVLT and the associated phosphoprotein, p53, by in situ electroporation
(lanes 5 and 6) compared to cells labeled in vivo with 200µCi [32P]orthophosphate (lanes 9 and
10). (B) Human 293 cells transformed with adenovirus DNA were labeled as described earlier and extracts
were preadsorbed with normal mouse IgG (lanes 2, 4, 6, 8, and 10) or immunoprecipitated using the M73,
anti-E1A antibody (lanes 1, 3, 5, 7, and 9). Bracket points to the position of the phosphorylated E1A bands.
M, molecular weight marker lanes.
The technique of in situ
electroporation is very versatile.
A large variety of molecules, such as peptides
(Boccaccio et al.
, 1998; Raptis et al.
, 2000), nucleotides
(Brownell et al.
, 1997), antibodies (Raptis and Firth,
1990), or drugs (Marais et al.
, 1997), can be introduced,
alone or in combination, at the same or different times,
in continuously growing or growth arrested cells or
cells at different stages of thefir division cycle (see
chapter 44 by Raptis et al.
- The slides can normally be washed with Extran-
300 and reused a number of times. However, in the
case of introduction of radioactive material, exposure
of personnel to irradiation is an important consideration.
The removal of 32P-labelled nucleotides from the
ITO-coated glass is difficult because the phosphate
group is attracted to this coating (Tomai et al., 2000),
hence the use of inexpensive slides and electrodes that
can be discarded after use is highly desirable. Slides
with a conductivity of 20Ω/sq are sufficiently inexpensive
that they can be discarded after use or stored
for the 32P to decay before washing. The use of less conductive
ITO-coated glass (100Ω/sq) would reduce the
cost of the slides further. However, in our experience,
the conductivity of this grade of glass is not sufficiently
consistent for electroporation experiments due to
problems related to uniformity of thickness encountered
with the thinner coating of the less conductive,
commercially available surface. On the other hand, the
use of more conductive glass (2Ω/sq) requires a
smaller electrode angle, which reduces the cost of the
material substantially. However, this glass is not a
regular production item, hence it is more expensive.
- The [α32P]GTP must be of the highest purity.
Because a number of lots were found to contain
varying amounts of [α32P]GDP, it is wise to test the
preparation by thin-layer chromatography before use.
- Determination of the optimal voltage and
capacitance. Electrical field strength has been shown
to be a critical parameter for cell permeation, as well
as viability (Chang et al., 1992). It is generally easier to
select a discrete capacitance value and then control the
voltage precisely. The optimal voltage depends on
the strain and metabolic state of the cells, as well as the
degree of cell contact with the conductive surface, possibly
due to the larger amounts of current passing
through an extended cell (Yang et al., 1995; Raptis and
Firth, 1990). Densely growing, transformed cells or
cells in a clump require higher voltages for optimum
permeation than sparse, subconfluent cells. Similarly,
cells that have been detached from their growth
surface by vigorous pipetting prior to electroporation
require substantially higher voltages. In addition, cells
growing and electroporated on collagen, poly-L-lysine,
or CelTakTM-coated slides require substantially higher
voltages than cells growing directly on the slide.
The margins of voltage tolerance depend on the size
and electrical charge of the molecules to be introduced.
For the introduction of small, uncharged molecules
such as Lucifer yellow or nucleotides, a wider range
of field strengths permits effective permeation with minimal damage to the cells than the introduction of
antibodies or DNA (Raptis and Firth, 1990; Brownell et
al., 1997). For all cells tested, the application of multiple
pulses at a lower voltage can achieve a better
permeation and is better tolerated than a single pulse.
This is especially important for the electroporation of
serum-starved cells where the margins of voltage tolerance
were found to be substantially narrower compared
to their counterparts growing in 10% calf serum
(Brownell et al.
, 1997). Results of a typical experiment
are shown in Fig. 4. Rat Flll cells were grown on slides
with a 50 × 30mm cell growth area (Figs. 1C and 1D)
and six pulses were delivered from a 10µF capacitor.
Following electroporation of the first strip of cells, the
negative electrode was translocated laterally (Fig. 1C,
arrow) so that the whole area was electroporated in
four strips. The application of six exponentially decaying
pulses of an initial strength of ~40-55 V resulted in
essentially 100% of the cells containing the introduced
dye, Lucifer yellow, whereas [α32
~50 V for a maximum signal.
|FIGURE 4 Effect of field strength on the introduction of nucleotides. Six pulses of increasing
voltage were applied from a 10µF capacitor to serum-starved 10T½ cells growing on a conductive surface
of 50 × 30mm in the presence of 10µCi [α32P]GTP () or 10µCi [γ32P]ATP (). Total protein labelling was
quantitated using a nitrocellulose filter-binding assay (Buday and Downward, 1993). Numbers refer to cpm
per 100µg of protein in clarified extracts. As a control, cells were electroporated with 5 mg/ml Lucifer yellow
(O) and its introduction was assessed by fluorescence measurement of cell lysates using a Perkin-Elmer
Model 204A fluorescence spectrophotometer. Cell killing () was calculated from the plating efficiency of the
cells 2h after the pulse [from Raptis et al. (2003) and Tomai et al. (2003), reprinted with permission].
Cell damage is microscopically manifested by the
appearance of dark nuclei under phase-contrast illumination.
For most lines, this is most prominent
5-10min after the pulse. Such cells do not retain
Lucifer yellow and fluoresce very weakly, if at all (Fig.
5). It was also noted that the current flow along the
corners of the window is slightly greater than the rest of the conductive area. For this reason, as the voltage
is progressively increasing, damaged cells will appear
on this area first. This slight irregularity has to be
taken into account when determining the optimal
Example of determination of the optimal voltage.
|FIGURE 5 Determination of optimal voltage. Rat Flll fibroblasts growing on conductive slides (Figs. 1A
and 1B, cell growth area, 32 × 10mm) were electroporated in the presence of 5mg/ml Lucifer yellow using
three pulses of 155 (A and B) or 190 (C and D) volts delivered from a 20µF capacitor. After washing of the
unincorporated dye, cells were photographed under phase-contrast (A and C) or fluorescence (B and D) illumination.
Arrows in C and D point to a cell that has been killed by the pulse. Note the dark, pycnotic and
prominent nucleus under phase contrast and the fiat, nonrefractile appearance. Such cells do not retain any
electroporated material, as shown by the absence of fluorescence (C). It is especially striking that cells at the
top of frames C and D, situated at the edge of the electroporated area, received a larger amount of current
and have been killed by the pulse. Magnification: 240 times.
Prepare a series of slides with cells plated uniformly in
a 32 × 10 mm window (Figs. 1A and 1B). Set the apparatus
at 20µF capacitance. Prepare a solution of 5 mg/ml
Lucifer yellow in phosphate-free DMEM and a solution
P]GTP or [γ32
P]ATP containing 5 mg/ml Lucifer
yellow in phosphate-free DMEM. Electroporate the
Lucifer yellow solution at different voltages, 20µF,
three pulses, to determine the upper limits where a
small fraction of the cells at the corners of the window
(usually the more extended ones) are killed by the
pulse, as determined by visual examination under
phase-contrast and fluorescence illumination 5-10 min
after the pulse (Fig. 5). Depending on the cells and
growth conditions, this voltage can vary from 130 to
190 V. Repeat the electroporation using the radioactive
nucleotide solution at different voltages starting at 20 V
below the upper limit and at 5V increments. The
Lucifer yellow offers a convenient marker for cell permeation
and it was found not to affect the results.
Other characteristics of the technique
- Serum was shown to facilitate pore closure
(Bahnson and Boggs, 1990).
- The slides come with the apparatus and are
sterile. If sterility is compromised or if the slides have
been washed to reuse, then place in petris and sterilize
by adding 80% ethanol for 20min and then removing
the ethanol by rinsing with sterile distilled water.
. Under the
appropriate conditions, in situ
electroporation does not
affect cell morphology or the length of the G1 phase
of serum-stimulated cells and does not induce c-fos
(Raptis and Firth, 1990; Brownell et al.
, 1997). In addition,
it does not affect activity of the extracellular
signal-regulated kinase (Erk½) or two kinases commonly
activated by a number of stress-related stimuli,
JNK/SAPK and p38hog
(Robinson and Cobb, 1997),
presumably because the pores reseal rapidly so that
the cell interior is restored to its original state
(Brownell et al.
Measurement of steady-state Ras activity is possible
using this method because, contrary to other methods
of cell permeabilization, such as streptolysin-O (Buday
and Downward, 1993), the cells are not detectably
affected by the procedure so that they can be incubated
for long periods of time before extraction. In addition,
electroporation does not appear to induce a rapid
breakdown of intracellular GTP in any of the lines
tested, even under conditions where a substantial fraction of the cells are killed by the pulse (Fig. 2). As
a result, determination of the Ras-bound, GTP/GTP +
GDP ratio is made easier by the fact that although the
optimal voltage must be determined empirically as in
all electroporation experiments, excessively high voltages,
despite the fact that they may kill a substantial
proportion of the cells, do not alter the ratios obtained
(Fig. 2B), presumably because such cells rapidly lyse,
without affecting the results.
- Care must be taken so that cells do not dry
during the procedure, especially during wiping of the
frame with a tightly folded Kleenex. It was found that
serum-starved cells were especially susceptible. The
morphology of cells that have been killed by drying is
very similar to cells that have been killed by the pulse
- Accurate determination of the optimal voltage
is very important. For most nucleotide introduction
applications, optimal labelling was observed in the
range of 140-160 V, whereas these margins were found
to be narrower for serum-starved cells. Nevertheless, the Ras-bound GTP/GTP + GDP ratios or the profile
of SVLT or EIA labelling obtained was found to be the
same even when a substantial proportion of the cells
were killed by the pulse, in which case merely 32P incorporation is reduced.
The financial assistance of the Canadian Institutes
of Health Research, the Natural Sciences and Engineering
Research Council of Canada, and the Cancer
Research Society Inc. to LR is gratefully acknowledged.
AV is the recipient of NSERC and Ontario
Graduate studentships, a Queen's University graduate
award, and a Queen's University travel grant. ET is the
recipient of a Queen's University graduate award, an
NSERC studentship, and awards from the Thoracic
Society and the Lemos foundation. HB was the recipient
of a studentship from the Medical Research
Council of Canada and Queen's University graduate
school and Microbix Biosystems Inc. travel awards.
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