PREPARATION OF PROTEIN EXTRACTS
INTRACELLULAR PROTEINS
L. monocytogenes EGDe
L. monocytogenes EGDe is cultured in an orbital
shaking water bath at either 20° or 37°C with Brain Heart Infusion
(BHI) medium or MCDB202 medium (Cryo Bio System, L'Aigle, France) supplemented
with 0.36% glucose, 0.1% trace elements and 0.1% of a 10% Yeast Nitrogen
Base solution.
The strain is grown until the mid-log or the stationary phase. Then,
20 ml of culture is centrifugated (7 500 g, 15 min), then the cell pellet
is washed twice with TE buffer (Tris-HCl 20 mM, pH 7.5, EDTA 5 mM, MgCl2
5 mM) resuspended in 1 ml of TE buffer at pH 9.0 and stored at -20°C.
500 ml of the bacterial suspension are sonicated with a Vibracell sonicator
(3 X 2 min at power level 5 and 50% of duty cycle).
After a 30 min-treatment with Dnase I/Rnase A, 500 µl of a solution
containing urea, thiourea, CHAPS and TBP are added in order to obtain
a final concentration of 4 M, 2 M, 2% and 2 mM of each constituent,
respectively.
After a 30 min-incubation on ice with intermittent agitation, the soluble
protein sample is separated from cell debris by centrifugation (13 000
g, 20 min).
The supernatant is collected and proteins are quantified by the method
of Bradford (1976) using the Bio-Rad protein assay kit with bovine serum
albumin as the standard. The protein sample is then precipitated with
three volumes of cold acetone at -20°C during at least 2 h and pelleted
by centrifugation (13 000 g, 40 min). The protein pellet is resuspended
in IEF buffer (urea 7 M, thiourea 2 M, CHAPS 4%, and trace of bromophenol
blue) at a final concentration of 5 µg protein/µl and stored
at -20°C.
EXTRACELLULAR PROTEINS
L. monocytogenes (Exoproteome 12 strains)
A pre-culture of each strain was carried out in liquid BHI medium, at 37°C and under shaking at 150 rpm during 12 h. Cultures were inoculated from pre-cultures at an initial OD600 nm of 0.1. Thus, the cultures grown with MCDB202 medium supplemented with 1% glucose, 0.1% trace elements and 0.1% of 10% Yeast Nitrogen Base solution, were incubated at 37°C under shaking at 150 rpm until late exponential phase (OD600 nm=0.9). After centrifugation of cell cultures (7500 x g, 15 min, 4°C), the supernatants were filtered through 0.22 μm membranes and treated by adding 1% (v/v) of phenylmethylsulfonyl fluoride (PMSF) (20mM) and 1% (v/v) of sodium deoxycholate (20mg/mL), then incubated for 30 min in ice. Proteins contained in the samples were precipitated overnight at 4°C with 10% trichloroacetic acid (TCA) (10% w/v). After centrifugation(20300 x g, 30 min, 4°C), precipitates were washed overnight at -20°C with 15 mL of ice-cold acetone. After three washes with ice-cold acetone, protein pellets were dryed in the open air, and then solubilized in IEF buffer (5 M urea, 2 M thiourea, 2% CHAPS, 10 mM TrisHCl, in 50% trifluoroethanol and traces of bromophenol blue). Proteins were quantified with the method of Bradford by using the Bio-Rad protein assay kit and bovine serum albumin as the standard.
FIRST
DIMENSION
INTRACELLULAR PROTEINS
- IPG Strips (Bio-Rad): pH 3-10, pH
3-6 and pH 5-8 gradients
- Passive rehydration with protein sample
in total volume of 400 µl for 18 cm IPG strips
Rehydration
buffer |
Sample
loading |
Urea |
7
M |
Thiourea |
2
M |
CHAPS
|
4% |
Bromophenol
blue |
trace |
TBP
|
2
mM |
Ampholytes*
|
1,5% |
|
|
|
Analytical |
Semi-preparative
(for MS identification) |
|
Silver
staining |
Coloïdal
blue staining |
Strip
3-10 |
60
µg |
800 µg |
Strip
5-8 |
50
µg |
800 µg |
Strip
3-6 |
50
µg |
800 µg |
|
*according to the
IPG strip gradient:
Strip 3-10: ampholytes 3-10
Strip 5-8: ampholytes 5-8
Strip 3-6: ampholytes 3-5
|
EXTRACELLULAR PROTEINS
- IPG Strips (Bio-Rad): pH 3-10 NL gradients
- Passive rehydration for 17.5 h with protein sample
in total volume of 400 µl for 17 cm IPTG strips
Rehydration
buffer |
|
Sample
loading |
Urée |
5
M |
|
|
Analytical |
Semi-preparative
(for MS identification) |
Thiourée |
2
M |
|
|
Silver
staining |
Coloïdal
blue staining |
CHAPS |
2% |
|
Strip
3-10 NL |
50
µg |
500
µg |
Tris-HCl dans 50% (v/v) TFE |
10
mM |
|
|
|
|
Bromophenol
blue |
trace |
|
|
|
|
TBP |
2
mM |
|
|
|
|
Ampholytes 3-10 |
0,3% |
|
|
|
|
- Protean IEF Cell (Bio-Rad)
|
Strip
3-10 NL (Bio-Rad, 17 cm) |
Running
conditions: 66450 Vhs |
|
Time |
Vhs |
Ambient temperature |
Réhydratation
passive |
17,5 |
0 |
Room temperature |
50 V* |
7 h |
350 |
19 °C |
200 V |
2 h |
200 |
19 °C |
1000 V |
2 h |
1200 |
19 °C |
1000 V |
1 h |
1000 |
19 °C |
8000 V |
5 h |
22500 |
19 °C |
8000 V |
about 5 h: until reaching the total Vhs |
41000 |
19 °C |
Total |
22 h |
66450 |
|
*After the first step (50 V for 7 h), changing paper wicks at each electrode.
EQUILIBRATION
|
Equilibration
buffer |
|
|
Step
1: Equilibration buffer + respectively TBP 5 mM or 2mM (Fluka) for intracellular or extracellular proteins. 15 min equilibration
in 10 ml.
|
Urea |
6
M |
|
|
SDS |
2%
(w/v) |
|
Step
2: Equilibration
buffer
+ 2,5 % iodoacetamide + bromophenol blue (trace). 15
min equilibration in 10 ml.
|
Tris-Cl,
pH 8.8 |
50
mM |
|
|
Glycerol |
30%
(v/v) |
|
SECOND
DIMENSION (SDS-PAGE)
- The
second dimension is carried out in 12,5% acrylamide gel (190 x 185 x
1 mm) in denaturing conditions (acryl/bis solution from Bio-Rad).
|
Resolving
gel
(0,375 M Tris, pH 8,8) |
|
For
silver staining, add sodium
thiosulfate 2 mM and 5 mM in gel
and in migration buffer, respectively. |
Monomer concentration
(% T, 3,3% C)
|
12.5% |
Acrylamide/bis
(40% T, 3,3% C) stock
|
31,2
ml |
1,5 M Tris-HCl,
pH 8,8
|
25
ml |
dd H2O
|
42,3
ml |
10 min. degazing
under vacuum
|
|
10 % SDS
|
1
ml |
10 % ammonium
persulfate (fresh preparation)
|
600
µl (0,06 %) |
Temed
|
60
µl (0,06 %) |
Final volume
|
100
ml |
- Place
IPG gel on top of the resolving gel and overlay with a hot 1% "low
melting" agarose solution. Carefully press the IPG strip onto the
surface of the acrylamide gel to achieve complete contact. Allow agarose
to solidify for at least 5 min. Repeat this procedure for each IPG strip.
Agarose
solution 1% |
|
|
-
dissolve agarose in a water bath without boiling
- aliquot per 1 ml, preserve at 4 °C
Before use, heat at 60°C in
a water bath until dissolution.
|
Agarose LMP
|
0,1g |
Tank buffer
1X
|
10
ml |
Bromophenol
blue
|
some |
- Migration:
Protean II XL Multicell (Bio-Rad)
Migration Program |
Per gel :
|
|
|
|
15 mA
(constant)
|
1000
V |
500
W |
1
h |
40 mA
(constant)
|
1000
V |
500
W |
~5
h |
OR
Per gel :
|
|
|
|
15 mA
(constant)
|
40
V (limitant) |
25
W |
1 h
|
15 mA
(constant)
|
150
V (limitant) |
25
W |
~15
h |
STAINING
(use Milli-Q water to prepare the
different solutions)
- silver staining based on Blum et al. (1987) modified
by Rabilloud (1992). From "Rabilloud, T. (Ed.), Proteome Research:Two-Dimensional
Gel Electrophoresis and Identification Methods, Springer, Germany 1997,
pp. 107-126".
Fast
Silver Nitrate Staining (see Note 1 first). This protocole
is based on the protocol of Blum et al. (1987), with modifications (Rabilloud
1992).
- Fix the gels (>3 X 30 min.) in 5 % acetic acid/30 % ethanol (v/v)
- Rinse in water for 4 X 10 min.
- To sensitize, soak gels for 1 min (1 gel at a time) in 0.8 mM sodium
thiosulfate (Notes 2 and 3).
- Rinse 2 X 1 min in water ( Note 3).
- Impregnate for 30-60 min in 12 mM silver nitrate (0.2 g/l). The gels
may become yellowish at the stage.
- Rinse in water for 5-15 s (Note 4).
- Develop image (10-20 min) in 3 % potassium carbonate containing 250 µl formalin and 125 µl 10 % sodium thiosulfate per liter (Note
5).
- Stop develpment (30-60 min) in a solution containing 40 g of Tris and
20 ml of acetic acid per liter.
- Rinse with water (several changes) prior to drying or densitometry.
Note
1: general practice: Batches of gels (up to five gels per box) can
be stained. For a batch of
three to five medium-sized gels (e.g. 160 x 200 x 1.5 mm), 1 l of the
requiered solution is used, which corresponds to a solution/gel volume
ratio of 5 or more; 500 ml of solution is used for one or two gels. Batch
processing can be used for every step longer than 5 min, except for image
development, where one gel per box is requiered. For steps shorter than
5 min, the gels should be dipped individually in the corresponding solution.
For changing solutions, the best way is to used a plactic sheet.
The sheet is pressed on the pile of gels with the aid of a gloved hand.
Inclining the entire setup allows the emptying of the box while keeping
the gels in it. The next solution is poured with the plastic sheet in
place, which prevents the solution flow from breaking the gels. The plastic
sheet is removed after the solution change and kept in a separate box
filled with water until the next solution change. This water is changed
after each complete round of silver staining. In this case, only one gel
per dish is required.A setup for multiple staining of supported gels has
been described elsewhere (Granier and De Vienne 1985).
When gels must be handled individually, they are manipulated with
gloved hands. The use power-free, nitrile gloves is strongly recommended,
as powdered latex gloves are often the cause of pressure
marks. Except for development or short steps, where occasional hand agitation
of the staining vessel is convenient, constant agitation is required for
all the steps. A reciprocal ("ping-pong") shaker is used at
30-40 strokes per minute.
Dishes used for silver staining can be made of glass or plastic.
It is very important to avoid scratches in the inner surface of the dishes,
as scratches promote silver reduction and thus artefacts. Cleaning is
best achieved by wiping with a tissue soaked with ethanol. If this is
not sufficient, use instantly prepared Farmer's reducer (50 mM ammonia,
0.3 % potassium ferricyanide, 0.6 % sodium thiosulfate). Let the yellow-green
solution dissolve any trace of silver, discard, rinse thoroughly with
water (until the yellow color is no longer visible), then rinse with 95
% ethanol and wipe.
Formalin stends for 37 % formaldehyde. It is stable for months at
room temperature. However, solutions containing a thinck layer of polymerized
formaldehyde must, not be used. Never put formalin in the fridge, as this
promotes polymerization, 95 % ethanol can be used instead of absolute
ethanol. Do not use denatured alcohol. It is possible to purchase 1 M
silver nitrate ready-made. The solution is cheaper than solid silver nitrate
on a silver weight basis. It is stable for months in the fridge.
Last, but not least, the quality of water is critical. Best results
are obtained with water treated with ion exchange resins (resistivity
higer then 15 mega ohms/cm). Distilled water gives more erratic results.
Note 2: 0.8 mM sodium thiosulfate corresponds to 2 ml/l of 10 %
sodium thiosulfate (pentahydrate). The 10 % thiosulfate solution is made
fresh every week and stored at room temperature.
Note 3: The optional setup for sensitization is following prepare
four staining boxes containing respectively the sensitixing thiosulfate
solution, water (two boxes), and the silver nitrate solution. Put the
vessel containing the rinsed gels on one side of this series of boxes.
Take one gel out of the vessel and dip it in the sensitizing and rinsing
solutions (1 min in each solution). Then transfer to silver nitrate. Repeat
this process for all the gels of the batch. A new gel can be sensitized
while the former one is in the first rinse solution, provided that the
1 minute time is kept (use a bench chronometer). When several batches
of gels are stained on the same day, it is necessary to prepare several
batches of silver solution. However, the sensitizing and rinsing solutions
can be kept for at least three batches, and probably more.
Note 4: This is very short step is intended to remove the liquid
film of silver solution carried over with the gel.
Note 5: When the gel is dipped in the developer, a brown microprecipitate
of silver carbonate should form. This precipitate must be redissolved
to prevent deposition and background formation. This is simply achieved
by immediate agitation of the box. Do not expect the appearance of the
major spots before 3 min of development. The spot intensity reaches a
plateau after 15-20 min of development; then background appears. Stop
development at the very beginning of background development. This ensures
maximal and reproducible sensivity.
- Silver nitrate staining compatible with mass spectrometer identification
- Fix the gel 2 X 30 min in 10% acetic acid/ 40 % ethanol (v/v).
- Sensitize the gel for 30 min in 30% ethanol/ 0.2% thiosulfate de sodium (prepared and added at the last time)/ 6.8% acetate de sodium.
- Rinse 3 X 5min in water.
- Impregnate for 20 min in 0.25% silver nitrate (2.5 g/L).
- Rinse 2 X 1 min in water.
- Develop image (10-20 min) in 2.5% carbonate de sodium/ 0.04% formaldehyde until obtaining the intensity desired.
Stop development (10 min) in a solution containing 14.6g of EDTA per liter.
Destaining of silver stained spots
- Excise spots from gel and put them into 0.5 mL tubes.
- Add 200 µL of destaining solution (30 mM Potassium Ferricyanid/100 mM Sodium Thiosulfate (1:1)) and incubate approximately 5 min.
- After removing the destaining solution, wash 2 X 15 min each spot with milliQ water under agitation. Remove the rinse water.
- Apply the destaining protocol used for colloidal Coomassie blue stained spots.
- colloidal
Coomassie blue
staining:
according to Neuhoff et al. (1988). From "Rabilloud, T. (Ed.),
Proteome Research:Two-Dimensional Gel Electrophoresis and Identification
Methods, Springer, Germany 1997, pp. 107-126".
- After electrophoresis, fix the gels 3 X 30 min in 30 % ethanol (v/v) phosphoric
acid (Note 1).
- Rinse 3 X 20 min in 2 % phosphoric acid.
- Equilibrate for 30 min in a solution containing 2 % phosphoric acid,
18 % (v/v) ethanol and 15 % (v/v) ammonium sulfate (Note 2).
- Add to the gels and solution 1 % (v/v) of a solution containing 20
g of Brilliant Blue G per liter (Note 3). Let the stain proceed for 24
to 72 h.
- If needed, destain the background with water. Avoid alcohol containing
solutions.
Note 1:
The concentrated phosphoric acid used is 85 % phosphoric acid. Percentages
are expressed in volumes. For example, the fixing solution contains 20
ml of 65 % phosphoric acid per liter.
Note 2: The solution is prepared as follows. For 1 l of solution,
place 500 ml of water in a flask with magnetic stirring. Add 20 ml of
85 % phosphoric acid, then 150 g of ammonium sulfate. Let dissolve, transfer
into a graduate cylinder and adjust to 800 ml with water. Add 20 ml additional
water, retransfer into the flask with stirring and add 180 ml ethanol
while stirring.
Note 3: The Brilliant Blue G solution is prepared by dissolving
2 g of pure Brilliant Blue (e.g. Serva Blue G) in 100 ml of hot water
with stirring. Dissolution is complete after 30 min. Let the solution
cool, then add 0.2 g/l sodium azide as a preservative. Store at room temperature.
On the whole, staining with dyes is compatible with most subsequent protein
analysis methods, provided that alterations to the protocols given here
are made to maximize the yields in the subsequent protein microanalysis
step (see Chapter 9 on microsequencing). We have, however, experienced
difficulties when trying to perform internal peptide sequencing from colloidal
blue-stained spots, while analysis by mass spectrometry (peptide mass
fingerprinting) did not give any problem. The main limitation, even for
colloidal staining, lies rather limited sensivity.
Destaining of colloidal Coomassie blue stained spots
- Destain each spot in 100 µL of 25 mM ammonium bicarbonate/5% acetonitrile. Incubate 30 min under agitation then remove the buffer.
- Add 100 µL of 25 mM ammonium bicarbonate/50% acetonitrile. Incubate 30 min then remove the buffer.
- Add 200 µL d'acetonitrile 100% and incubate 10 min.
- Remove acetonitrile and dry spots under vacuum and hot.
|