Analysis of bacterial proteomes - INRA de Clermont-Ferrand - Theix, UR454 Microbiologie
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protein extracts1st dimension
equilibration
2nd dimension
staining

 

     

2-D PROTOCOLS

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

 

  • Multiphor II isoelectrofocalisation (Amersham Bioscience)
Strip 3-10 (Bio-rad, 18 cm) Running conditions: 40000 Vhs
Time

Vhs

Ambient temperature

Passive rehydration

12-16 h

0

 

from 0 to 40

1

19 °C

40

7

19 °C

from 40 to 500

910

19 °C

500

1500

19 °C

from 500 to 3500

6000

19 °C

3500

28000

19 °C

Total

17h

36418

Strip 5-8 (Bio-rad, 18 cm) Running conditions: 80000 Vhs
Time

Vhs

Ambient temperature

Passive rehydration

16-24h

0

 

from 0 to 50

10

19 °C

50

450

19 °C

from 50 to 200

10

19 °C

200

200

19 °C

from 200 to 1000

600

19 °C

from 1000 to 3500

33000 (33 kVhs)

19 °C

3500

45000 (45 kVhs)

19 °C

Total

44h

79270

Strip 3-6 (Bio-rad, 18 cm) Running conditions: 55000-60000 Vhs
Time

Vhs

Ambient temperature

Passive rehydration

16-24h

0

 

from 0 to 50

10

19 °C

50

450

19 °C

from 50 to 200

10

19 °C

200

200

19 °C

from 200 to 1000

600

19 °C

from 1000 to 3500

33000 (33 kVhs)

19 °C

3500

25000 (25 kVhs)

19 °C

Total

34h

59270

After migration, IPG strips are preserved in plastic bags at -20°C.

 

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).

  1. Fix the gels (>3 X 30 min.) in 5 % acetic acid/30 % ethanol (v/v)
  2. Rinse in water for 4 X 10 min.
  3. To sensitize, soak gels for 1 min (1 gel at a time) in 0.8 mM sodium thiosulfate (Notes 2 and 3).
  4. Rinse 2 X 1 min in water ( Note 3).
  5. Impregnate for 30-60 min in 12 mM silver nitrate (0.2 g/l). The gels may become yellowish at the stage.
  6. Rinse in water for 5-15 s (Note 4).
  7. Develop image (10-20 min) in 3 % potassium carbonate containing 250 µl formalin and 125 µl 10 % sodium thiosulfate per liter (Note 5).
  8. Stop develpment (30-60 min) in a solution containing 40 g of Tris and 20 ml of acetic acid per liter.
  9. 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
  1. Fix the gel 2 X 30 min in 10% acetic acid/ 40 % ethanol (v/v).
  2. 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.
  3. Rinse 3 X 5min in water.
  4. Impregnate for 20 min in 0.25% silver nitrate (2.5 g/L).
  5. Rinse 2 X 1 min in water.
  6. 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

  1. Excise spots from gel and put them into 0.5 mL tubes.
  2. Add 200 µL of destaining solution (30 mM Potassium Ferricyanid/100 mM Sodium Thiosulfate (1:1)) and incubate approximately 5 min.
  3. After removing the destaining solution, wash 2 X 15 min each spot with milliQ water under agitation. Remove the rinse water.
  4. 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".
  1. After electrophoresis, fix the gels 3 X 30 min in 30 % ethanol (v/v) phosphoric acid (Note 1).
  2. Rinse 3 X 20 min in 2 % phosphoric acid.
  3. Equilibrate for 30 min in a solution containing 2 % phosphoric acid, 18 % (v/v) ethanol and 15 % (v/v) ammonium sulfate (Note 2).
  4. 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.
  5. 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

  1. Destain each spot in 100 µL of 25 mM ammonium bicarbonate/5% acetonitrile. Incubate 30 min under agitation then remove the buffer.
  2. Add 100 µL of 25 mM ammonium bicarbonate/50% acetonitrile. Incubate 30 min then remove the buffer.
  3. Add 200 µL d'acetonitrile 100% and incubate 10 min.
  4. Remove acetonitrile and dry spots under vacuum and hot.

Contacts :  Michel Hébraud - Ingrid Chafsey

Last update: 3 Septembre, 2012

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