PREMIERE DIMENSION

PROTEINES INTRACELLULAIRES

• IPG Strips (Bio-Rad) : gradients pH 3-10, pH 3-6 et pH 5-8
• Réhydratation passive d'IPG Strips de 18cm dans un volume total de 400 µl en présence de l'échantillon

*En fonction du gradient de l'IPG Strip :
Strip 3-10 : ampholytes 3-10
Strip 5-8 : ampholytes 5-8
Strip 3-6 : ampholytes 3-5

Après la migration, les strips sont conservés dans des sacs plastiques à -20°C.

PROTEINES EXTRACELLULAIRES

• IPG Strips (Bio-Rad) : gradients pH 3-10 NL
• Réhydratation passive durant 17,5 h dans un volume total de 400 µl en présence de l'échantillon

*Après la première étape (50 V pendant 7 h), changer les mèches en papier à chaque électrode.

EQUILIBRATION

DEUXIEME DIMENSION (SDS-PAGE)

• La deuxième dimension est réalisée en gel d'acrylamide à 12,5% (190 x 185 x 1 mm) en conditions dénaturantes (solution acryl/bis commercialisée par Bio-Rad).

• Déposer l'IPG strip sur le gel d'acrylamide, et recouvrir d'une solution d'agarose " Low Melting Point " à 1%. Presser avec soin l'IPG strip sur la surface du gel d'acrylamide pour un contact complet. Attendre la prise en masse de l'agarose au moins 5 min. Répéter pour chaque strip.

• Migration : cuve Multicell Protean II XL (Bio-Rad)

COLORATION (utiliser de l'eau Milli-Q pour préparer les différentes solutions)

• Coloration au nitrate d'argent basée sur le protocole de Blum et al. (1987) modifié par Rabilloud (1992). Tiré de "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.

• Coloration au nitrate d'argent compatible pour l'identification par spectromètre de masse : Protocole de Yan et al. Electrophoresis 21 (2000) 3666-3672

1. Fixer le gel 2x15min dans 10% acide acétique / 40% éthanol (v/v).
2. Sensibiliser le gel 30 min dans 30% éthanol/ 0.2% thiosulfate de sodium (préparé et ajouté extemporanément)/6.8% acétate de sodium.
3. Laver 3x5min le gel à l'eau milliQ.
4. Laisser le gel 20min dans 0.25% nitrate d'argent.
5. Laver 2x1min le gel à l'eau milliQ.
6. Laisser dans la solution de révélation : 2.5% carbonate de sodium/0.04% formaldéhyde (ajouté extemporanément) jusqu'à obtention de l'intensité désirée (estimation visuelle du temps ~10-20min).
7. Stopper le développement en laissant le gel 10 min dans 1.46% EDTA.
8. Rincer 3x5min le gel à l'eau milliQ

Décoloration des spots protéiques colorés au nitrate d'argent (d'après : montage in gel DigestZP kit, Millipore )

1. Prélever les spots du gel et les placer dans des tubes de 0.5 mL.
2. Sous agitation, décolorer chaque spot dans 200 µL de solution de décoloration (30mM de ferricyanide de potassium/100 mM de thiosulfate de sodium (1 :1)). Laisser incuber plus ou moins 5 min puis éliminer la solution de décoloration.
3. Sous agitation, laver 2 X 15 min chaque spot avec 150 µL d'eau milliQ puis éliminer l'eau en la pipetant.
4. Appliquer le protocole de décoloration des spots colorés au bleu de Coomassie colloïdal.

• Coloration au bleu de Coomassie colloïdal selon Neuhoff et al. (1988). Tiré de "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.

Décoloration des spots protéiques colorés au bleu de Coomassie colloïdal

1. Sous agitation, décolorer chaque spot à l'aide de 100 µL de tampon 25 mM bicarbonate d'ammonium/5% acétonitrile. Laisser incuber 30 min puis éliminer le tampon en le pipetant.
2. Ajouter 100 µL de tampon 25 mM bicarbonate d'ammonium/50% acétonitrile et laisser incuber 30 min. Eliminer le tampon et répéter cette deuxième étape.
3. Ajouter 200 µL d'acétonitrile 100% et incuber 10 min.
4. Eliminer l'acétonitrile puis faire sécher au speed-vac.