Service Offered: 2D-gel Electrophoresis (Isoelectric focusing)

First Dimension (separation of proteins based on their pI):

It is important that proteins are completely solubilized before focusing is started otherwise partially solubilized proteins will inevitably appear at additional spots in the gel. For the present application, we will be using cytosolic fractions that have been extracted in the presence of milli-Q water. Alliquotes (about 200 µg) are lyophilized and stored until use.

Samples are suspended in 5 µl of 5%SDS, 0.1%DTT and heated for 10 min at 60°C. This step will ensure that any insoluble components in the sample will be solubilized and thus allow for a better focusing step.

To the above sample, add 125 µl rehydration buffer consisting of 8M urea, 2% CHAPS, trace of bromophenol blue and freshly added 0.4 mg DTT and 6.5 µl IPG buffer. Allow the suspension to sit at room temperature for 30 min. Centrifuge to remove insoluble debris.

Apply the soluble fraction into one lane in the rehydration/focusing tray (7 cm bed length). Add a 7-cm IPG dry strip to the solution (strip gel side should be face down). Strip rehydration is allowed to take place for 12 hours. Electro focusing is then carried out for 8000 volt hours. Depending on strip length and the presence of salts and detergent in the original sample, focusing could take two hours to many hours.

Preparation of Focusing Strip for the Second Dimension:

At the end of the first dimensional separation (focusing) the strips are equilibrated for 20 min in equilibrating buffer (50mM Tris-HCl, pH 8.8, 6M urea, 30% glycerol, 10 mg DTT in 1ml buffer), followed by 20 min equilibration in the above buffer containing 25 mg iodoacetamide per ml buffer.

Second Dimension (separation of proteins based on their molecular size):

The second dimension is usually an SDS-polyacrylamide gel electrophoresis, which allows the separation of the proteins based on their molecular size. The percentage of acryamide in the separation gel is chosen based on the size of the proteins to be separated. The stacking gel is poured on top of the separation gel. A comb, which consists of a broad one lane to accommodate the focusing strip and a lane on the side to apply marker proteins, is placed in the stacking gel. The comb is removed from the gel after it has polymerized and the strip from above is applied horizontally on top of the stacking gel. The proteins in the strip are allowed to separate in the second dimension based of their apparent molecular weight in SDS.

Coomassie Staining of the proteins:

1. Place gel in staining solution (0.2% coomassie blue, 40%methanol and 7% acetic acid). Allow to stain while shaking for 1 - 2 hours
2. Destain the gel in 45% methanol 7% acetic acid Change the destaining solution until the protein spots are completely visualized

Silver staining and identification of individual protein spots

1. Gel is first fixed in 50% methanol, 7% acetic acid for 30 min followed by 50% methanol for another 30 min.
2. Gel is then washed in clean water 3 - 4 times
3. Gel is stained in pre-chilled 0.1% silver nitrate in water for 30 min then washed thoroughly in water.
4. Develop the gel in 2% ammonium acetate containing 0.04% formaldihyde (if the developer becomes colored, change it until the protein spots are visible).

Data Provided: Picture and actual gel

Cost:	on-campus:	$150.00/sample
	off-campus:	$300.00/sample

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