The ISO-DALT system is a high-throughput approach to analysis of proteins by two-dimensional gel electrophoresis (2DE). Originally built at Argonne National Laboratory under the direction of Norman and Leigh Anderson (1978a, 1978b), the ISO-DALT system provides the capability to generate and run first-and-second-dimension gels in multiples of 20.

Modifications of the chemistry used for first-dimension separation in the ISO apparatus have been developed to allow the resolution of very acidic and very basic proteins, in addition to the classical use of isoelectric focusing for the separation of neutral proteins. Modifications of the polyacrylamide gel composition for the second-dimension separation in the DALT apparatus can be made to adjust the molecular weight range of proteins resolved. Thus, the spectrum of proteins that can be surveyed by 2DE has been broadened beyond that originally described by Patrick O’Farrell in 1975.

The Argonne ISO-DALT system has stood the test of time, still producing 2DE patterns of superior quality. The procedures described in this Web-based manual have evolved over almost three decades of use and include the most current recipes and electrophoresis procedures used by the Argonne Protein Mapping Group. The details of the procedures are specific to the Argonne ISO-DALT system and may require some modification for use with commercially available electrophoresis systems. The recipes are generally applicable independent of the equipment used.

Although the vendors and products described pertain to the work done by the Argonne Protein Mapping Group, they are not specifically endorsed or recommended by Argonne National Laboratory. The authors of this manual have 50 combined years of work with 2DE and have run over 55,000 gels. Thus, the procedures outlined in this document have been well tested and fine-tuned.

1. SAMPLE PREPARATION

1.1 GENERAL SAMPLE PREPARATION

The solutions for sample preparation can be made in fairly large quantities (up to 1 L) and stored frozen at -70 °C in small aliquots. The following four solutions have been optimized for the samples specified. Experimentation with these methods and chemicals is advised to find the best conditions for other types of samples.

For all of the solubilization conditions described below, samples should be centrifuged at 22 °C for 1 h at approximately 100,000 ´ g in an ultracentrifuge or for 10 min at 100,000 rpm (435,000 ´ g) in a Beckman TL‑100 centrifuge. For mixes containing 9 M urea, use 1.5 ml polyallomer centrifuge tubes, as polycarbonate tubes may crack. The supernatant is recovered for analysis by 2DE. Samples may be analyzed immediately or stored frozen (-70 °C) for future analysis. The small translucent pellet that contains DNA and cell debris is discarded.

1. SDS mix (body fluids such as serum, plasma, or amniotic fluid). To a 10 ml sample, add 20-30 ml of SDS mix. Next, heat the sample on a 95 °C heating block for 5 min to achieve maximum solubilization and to inactivate any proteolytic enzymes.

2. NP-40/urea mix (solid tissue samples, isolated cells, and pure proteins). For a wet tissue sample, use a volume of mix that is eight times the blotted wet weight of the sample (e.g., for a 100 mg sample, use 800 ml of mix). For a frozen tissue sample, pulverize the sample on a platform chilled on dry ice and use a volume of mix that is four times the weight of the powdered sample. Homogenize (or sonicate) before centrifuging the sample. For solubilizing isolated cells, use 5 ´ 10⁶ cells per 50 µl of mix when Coomassie blue detection will be used. When silver‑stain will be used, use 5 ´ 10⁵ cells per 50 µl of mix. Disperse the cell pellet by tapping the bottom of the tube gently prior to addition of the solubilization mix. After the mix is added, mix the cell lysate well by drawing the solution up and down in a pipette tip. For pure protein samples, mix with solubilization mix to obtain a final protein concentration of 1 mg/mL. Do not heat any protein sample in the presence of urea, because carbamylation of the proteins will occur.

3. Urea mix without NP-40 (urine). Mix 10 mg of lyophilized urinary proteins with 100 ml of the mix. Load 10 ml (or less) of the solution onto the ISO gel.

4. NP-40/urea/DTE mix (muscle). For a wet tissue sample, use a volume of mix that is eight times the blotted wet weight of the sample (e.g., for a 100 mg sample, use 800 ml of mix). For a frozen tissue sample, pulverize the sample on a platform chilled on dry ice and use a volume of mix that is four times the weight of the powdered sample. Homogenize (or sonicate) before centrifuging the sample.

1.2 RADIOACTIVE SAMPLE PREPARATION

1. Samples (cells or tissue pieces) are incubated with either l‑[³⁵S]‑methionine (approximately 50 mCi per sample) or ³²P‑orthophosphate (200 mCi per sample) in methionine- or phosphate-free tissue culture media for 1-18 hours.

2. Cells or tissue pieces are collected from the labeling media by centrifugation, and the media is discarded. Samples are washed at least twice with phosphate-buffered saline.

3. The radioactively labeled cells or tissues are solubilized in approximately 50 ml of NP40-urea mix and centrifuged in a Beckman microfuge for 8 min at 100,000 rpm. After centrifugation, the supernatants are stored at -70 °C.

1.3 PROTEIN ANALYSIS

The protein concentrations of samples in solubilization mixtures containing NP-40/urea and mercaptoethanol can be determined using a modified Bradford protein assay (Ramagli and Rodriguez 1985). The optimal protein load for Coomassie blue staining of whole cell lysates is 150-300 mg protein, while 20-60 mg of protein is recommended for silver-stained gels.

CHAPTER 2: THE ISO SYSTEM FOR FIRST-DIMENSION SEPARATION

2.1 CASTING GELS IN THE ISO APPARATUS

There are two ISO formats, the 7-in. (18 cm) and the 10-in. (25 cm). The selection of ISO format should be based on optimal resolution of the proteins being analyzed. At Argonne, the 10-in. format is used for isoelectric focusing of mouse liver and tissue culture cell proteins, and the 7-in. format is used for BASO-DALTs and isoelectric focusing of microbial proteins. The entire system must be clean and dry.

1. Place a metal retainer on the bottom of the gel tubes.

2. Fill the bottom chamber of the ISO apparatus with tap-distilled water (2 l for 7‑in. systems and 3 l for 10-in. systems). Place the Lucite trough on the base of the support stand, and position the rack containing the upper chamber and the gel tubes into the acrylic trough.

3. Prepare the polyacrylamide gel solution by mixing the following in a 150‑ml lyophilizer flask. (Note: acrylamide monomer must be handled as a suspected human carcinogen.)

Compound	Amount
Ampholytes (the majority of ANL isoelectric focusing gels are 50:50 pH 5-7:pH 3-10; P. furiosus and S. oneidensis gels are an exception)	0.8 mL
Double-distilled water	6.0 mL

Compound

Amount

Urea

8.25 g

Acrylamide (30% solution)

2.0 mL

Ampholytes (the majority of ANL isoelectric focusing gels are 50:50 pH 5-7:pH 3-10; P. furiosus and S. oneidensis gels are an exception)

0.8 mL

Double-distilled water

6.0 mL

Dissolution, an endothermic process for urea, is aided by warming the flask and its contents in a water bath, but do not heat the urea solution beyond room temperature.

4. Degas the solution briefly using a vacuum pump dedicated for degassing acrylamide solutions. (If the degassing is too long, the urea will come out of solution; if this happens, warm the flask slightly until the urea goes back into solution.)

5. Add 0.3 ml NP-40 detergent (except with gels for urine proteins, where only two drops should be added) and swirl gently (vigorous swirling introduces bubbles). Then carefully add:

Compound

Amount

Ammonium persulfate (catalyst; 10% solution)

50 mL

TEMED (N,N,N’,N’

-tetramethylethylenediamine; an accelerator)

5 mL

6. Pipette the acrylamide solution (approximately 15 mL) into the trough. Carefully layer 3-4 ml of double-distilled water over the acrylamide solution to bring the fluid level up to the top of the trough.

7. Slowly lower the upper chamber/tube assembly with the acrylamide trough into the bottom chamber containing the appropriate volume of water. Allow the acrylamide to rise evenly by displacement in all tubes. Examine the tubes for bubbles. If there are any, use a 1 ml syringe with a cut off yellow pipette tip attached (the wide top end cut off) to suck acrylamide out the top of the tube until the bubble is removed. Allow the fluid to fall back to the proper level.

8. Allow the gels to polymerize for at least 2 hours.

2.2 PREFOCUSING

1. After the gels have polymerized, remove the upper chamber/gel tube assembly with the support stand from the bottom chamber. Empty the water from the bottom chamber and fill with 10 mM phosphoric acid.

2. Carefully place the tube stand on its side and hold on to the sides of the metal retainer. Pull off the trough by wiggling it free of the polymerized acrylamide and retainer. Do not bend the metal retainer or stretch the gels in the tubes. Cut with a razor blade between the end of the tubes and the metal retainer. Remove the retainer and rinse the outside of the tubes. Reinsert the upper chamber with attached tubes into the lower chamber containing the acid solution.

4. Degas 200 ml of double-distilled water. Add 0.4 ml of 10 N NaOH and pour the solution into the upper chamber. Use a 100 ml Hamilton syringe containing the NaOH solution to displace the air pocket that forms between the top of the gels and the NaOH in the upper chamber. Be careful not to disturb the tops of the gels.

5. Prefocus the 7-in. ISO for 1 h at 200 V, or the 10‑in. ISO setup at 300 V.

2.3 ISOELECTRIC FOCUSING WITH CARRIER AMPHOLYTES [ISO]

Using a Hamilton syringe, underlay 5 to 25 ml of sample on top of each isoelectric focusing gel. Larger volumes result in poor resolution and can cause the ISO gel to break. The optimum protein loading level is 100-300 mg for Coomassie blue detection or 20-60 mg for silver stain. If isoelectric focusing standards (e.g., Carbamalyte Calibration Kit, Amersham Pharmacia Biotech, Catalogue 17‑0582‑01) are to be run with the samples, add 2-4 ml to each tube above the sample.

Although the optimal separation time depends on the sample type, most cellular proteins are well separated in the 7-in. ISO apparatus with a run time of approximately 14,000 Vh (e.g., 800 V for 17.5 h), while plasma or amniotic fluid proteins require approximately 12,000 Vh. When using the 10-inch ISO apparatus, the optimum run for tissue and cellular protein is 30,000 Vh.

2.4 ISOELECTRIC FOCUSING WITH IPG STRIPS [IPG]

IPGs are immobilized pH gradient strips that are fixed on a solid plastic support. They must be rehydrated before isoelectric focusing. The apparatus with corollary equipment is purchased commercially (e.g., Bio-Rad), and instructions for setup are included.

Add 125-250 ml (7 cm IPG strip) or 300-600 ml (17 cm IPG strip) of the rehydration buffer to each channel of the isoelectric focusing tray, making sure that each strip is completely wet to prevent uneven rehydration. Volume of the sample should not exceed 30 ml (5-100 mg protein for silver staining, and up to 1 mg for Coomassie blue) per IPG strip. Apply mineral oil to each channel containing a strip, covering the entire strip. Put the lid on the tray and place the entire assembly on the Peltier platform. Be sure to align the electrodes of the focusing tray with the color-coded Peltier platform’s electrode connections. Program or select the desired method, and start the run.

Isoelectric focusing is immediately done after rehydration. IPG strips may be stored indefinitely at -70 °C after isoelectric focusing or may be loaded on to second dimension gels. Before running in the second dimension, IPG strips are equilibrated first with DTT-equilibration buffer for 10 min and then with iodoacetamide-equilibration buffer for an additional 10 min. After equilibration, IPG strips are placed on top of the second dimension slab gels.

2.5 NON-EQUILIBRIUM pH GRADIENT ELECTROPHORESIS

(NEPHGE/BASO)

Proteins with pI values higher than 8.0 do not focus well or fail to enter isofocusing gels. To study such basic proteins, the technique of running a BASO under nonequilibrium conditions was devised as a modification of the NEPHGE (non-equilibrium pH gradient electrophoresis) system (O’Farrell et al., 1977).

1. The BASO gels are cast in the 7-in. ISO apparatus using the same procedure as for ISOs. However, the BASO gel separation requires the use of the wide range pH 3-10 ampholyte (e.g., Biolyte 3/10).