created: 20th April 1998, last updated: 11th May 1998,© 1998 ABRF

 

Setting Up Two-Dimensional Gel Electrophoresis for Proteome Projects

BRADLEY J. WALSH AND BENJAMIN HERBERT, MACQUARIE UNIVERSITY, NSW AUSTRALIA


Introduction

Two-dimensional polyacrylamide gel electrophoresis (2DE) has come into widespread use since the publication, in the early 1970's, of methods combining isoelectric focusing (IEF) in the first dimension and SDS-PAGE in the second dimension. In 1975, three separate papers by O'Farrell (1), Klose (2), and Schele (3) demonstrated that it was possible to combine IEF with the pore gradient SDS-PAGE gels of Kenrick and Margolis (4) published five years earlier. O'Farrell used autoradiographic detection to reveal more than a thousand spots on a 2DE gel, thus demonstrating the resolving power of the technique.

The technique enables the separation of extremely complex protein mixtures. More than 7,500 components have been separated by 2DE, compared with a maximum of about 100 by reversed-phase HPLC. For the most up-to-date review of the area see Proteome Research: New Frontiers in Functional Genomics, 1997(5).

Setting Up a Two-Dimensional Electrophoresis Laboratory

Tables 1 and 2 give a list of equipment, chemicals and reagents that we recommend for 2D electrophoresis. While other suppliers are available, one must be certain of obtaining high purity chemicals and using Milli-Q or equivalent water for all reagents. The total budget for setting up the laboratory, as per Tables 1 and 2, is about $60,000.

Table 1: Recommended Equipment

Catalogue No.

Item

Company

18-1004-30

Immobiline DryStrip kit

Pharmacia Biotech

18-1013-74

Reswelling cassette

Pharmacia Biotech

18-1018-06

Multiphor II electrophoresis unit

Pharmacia Biotech

18-1102-77

Multitemp III thermostatic circulator

Pharmacia Biotech

165-1790

HydroTech gel drying system

BioRad

165-1816

Protean II stretch kit

BioRad

165-1951

Protean II Xi multicell

BioRad

165-1956

Protean II Xi multicell 2D conversion kit

BioRad

165-1991

Plate Washer for Protean IIxi glass plates

BioRad

165-2001

Model 395 gradient former

BioRad

165-2025

Protean IIxi multicasting chamber

BioRad

165-5053

PowerPac 200

BioRad

165-5057

PowerPac 3000

BioRad

170-LARGE

Trans Blot SD system (custom large unit)

BioRad

Peristaltic pump

Cole Palmer Instr.

E862

Consort 6,000 V power supply

Gradipore Ltd

Table 2: Recommended Chemicals and Reagents

Catalogue No.

Item

Company

161-0719

Tris

Bio-Rad

161-0724

Glycine

Bio-Rad

161-0107

Acrylamide

Bio-Rad

161-0730

Urea

Bio-Rad

161-0713

Tricine

Bio-Rad

161-0318

Pre-stained broad range markers

Bio-Rad

162-0115

Nitrocellulose roll

Bio-Rad

165-0963

Cellophane, 50 Sheets

Bio-Rad

165-1824

Outer Protean II Plate

Bio-Rad

161-0202

Piperazine diacrylamide

Bio-Rad

161-0325

Kaleidoscope polypeptide standards

Bio-Rad

161-0400

Coomassie Brilliant Blue R250

Bio-Rad

161-0402

Amido Black 10B

Bio-Rad

165-0963

Cellophane membrane backing

Bio-Rad

161-0460

CHAPS

Bio-Rad

161-0700

ammonium persulfate (APS)

Bio-Rad

161-0800

TEMED

Bio-Rad

165-1823

Protean II inner plate

Bio-Rad

170-3960

Extra thick blot paper

Bio-Rad

732-2010

Econ-Pac 10DG desalting column

Bio-Rad

90827

Tributylphosphine, 97%

Fluka Chemicals

RG-602

Yellow electrical tape

Gradipore

10236 BA

Sodium acetate

Merck P/L

various

Immobiline DryStrip, 11 cm, pI 4-7 and pI 3-10

Pharmacia Biotech

various

Immobiline DryStrip, 18 cm, pI 4-7 and pI 3-10

Pharmacia Biotech

10233 4K

BDH AgNO3

Selby-Biolab

10011 4P

BDH AnalaR ammonia solution

Selby-Biolab

3016

BDH AnalaR 20 L methanol

Selby-Biolab

A 2504

Aminocaproic acid

Selby-Biolab

C 0759

Citric acid

Sigma-Aldrich

E 8263

Endonuclease, 5000 U

Sigma-Aldrich

G 6403

Glutaraldehyde, 50% solution

Sigma-Aldrich

-

Medical grade paraffin oil

Local supplier

Immobilized pH Gradients

The traditional ampholyte pH gradients are formed during electrophoresis and are beset by the problems of gradient drift and batch variability. These problems can be obviated by the use of Immobilines, a set of buffering acrylamide derivatives that contain either a free carboxylic acid or a tertiary amino group. Using the appropriate combination of Immobilines and a gradient maker, it is possible to generate any pH gradient in the range of pH 2.5 to 12. The Immobilines are co-polymerised with acrylamide and Bis so that the pH gradient exists before electrophoresis and is immobile (6,7). Immobilized pH gradients (IPGs) ensure highly reproducible pH gradients that are insensitive to disturbances from sample components and, due to their stability, permit focusing times of sufficient duration for proteins to attain their isoelectric points (8). As a consequence, focusing reproducibility is greatly improved, thus simplifying spot identification, pattern matching, and inter-laboratory comparisons. The sample load capacity of IPGs is much higher than in carrier ampholyte IEF, because the focusing protein zones cannot displace the pH gradient. Moreover, intrinsically different samples (plasma, cerebrospinal fluid, biopsies) will give the same focusing positions for common proteins allowing direct comparison of resulting 2-D maps. The gradients can vary from broad range, covering 7 pH units, to ultra-narrow 0.1-pH unit gradients, allowing increased resolution of closely related proteins. The pH gradient is also stable thus allowing storage of samples separated on IPG strips at -80oC prior to running second dimension gels. Due to these factors the IPG system is now the method of choice for 2DE. The methods discussed in this paper are optimized for use with IPGs.

The First Dimension of 2D Electrophoresis

Sample Preparation for 2DE

Pretreatment of samples for IEF involves solubilization, denaturation, and reduction to completely break the interactions between the proteins and to remove non-protein sample components such as nucleic acids (9). Ideally, one would achieve complete sample solubilization in a single step and thus avoid unnecessary handling, as is the case for soluble samples that can be readily taken up in the typical IEF sample solution of 8 M urea, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 50-100 mM dithiothreitol (DTT), and 40 mM Tris. The challenge for 2D-PAGE is the solubilization and separation of insoluble samples such as membrane associated proteins and proteins from highly resistant tissues like hair and skin. In an ideal world, samples could be applied to 2D gels unfractionated; but, this is not always practical, especially with very complex mixtures or when abundant proteins dominate the sample as in the case of albumin in plasma. Organelle and plasma membrane fractions can be used to considerably reduce the complexity of cellular samples.

Many samples such as wool proteins and membrane proteins are solubilized in the standard sample solution only to become insoluble during the course of the prolonged focusing required during the first dimension separation in IPGs. Rabilloud et al. (10) reported the selective adsorption of hydrophobic membrane proteins to the IPG during the IEF, resulting in these proteins being under-represented on the second-dimension gel. The incorporation of thiourea, up to 2 M, and mixtures of CHAPS and other sulphobetaine surfactants improved the solubilization of the proteins during the IEF and increased the transfer of proteins to the second -dimension gel. This suggests that hydrophobic interactions between the proteins and the IPG gel matrix are responsible for the losses. Other recent studies have shown that some of the acrylamide buffers used to form IPGs are hydrophobic, especially the basic buffers, which leads to hydrophobic interactions between the gel matrix and the sample proteins (11,12).

Aside from the issue of protein losses due to hydrophobic interactions, a separate problem is inherent in the choice of reducing agents used in sample solutions for IEF. In order to completely solubilize complex mixtures of proteins for IEF it is necessary to completely break inter- and intra-chain disulphide bonds by reduction. This is usually achieved with a free-thiol-containing reducing agent such as 2-mercaptoethanol or DTT (9). However, reagents such as DTT are charged and thus migrate out of the pH gradient during the IEF, which results in a loss of solubility for some proteins, especially those that are prone to interaction by disulphide bonding, such as the keratins and keratin-associated proteins from hair and wool. Replacing the thiol-containing reducing agents with a non-charged reducing agent such as tributyl phosphine (TBP) greatly enhances protein solubility during the IEF and results in increased transfer to the second dimension (13). Because thiourea and TBP increase protein solubility through different routes, they can be seen as complementary reagents. Thiourea increases the chaotropic power of the sample solution, and TBP ensures complete reducing conditions during the IEF; thus, it is advantageous to combine the two in a modified sample solution (10,13).

The presence of nucleic acids, especially DNA, has a detrimental effect on the separation of proteins by IEF for a number of reasons. Under denaturing conditions, such as in the sample solutions described above, DNA complexes are dissociated and cause a marked increase in the viscosity of the solution that inhibits protein entry and slows migration in the IPG. In addition, DNA binds to proteins in the sample and causes artifactual migration and streaking (9). There are two methods of nucleic acid removal that are suitable for maintaining sample integrity for IEF. The first is to utilize the ability of carrier ampholytes to form complexes with nucleic acids and then remove the complexes using ultracentrifugation. If the extraction is done at high pH, proteins behave as anions and binding to the anionic nucleic acids is minimized (9). The second method is enzymatic digestion, which is achieved by adding endonuclease to the sample after solubilization in sample solution at high pH (40 mM Tris, pH 9.5), thus minimizing the action of contaminating proteases. The advantage of the endonuclease method is that sample preparation can be achieved in a single step, by the addition of the enzyme prior to loading the IPG.

The total salt concentration should not exceed 300 mM in the sample, otherwise desalting is required. Desalting can be achieved with the use of disposable desalting columns such as the BioRad Econ-Pac 10DG. Our preference is to then freeze-dry the sample and redissolve in sample solution to allow ìin-gelî rehydration of the sample (see below). All solutions should be prepared with Milli-Q water. To avoid protein contamination, it is important to wear gloves when preparing solutions and handling equipment and samples. Write the date of preparation on all stock solutions.

Standard Solutions.

Prepare stock solutions as follows:

Urea Stock Solution.

Reagents

Amount

8.5 M urea

510.51 g

Water

Q.S. to 1 L

Dissolve in a Schott bottle with gentle heating (less than 30oC) and sonication. Add 5 g of Bio-Rad deionizing resin and stir for 10 minutes. If the resin de-colorizes, add a further 5 g and repeat until resin is not de-colorized. Filter the solution through Whatman No. 1 paper using a Buchner funnel.

Store 50-mL aliquots of this urea solution at -80oC until required, and discard any unused solution after 3 months. Use deionized 8.5 M stock to prepare all urea containing solutions. Do not store urea solutions at room temperature (or 4oC) for any longer than necessary. Urea in solution exists in equilibrium with ammonium cyanate, which can cause irreversible protein modification and interfere with IEF.

Tributyl Phosphine Stock Solution.

Reagents

Amount

200 mM TBP

1 mL

Isopropanol

Q.S. to 20 mL

Take care: concentrated TBP reacts violently with organic matter. Clean spills with a wet paper towel.

All the procedures in making TBP solutions should be done in a fume cupboard. Flush the concentrated TBP and the 200 mM stock with oxygen-free nitrogen after use. Store the TBP concentrate and stock in the dark at 4oC. Do not store the 200 mM stock for longer than two weeks.

Sample Solution. This solution is used for sample application by in-gel rehydration.

Reagents

Amount

8 M Urea

18.8 mL of 8.5 M stock

100 mM DTT

308 mg

or

or

2 mM TBP

200 mL of 200 mM TBP stock

4% CHAPS

800 mg

0.5% Carrier (3-10 Biolytes) ampholytes

100 mL

40 mM Tris

0.1 g

0.001% Orange G dye

20 mL of 1% stock

water

Q.S. to 20 mL

Dissolve the CHAPS and DTT in the urea, add the Biolytes and Orange G and make up to a final volume of 20 mL. When using TBP, add last, flush the solution with nitrogen and seal. Use carrier ampholytes that match the pH range of the IPG. For pH 4-7 and 3-10 IPGs use 3-10 Biolytes. Aliquot solution into 1-mL lots and freeze at -80oC until required for up to three months. Thaw only the required number of aliquots and discard leftover solution. Add sample solution to dry, de-salted protein sample and use for rehydration of IPG strips or cup loading.

Removal of Nucleic Acids.

Add 150 units of endonuclease to the sample (up to 2 mL) after solubilization in the sample solution.

Endonuclease Solution

Reagents

Amount

Sigma endonuclease

Differs with each batch

Manufacturer's buffer

Stored frozen, ready made

Dilute endonuclease with manufacturer's buffer (50% glycerol, 20 mM Tris HCl, pH 8.0, 2 mM MgCl2, 2 mM NaCl) until 5 mL is equal to 150 units.

Sample Application During Rehydration.

The IPG strips are rehydrated, individually, in solutions of the sample (12). As the strips rehydrate the proteins in the sample are absorbed and distributed over the entire length of the strip. This sample application method works because IEF is an equilibrium technique.

The method has a number of major advantages over cup loading. First, sample application during rehydration removes the problems of sample precipitation during cup loading. This allows for more accurate quantitation because more complete entry of the sample has occurred. Second, shorter focusing times can be used because the sample proteins are in the IPG prior to the commencement of IEF. Very large sample amounts (up to 5 mg of a single protein for broad gradients, 20 mg for narrow gradients) can be loaded using this method (14). High sample loads do not give good separation using the conventional method of loading in a cup. Thus, for both preparative and analytical separations, we prefer applying samples by gel rehydration rather than cup loading.

For sample application by rehydration, the maximum volume of solution is 500 mL per 18 cm IPG (250 mL for 11 cm IPG). Remove the protective plastic cover from the IPG strip and place each strip in a 2-mL plastic pipette (cut to the correct length). Seal the tip end of the plastic pipette with parafilm, and introduce the protein solution using an autopipette. Ensure that the solution is dispensed on the gel side of the IPG. Seal the open end of the plastic pipette with parafilm, leave on a flat surface, and allow the rehydration to proceed overnight at room temperature. Check the progress of the rehydration after overnight rehydration and invert the pipette a number of times to re-distribute the remaining liquid. After a total of 24 hours, the IPG should be completely rehydrated and no solution should be left outside the IPG.

Sample ApplicationóCup Loading.

As a general rule cup loading is not the best method of loading IPGs. However, cup loading may be used when the sample contains high levels of DNA or RNA or other large molecules such as cellulose. In these cases the proteins in the sample enter the gel more easily with an applied potential difference. This is performed according to the instructions supplied with the DryStrip kit.

Preparing Second-Dimension Gels

Second dimension gels can be run horizontally in flat bed systems or vertically in tank systems. Generally, vertical slab gels are larger and thus offer better resolution. Additionally, multiple vertical slab gels can be run in large tanks (5-20 gels per tank). A stacking gel is not normally used, especially if the separating gel is a pore gradient (15).

Why Use Gradient Gels?

The total monomer concentration in the gel is denoted as %T and the percentage of total monomer that is cross-linker, such as bis-acrylamide or piperazine diacrylamide, is denoted as %C. Second dimension gels can be of two sorts: homogeneous gels, with constant %T and %C, and gradient gels, with increasing %T, usually with constant %C. The choice of %T is determined by the molecular weight of the protein to be separated. If %T is too low, there will be insufficient retardation; while, if it is too high, the molecules will not penetrate the gel sufficiently.

When separating components in a narrow molecular weight range, homogeneous gels generally give better separation. A suitable %T can be estimated from charts of mobility in these gels for molecules of different molecular weight, but mobility is highly dependent on the buffer used, particularly its pH. Typically, a homogeneous 12% or gradient 8-18% gel is best for crude samples, such as a whole cell lysate.

Gradient gels have two advantages: they allow proteins with a wide range of molecular weights to be analyzed simultaneously and the decreasing pore size functions to sharpen the bands, that is, improves resolution. For many applications, the use of a gradient gel gives a superior result.

Casting Gels Using a Multi-Casting Chamber.

SDS PAGE gels should be poured in a multigel casting chamber to ensure that they are uniform in composition. This is especially important for gradient gels. These gels should be prepared the day before they are required.

Prior to assembling the casting chamber, the glass plates should be carefully cleaned. Soak the plates in hot Pyroneg detergent for at least 2 hours and then scrub them with a scouring pad. After scrubbing, the plates should be given a final cleaning in a dishwasher using the pot scrub setting without Pyroneg or other detergents. After drying, the plates should be stored in the glass plate drawer until required.

When assembling the glass plates for casting, each one should be wiped with a methanol soaked tissue and then dried with a lint free tissue. Two glass plates should be placed together, with the bevelled top edges facing in, and separated by two 2-mm thick spacers positioned along the sides. Each set of plates should be taped on the sides to ensure that the plates and spacers do not move during gel casting. Small squares of filter paper, which can be used as labels, are placed inside each gel's glass plate assembly, near the spacers, before the gels are cast. They do not interfere with the running of the gel and are very useful for keeping track of samples when running several gels at a time.

Acrylamide Stock - 40% T/ 2.5% C

Reagents

Amount

acrylamide

780 g

piperazine diacrylamide

20 g

water

Q.S. to 2 L

Add 5 g of Bio-Rad deionizing resin (AG 501 X8 (D), Cat No. 142-6425) and stir for 10 minutes. If the resin de-colorizes, add a further 5 g and repeat until resin is not de-colorized. Filter the solution through Whatman No. 1 paper using a Buchner funnel. The acrylamide stock should be stored in the fridge and not kept for more than two weeks. For the cross-linker, we use piperazine diacrylamide instead if bis-acrylamide because it is more compatible with our silver-staining protocol.

Gel Buffer

Reagents

Amount

1.875 M Tris

227 g

water

Q.S. to 1 L

adjust pH to 8.8

use concentrated HCl

The buffer stock should be stored in the refridgerator and not kept for more than two weeks.

SDS-PAGE Anode Electrode Buffer

 

Reagents

Amount

0.75 M Tris

908.6 g

0.005% Na azide

0.5 g

water

Q.S. to 10 L

adjust pH to 8.8

use concentrated HCl

The buffer stock should be discarded after 20 gel runs.

SDS-PAGE Cathode Electrode Buffer

Reagents

Amount

192 mM Glycine

43.2 g

0.1% SDS

3 g

water

Q.S. to 3 L

adjust pH to 8.3

use Tris

The buffer should be made fresh for each gel run.

Preparing the gel solutions

Use the following table to find the correct volumes of buffer stock, acrylamide stock, and water to use for preparing gradient gels. If homogeneous gels are to be used, the volumes for %T and initiators can be doubled.

Use a measuring cylinder to dispense the appropriate volumes into conical flasks. When preparing the heavy (higher %T) solution for gradient gels, add half of the volume as glycerol and the other half as Milli-Q water to increase the density.

Formulations for six Protean II gels (2-mm thick)

% T

Buffer(mL)

Stock Acrylamide (mL)

Water (mL)

8

55

55

165

12

55

83

138

16

55

110

110

20

55

138

83

Mix the solutions thoroughly and then remove the dissolved air from the solutions under vacuum. Place the gel solutions into a vacuum dessicator and evacuate using an oil pump until both solutions have stopped bubbling. This will take between 30 minutes to 1 hour depending on the volume of solution. This will remove any air and prevent oxygen from inhibiting the polymerization. Dissolved gas in the gel solutions may also cause bubbles in the gels during polymerization.

While the solutions are in the desiccator, assemble the gradient maker and peristaltic pump. The gradient maker and peristaltic pump tubing should be rinsed with Milli-Q water. Set the peristaltic pump to 25 rpm, and check that the flow rate is between 60 and 75 mL/minute. After the pump and gradient maker have been assembled and checked, empty the tubing and connect the inlet tubing of the casting unit to the pump tubing. Ensure that the clamp is in place, and open, on the casting unit inlet tubing.

When the solutions have stopped bubbling, the desiccator should be vented. Prepare the ammonium persulphate (APS) initiator solution. Weigh 100 mg of APS into an Eppendorf tube, add 1 mL of Milli-Q water and dissolve with shaking. Add the initiators to the gel solutions and mix by gentle swirling, taking care not to introduce air bubbles. It is very important to prepare this solution fresh each time gels are polymerized since old solutions will deteriorate and the rate of polymerization, and hence the pore size, will be different. The sodium thiosulphate is added to the gel mixture at this time to allow it to be incorporated into the gel matrix. Thiosulphate is added to gel solutions because it reduces background when diamine silver stain is used.

Sodium Thiosulfate Stock Solutions

Reagents

Amount

100 mM Na thiosulphate

0.32g Na thiosulphate

water

Q.S. to 20 mL

Add the following volumes of initiators to both the high and low %T solutions: 0.165 mL of TEMED, 1.65 mL of 10% APS, and 0.20 mL of sodium thiosulphate solution per mL of gel solution.

Pour the gel solutions into the gradient maker. Check that the gradient maker valves are in the closed position. Pour the light, low %T solution into the mixing chamber and the heavy solution into the reservoir chamber. Start the magnetic stirrer in the mixing chamber, open the outlet valve on the gradient maker, and turn on the pump. Allow the tubing to fill with gel solution and then open the valve between the mixing and reservoir chambers. When the gels are being poured, it is important to constantly check that the magnetic stirrer is mixing correctly.

Allow the gel solution to fill the chamber until there is a gap of 0.5 cm at the top of the glass plates. This should require nearly all of the solution in the gradient maker. Do not allow air bubbles to enter the casting chamber. When the solution has filled the casting chamber, switch off the pump and shut the clamp on the inlet hose of the casting chamber. Remove the pump tubing from the casting chamber inlet and flush the gradient maker and pump tubing with water.

Overlay the gel solutions with 15 mL of water saturated isobutanol. Cover the casting unit with a plastic bag and allow the gels to polymerize overnight. Do not move the gels until polymerization has taken place. After overnight polymerization, remove the isobutanol overlay and replace it with 1X gel buffer. If the gels are not going to be used immediately, they should be stored in the refrigerator (4oC) until required. Do not store gels for longer than two days after polymerization.

Running a Two-dimensional Gel

Performing IEF

Ensure that the IPG tray, strip aligner sheet, sample cup bar (if required), and electrodes are clean and dry prior to assembling the apparatus. The cleaning should be done in a solution of hot Pyroneg laboratory detergent. This removes paraffin oil and protein from the previous IEF run. After cleaning the tray, strip aligner, sample cup bar, and electrode should be rinsed thoroughly in Milli-Q water and dried.

Set the MultiTemp II thermostatic circulator at 25oC and switch it on 15 minutes before starting the experiment.

Pour 50 mL of paraffin oil onto the cooling plate and place the IPG tray so that no air bubbles are trapped between the cooling plate and the tray. This ensures good contact between the Multiphor cooling plate and the IPG tray. Plug the IPG tray connectors into the appropriate holes, and check that the anode wire is connected at the front of the Multiphor. Place 20 mL of paraffin oil into the tray and insert the strip aligner sheet (ensure that the sheet is placed correctly, i.e., grooves down). Press the sheet in a rubbing motion to remove air bubbles between the sheet and the tray. Cut the electrode paper strips into 2-cm lengths (2 for each IPG to be run), soak in tap water, and blot firmly between sheets of tissue paper to remove excess water. Replace the rehydrated, unrinsed IPGs (gel side up) in the grooves of the strip aligner sheet. Use the grid on the cooling plate to ensure that the IPGs are straight. The papers should be placed (one at each end) on the IPGs overlapping the gel by 0.5 cm. The remaining 1.5 cm should protrude longitudinally from the IPG. The electrodes should be placed across the far ends of the electrode papers. Cover the strips completely with paraffin oil (approximately 100 mL).

Running conditions for IPGs

Power should be supplied to the Multiphor using a power supply capable of delivering 6,000 V and currents of µA. The following table gives the running conditions for cup loaded gels. Times for rehydrated gels are given in parentheses.

Running Conditions for IPGs

Phase

V

mA (max)

W (max)

Time (h)

Volt-hours

1

300

1.0

5.0

5 (2)

1,500

2

1,000

1.0

5.0

5 (1)

5,000

3

2,500

1.0

5.0

5 (1)

12,500

4

5,000

1.0

5.0

up to 10 (20)

to 100,000

Storage of IPGs after IEF

Because the pH gradient is fixed in the gel the focused proteins are more stable at their isoelectric points than in conventional carrier ampholyte IEF gels. The IPGs can be stored at -80oC indefinitely without having a detrimental effect on the final 2D pattern. The IPGs are bound to a plastic sheet, so gel cracking resulting from expansion and contraction, associated with freezing and thawing, is avoided. Hence, the IPGs retain their original dimensions after thawing. It is convenient to store the IPGs in 15-mL screw top plastic tubes, (e.g., Falcon tubes) which can then be used for the equilibration step. It is not necessary to wash or equilibrate the IPG strips before freezing them.

Equilibration of First Dimension

IPG strips must be equilibrated in SDS buffer prior to loading on the second-dimension gel. The intrinsic charges on the protein are insignificant compared to the negative charges provided by the saturation with the SDS contained in the sample buffer. The SDS-protein complexes are of uniform charge density (in the pH range 7ñ10) and will separate according to polypeptide size in polyacrylamide gels of the correct porosity.

The vast majority of 2DE systems employ dissociating conditions in which the proteins are treated with an ionic detergent, usually SDS, to solubilize protein complexes and with a thiol reagent such as mercaptoethanol or DTT. Thiol reducing agents cleave disulfide bonds thus disrupting complexes (intermolecular bonds) and compacted structures (intramolecular bonds). Almost all proteins can be solubilized under these conditions. Alkylating reagents modify thiol groups to derivatives that are stable during electrophoresis. They also scavenge unreacted reducing agent, which causes vertical streaking in the second-dimension gel.

The IPG strips are soaked twice for 10 minutes in a solution containing urea, SDS, glycerol, and gel buffer. The urea and glycerol are present to increase the solubility of the focused proteins in the IPG strip and also to minimize protein diffusion. In the first 10 minutes, DTT is added to reduce and solubilize the proteins and to allow correct SDS binding. The DTT is removed, and some protein alkylation occurs in the second 10 minutes by the addition of acrylamide.

After equilibration, the strips are embedded on the tops of the SDS gels using molten agarose (1% in electrode buffer).

Equilibration Buffer

Reagents

Amount

6 M Urea

36 g

2% SDS

2 g

5X SDS gel buffer

20 mL

20% glycerol

20 mL

water

Q.S. to 100 mL

For the TBP protocol, add 2.5 mL of 200 mM TBP stock and 2.5 g of acrylamide monomer to the 100 mL of equilibration solution. Equilibrate IPG strips in the screw top tubes used for storage. Add 10 mL of the equilibration solution to each tube and place on a shaker for 10-20 minutes. The optimal time of equilibration will vary for different samples and protein loads.

For the DTT protocol, divide the equilibration solution into two 50-mL aliquots. Add 1 g of DTT to the first aliquot and 1.25 g of acrylamide monomer to the second. Equilibrate IPG strips in the screw top tubes used for storage. Add 10 mL of the DTT equilibration solution to each tube and place on a shaker for 10 minutes. Replace the solution with 10 mL of acrylamide monomer equilibration solution and shake for another 10 minutes.

Running the Second Dimension

Gels should be run with cooling (5-10oC). The bromophenol blue marker dye should be allowed to migrate to the end of the gels (for vertical slabs) or the anode strip (for horizontal gels). Gels must be fixed as soon as a run is completed.

Following polymerization of the slab gels and equilibration of the IPG strips, apply the strips to the top of the glass plate 2-D assembly using forceps. Seal the strips to the top of the gel using hot 1% agarose in 1x running buffer. Place the gels into the Protean II Xi multicell and add running buffer to the upper chambers. Set the thermostatic circulator to 10oC and run gels at 5 mA/gel for 2 hours and then at 15 mA/gel overnight.

Molecular Weight Markers

Colored markers can be used to monitor the progress of the electrophoresis run. If each gel has a lane of markers, they are easy to overlay for later comparison. A lane to load the markers is formed next to the IPG strip by simply placing a small piece of a plastic drinking straw next to the strip prior to overlaying with agarose. The straw is cut so that it protrudes slightly above the glass plates and thus forms a well that does not fill with agarose.

It is wise to perform a one-dimensional SDS gel experiment (using the same system as your second dimension for 2DE) with an unknown sample to determine which markers to use. It is sometimes useful to add a known protein to a sample as an internal standard. This can be useful as a control between runs and to monitor the reproducibility of the 2DE patterns.

Fixing Gels

At the end of the run, gels are fixed in 40% methanol, 10% acetic acid in water for 30 minutes. The bromophenol blue marker will turn a green/yellow color as the fixing solution soaks into the gel.

When using autoradiography, fixing should be carried out in 30% isopropyl alcohol, 10% acetic acid for 30 minutes, as methanol interferes with the fluors.

Visualization of Results

Methods of Visualization

The analytical nature of 2DE makes it more appropriate to use high sensitivity detection, such as silver stain or autoradiography, rather than the more common staining with Coomassie Brilliant Blue (CBB).

Silver staining can be as much as 200 times more sensitive than CBB, permitting analysis of dilute samples. As a result, sample overload usually not a problem. Silver staining may be made as much as 8 times more sensitive by prior staining with CBB (16).

Silver stains can be classified into two families according to the nature of the silver reagent used for binding silver to the proteins. The first and simplest type of stain is the silver nitrate stain. In this procedure, the gel is soaked in a solution of silver nitrate, and the color is developed by reduction with formaldehyde at alkaline pH. The second, more sensitive, silver stain is the diamine stain in which the silver is complexed with ammonia.

For optimal results with diamine stains, the gels should be crosslinked with piperazine diacrylamide (PDA) instead of bisacrylamide, because PDA is more resistant to alkaline hydrolysis (17). Our laboratory commonly uses a diamine-type stain. However, for staining pre-made gels or homemade gels crosslinked with bisacrylamide, it is advisable to use a silver nitrate stain to minimize background staining.

Autoradiography of radiolabelled proteins requires exposure of the dried gels at ñ70oC for durations between 2 hours and 20 days, depending on the amount and energy of the radiolabel.

Common Problems in Visualization

The most common problems experienced with silver staining are due to its high sensitivity. High background staining and streaking are commonly due to insufficient washing or impure reagents and water of poor quality. Overloading of protein samples results in large black blotches. A detailed procedure is provided in Table 3.

 

Table 3. Silver-Staining Solutions (diamine silver stain) and Procedure.

Step

Time

Reagents

Fixer 1

1 h

40% methanol, 10% acetic acid

Fixer 2

1 h

45 g sodium acetate (anhydrous), 30% methanol, 5 mL/L glutaraldehyde

Wash

3 x 15 min

400 mL Milli-Q water/wash

NDS

30 min

0.5 g/L 2,7 Naphtalene-disulfonic acid solution (NDS)

Wash

4 x 20 min

400 mL Milli-Q water/wash

Silver

2 h

6g/L silver nitrate,15 mL/L ammonia solution, 0.8 g/L NaOH*

Wash

2 x 10 min

400 mL Milli-Q water/wash

Developer

about 10 min

0.1 g/L citric acid, 1 mL/L formaldehyde

Stop

10 min

5% acetic acid

Wash

2 x 15 min

400 mL Milli-Q water/wash

*Combine the ammonia and NaOH in 900 mL water and stir well. Dissolve the silver nitrate in 10 mL water and add slowly to the 900 mL whilst stirring rapidly. A brown precipitate will form but will redissolve immediately.

For optimal silver staining, it is essential to use high quality reagents and to prepare the solutions fresh each time. It is also desirable to do the staining in glass trays because this results in less background staining than plastic trays. The gels should be transferred to clean trays for the development step to minimize background from silver that has deposited on the trays during the silver impregnation step.

Rapid Coomassie Staining Solution

Reagents

Amount

0.1 % Coomassie R250

1 g Coomassie R250

30% methanol

300 mL methanol

5% acetic acid

50 mL acetic acid

water

Q.S. to 1 L

Add the methanol and acetic acid to the water and then add the R250. Stir the solution for 2 hours and then filter through Whatman No. 1 paper. The solution can be stored indefinitely and can be re-used. For staining, place the gels into the solution and place on a rocker for up to 2 hours. Replacing the stain with 5% acetic acid will enhance the sensitivity.

Gel Drying for Preservation and Analysis

A gel must be dried before autoradiography or storage as a permanent record. This is preferable to photographing the gels, which requires additional equipment and expense, and does not always give an accurate record of the results. Furthermore, many journals are requiring more accurate quantitation of results, such as those obtained by densitometry or phosphorimaging.

Wet gels that have been stained with CBB or silver may be scanned at this stage and then dried to give a permanent record. This record is most important, and some care should be taken when using gel driers with the thicker pore gradient gels, which are more prone to cracking.

It is preferable not to stain gels that will be autoradioraphed, as it will cause quenching of the samples. This is less of a problem with CBB than with silver staining. Gels should be soaked in an autoradiography reagent (if using 35S and 14C labelled proteins) for 30 minutes, with rocking at room temperature.

To dry gels, first soak them for 5 hours in the gel drying solution. Vertical slab gels are laid on cellophane (for scanning), or filter paper (for autoradiography), covered with cellophane and dried using the gradient cycle on a gel drier for 10 hours under vacuum at 55oC.

Gels labelled with 32P do not need to be treated with a fluor before autoradiography, but are fixed in the usual way and dried as above.

Gel Drying Solution

Reagents

Amount

50% methanol

1 L

2% polyethylene glycol 4000

40 g

water

Q.S. to 2 L

Soak the gels to be dried in this solution for at least 5 hours prior to drying. The gels are dried between 2 sheets of cellophane. The cellophane must be soaked in the drying solution for at least 30 minutes before drying. Ensure that the HydroTech pump is filled with clean tap water and connected to the drier. When the gels and the cellophane are ready for drying, place one sheet of cellophane on the porous base plate of the gel drier. Smooth out any air bubbles in the cellophane and then place the gels to be dried on top, and smooth out air bubbles. Place the top sheet of cellophane over the gels and remove air bubbles. Cut the excess cellophane away from around the gels leaving a border of at least 2 cm. Place the silicone rubber sheet on the drier over the gels, shut the lid and turn on the vacuum pump. Open the lid after 10 seconds, the silicone sheet should be adhering to the grooves around the base plate of the drier. If correct suction is not occurring, remove the obstruction, and reapply the vacuum. Dry the gels for 10 hours at 55oC, using the gradient setting.

Electroblots

While estimation of molecular weight and isoelectric point may assist in the identity of a particular spot, the technique of 2DE has its fulfilment in the definite identification of the spot. In the past, proteins in two-dimensional gels were identified by co-migration with known proteins or by blotting with antibodies of known specificity. The ability to chemically analyse proteins from single gels has resulted in an exponential leap forward for the field of proteomics. This allows one to relate observed functional changes (e.g., cellular activation) to the structural identities of proteins. Previously, the major drawback of 2DE as a preparative technique has been in the purification procedures and manipulations required to remove the protein from the gel matrix and present it in a form suitable for subsequent chemical analysis. Blotting to a solid support that is able to withstand the harsh chemicals used in Edman sequence or amino acid compositional analyses (such as PVDF) allows rapid protein identification.

Electroblotting Buffers

Buffer (1 L)

Composition

Amount

Anode Buffer

0.3 M Tris

36 g

20% methanol

200 mL

Cathode Buffer

0.04 M 6-amino -n-hexanoic acid

5.2 g

0.025 M Tris

3 g

20% methanol

200 mL

Prior to completing the second-dimension gel run, prepare the filter paper and membrane for the blot. Cut four pieces of BioRad extra thick blot paper and one piece of PVDF to the dimensions of the gel. Wash the filter paper three times (5 minutes each) in Milli-Q water and then wash two pieces in cathode buffer and two pieces in anode buffer until the blot is assembled. Wet the PVDF with 100% methanol and then wash it in cathode buffer until the blot is assembled.

Remove the second-dimension gel from the glass plates and wash for 5 minutes twice in Milli-Q water and for 5 minutes twice in cathode buffer. The filter paper should be rolled to remove excess buffer before assembling the blot. Assemble the blot from the bottom up, as follows. Place the two pieces soaked in anode buffer onto the anode electrode. Use a roller to remove air bubbles as each new layer of the blot is added. Place the sheet of PVDF onto the stack of anode paper. The gel is then placed onto the PVDF and rolled to ensure that no air bubbles are present. The remaining two pieces of paper that were soaked in cathode buffer are added to complete the assembly. Roll each piece as it is added. Close the cathode electrode over the tranfer unit. Close the safety lid, and blot at 0.8 mA per square centimetre of the gel. The blot should be run for 1 to 2 hours depending on the thickness of the gel.

Blot Staining (Amido Black)

After the blot has been completed, turn off the power supply and remove the PVDF from the blotting apparatus. Wash the PVDF in Milli-Q water for 5 minutes twice and then stain with 1% Amido Black in Milli-Q water for 1 minute. Destain the blot with Milli-Q water until the background is light blue. Attach a clip to one corner of the blot and hang it up to dry. The contrast will improve markedly when the blot is dry.

Blot Staining Solution

Reagents

Amount

amido black

10 g

water

Q.S. to 1 L

Spot Elution

Passive Elution of Spots

Spots stained with the rapid Coomassie stain are suitable for either passive elution or in-gel digestion to make them suitable for chemical analysis by Edman sequencing or mass spectrometry.

Passive Elution Buffer

Reagents

Amount

100 mM sodium acetate

82 mg

0.1% SDS

10 mg

10 mM DTT

15 mg

water

Q.S. to 10 mL

Store at 4oC.

Place stained, excised spots in Eppendorf tubes (these can be stored at -20oC at this stage). Just prior to elution wash gel pieces for 30 minutes in Milli-Q water. Add 300 µL of elution buffer and incubate at 37oC overnight. Collect supernatant and apply to Prosorb cartridge for 15 minutes. Wash twice in 50% methanol and store at -20oC.

Enzymatic Digestion of Spots

Enzyme Buffer

Reagents

Amount

200 mM ammonium bicarbonate

15.8 g

water

Q.S. to 1 L

Excise the spot to be digested (1mm x 1mm) from a CBB-stained gel and place in a microfuge tube. Remove most of the Coomassie stain with two 30-minute washes in 200 µL of 0.2 M ammonium bicarbonate, 50% acetonitrile at 30oC (18). Discard supernatant and dry band completely by centrifugal lyophilization (importantógel piece should have shrunk). Rehydrate gel in 50-100 µL of 0.2 M ammonium bicarbonate (rehydration solution) containing 0.5 µg of trypsin (Boehringer Mannheim) in rehydration solution and incubate overnight at 37oC. While it is important to keep the volume as small as possible, the gel piece should be completely covered. Collect the incubation buffer and add 200 µL of 1% TFA. Sonicate for 30 minutes at 35oC and collect the extract. Add 200 µL of 0.1% TFA, 60% acetonitrile, and sonicate at 35oC for 30 minutes. Pool extracts and dry to about 20 µL. Do not dry completely as peptides will stick to the tube. Separate peptides by reversed-phase HPLC for mass spectrometry or Edman sequencing, or store at -20oC.

Concluding Remarks

Two-dimensional gel electrophoresis is a powerful technique allowing separation of complex mixtures in one step. Such a technique that allows parallel processing of proteins in a form ready for chemical analysis has opened up the field of proteomics for a multitude of biomolecular resource facilities. The challenge is to be able to have high throughput instrumentation capable of identifying hundreds of proteins per week. Our laboratory has addressed this by building automation into some of the analysis tools. These include automated tagging for Edman sequencing, robotic workstation for amino acid analysis, and robotic gel/blot cutout for peptide digests used in mass spectrometry.

We have tried to give helpful tips for incorporating 2DE in a laboratory. If careful attention is paid to the key steps in 2DEósample preparation, isoelectric focusing, IPG strip equilibration, SDS PAGE, staining and blottingó many satisfying results should ensue.

References

1. O'Farrell PH. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 1975;250:4007-21.

2. Klose J. Protein mapping by combined isoelectric focusing and electrophoresis in mouse tissues. A novel approach to testing for induced point mutations in mammals. Humangenetik 1975;26:231-43.

3. Schele GA. Two-dimensional gel analysis of soluble proteins. Characterisation of guinea pig exocrine pancreatic proteins. J. Biol. Chem. 1975;250:5375-85.

4. Kenrick KG, Margolis J. Isoelectric focusing and gradient gel electrophoresis: a two-dimensional technique. Anal. Biochem. 1970;33:204-7.

5. Wilkins MR, Williams KL, Appel RD, Hochstrasser, DF, editors. Proteome Research: New Frontiers in Functional Genomics. Berlin:Springer-Verlag;1977.

6. Bjellqvist B, Ek K, Righetti PG, Gianazza E, Gorg A, Westermeier R, Postel W. Isoelectric focusing in immobilized pH gradients: principle, methodology and some applications. J. Biochem. Biophys. Meth. 1982;6:317-39.

7. Righetti PG. Immobilized pH gradients: theory and methodology. In: Burdon RH. van Knippenberg PH, editors. Laboratory techniques in biochemistry and molecular biology. Amsterdam:Elsevier;1990.

8. Bjellqvist B, Pasquali C, Ravier F, Sanchez JC, Hochstrasser D. A nonlinear wide-range immobilized pH gradient for two-dimensional electrophoresis and its definition in a relevant pH scale. Electrophoresis 1993;14:1357-65.

9. Rabilloud T. Solubilization of proteins for electrophoretic analyses. Electrophoresis 1996;17:813-29.

10. Rabilloud T, Adessi C, Giraudel A, Lunardi J. Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 1977;18:307-16.

11. Esteve-Romero J, Simo-Alfonso E, Bossi A, Bresciani F, Righetti PG. Sample streaks and smears in immobilized pH gradient gels. Electrophoresis 1966;17:704-8.

12. Rabilloud T, Valette C, Lawrence JJ. Sample application by in-gel rehydration improves the resolution of two-dimensional electrophoresis with immobilized pH gradients in the first dimension. Electrophoresis 1994;15:1552-58.

13. Herbert BR, Molloy MP, Gooley AA, Walsh BJ, Bryson WG, Williams KL. Improved protein solubility and alkylation in 2D electrophoresis using tributyl phosphine and acrylamide. Electrophoresis 1998; (submitted).

14. Bjellqvist B, Sanchez JC, Pasquali C, Ravier F, Paquet N, Frutiger S, Hughes GJ, Hochstrasser D. Micropreparative 2-D electrophoresis allowing the separation of milligram amounts of proteins. Electrophoresis 1993;14:1375-78.

15. Walsh BJ, Herbert BR. Casting and Running Vertical Slab Gel Electrophoresis for 2D-PAGE In: Link AJ, editor. Methods in Molecular Biology. New Jersey: Humana Press;1977.

16. De Moreno MR, Smith JF, Smith RV. Silver staining of proteins in polyacrylamide gels: increased sensitivity through a combined Coomassie blue-silver stain procedure. Anal. Biochem. 1985;151:466-70.

17. Hochstrasser DF, Harrington MG, Hochstrasser AC, Miller MJ, Merril CR. Methods for increasing the resolution of two-dimensional protein electrophoresis. Anal. Biochem. 1988;173:424-35.

18. Moritz RL, Eddes JS, Reid GE, Simpson RJ. S-pyridylethylation of intact polyacrylamide gels and in situ digestion of electrophoretically separated proteins: a rapid mass spectrometric method for identifying cysteine-containing peptides. Electrophoresis 1966;17:907-17.

 


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