High performance two-dimensional gel electrophoresis using a wetting agent Tergitol NP7

Two-dimensional electrophoresis is an efficient method for the analysis of a broad range of complex protein samples. Current two-dimensional gel techniques are not suited for analysis of the small amount of proteins from tissue samples in the presence of high concentration of salts. Here we describe an improved two-dimensional gel electrophoresis procedure based on the use of a nonionic wetting agent, Tergitol NP7, in rehydration solution combined with the application of a linear potential sweep during isoelectrofocusing. This experimental approach yields a dramatic increase in the resolution and focusing of proteins visualized on two-dimensional gels. This technique is less time-consuming and laborious than the current techniques and can be used for a variety of two-dimensional electrophoresis applications, including proteome analysis.


Introduction
Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is the method of choice for large scale protein microcharacterization as it allows both qualitative and quantitative analysis of proteins [1][2].2-DE performed with immobilized pH gradient (IPG) isoelectric focusing (IEF) has higher resolution, as compared to classical 2-DE with carrier ampholytes [3][4], due to use of well-defined narrow pH gradients [5], an improved reproducibility [6][7] and higher loading capacity for micropreparative runs [8][9].Consequently, IPG 2-D gels have become the universally used technology for separating complex protein mixtures allowing identification and characterization of single spot proteins.Indeed, the remarkable improvements in 2-DE resulting from IPG, together with convenient new instruments for IPG-IEF, have largely contributed to recent advances in proteome analysis.Different options exist in terms of methods and equipment for IEF.Amersham Biosciences offers two different systems for the first dimension separation: the Multiphor II System and the IPGphor isoelectric focusing system.Many laboratories use the IPGphor isoelectric focusing system which simplifies the first separation run by using IPG strips.The system is comprised of IPG strip holders that are used for both rehydration and IEF, and the IPGphor unit, which includes a 8 000 V power supply.Appropriate sample preparation is crucial for obtaining correct 2-D results.The presence of salts, residual buffers, and other charged small molecules in a sample preparation leads to high strip conductivity and prolongs the time required for IEF and can result in large regions at each end of the IPG strip in which proteins do not focus.Salt concentration can be reduced by precipitation of proteins with TCA or acetone or, alternatively, by sample dilution but, the problem of protein concentration becomes rapidly insurmountable in this latter case.
In this study we describe an alternative, rapid and simple strategy for sample preparation, based on the use of a nonionic detergent, Tergitol NP7.It is included in the rehydration solution to avoid problems with protein concentration or interfering substances.The use of NP7 in the rehydration solution combined with a series of voltage steps or gradients allows us to visualize a much larger proportion of a protein, even when present at a low level.This procedure provides an improvement when used with samples from yeast, gram positive bacteria and human cell culture.

Apparatus and chemicals
Immobilines (4-7), IPG buffer 4-7 and the equipment for running the IPG gels (IPGphor ) were from Amersham Biosciences (Saclay, France).Second dimension SDS gels were cast and run in a Hoefer Dalt System (Amersham Biosciences).Tergitol NP7 was purchased from Sigma (Saint Quentin Fallavieur, France).All other chemical reagents were purchased from Amersham Biosciences.Silver stained gels were scanned using a Epson 1600 Pro scanner in a transparency mode at 228 dpi.The image analysis, including pI, molecular masses, spot quantification and matching between gels, was carried out using PDQuest software (Bio-Rad, Hercules, CA, USA).A Krüss (Palaiseau, France) contact angle measuring system was used for contact angle visualization and measurements.

Sample preparation
Human muscle was ground in a liquid nitrogen cooled mortar and the powder obtained was immediately resuspended in a lysis buffer (8 M urea, 4% CHAPS, 65 mM DTT and 40 mM Tris base).The sample was vortexed for 1 min, frozen with liquid nitrogen and thawed at room temperature.The vortexing, freezing and thawing were repeated three to four times.The sample preparation was stored in liquid nitrogen since proteases are less active at lower temperatures.40 mM Tris base has been used because most tissue proteases are inactive above pH 9. Samples were spun at 35 000 g for 30 min to remove solid debris.Samples were incubated with DNase and RNase to separate proteins from nucleic acids, for 1 h at 47C, and then were spun at 35 000 g for 30 min.Protein concentration in the dissolved samples was determined using the dotMetric 1 mL protein assay from Chemicon (Valbiotech, Paris, France).

Electrophoresis
IPG gels with pH gradients 4-7 (13 cm long) were used.The reswelling solution contained 8 M urea, 2% CHAPS, 7 mg DTT and 0.5% IPG buffer pH 4-7.The sequence was as follows: 250 mL of reswelling solution was first delivered into the strip holder; a 13 cm strip was carefully put down on this solution; sample solution (30 mL) was then injected with a Hamilton syringe on one side and underneath this strip, on the anodic side; mineral oil was added in order to recover all the strip; the hydration was active since the IPG strip was rehydrated at 30 V overnight (13 h) in the presence of proteins, at 207C.Two different programs were run with, in all cases using the same Vh number, i.e. 20216.Focusing was performed through a series of voltage gradients (linear potential sweep) as follow: 13 h at 30 V, 1 h ramp to 200 V, 1 h ramp to 500 V, 1 h ramp to 1000 V, 2 h 45 min ramp to 3500 V and 2 h ramp to 5000 V; or through a series of voltage steps that began at a relatively low value, 13 h at 30 V, 1 h at 200 V, 1 h at 500 V, 1 h at 1000 V and continued with a maximum of 5000 V steady step.Current was limited to 0.05 mA per IPG gel strip.After the IEF, the IPG strips were stored between two plastic sheets at -787C until use or equilibrated immediately for 2612 min in 10 mL of a solution containing Tris-HCl buffer (50 mM pH 8.8, 6 M urea, 30% w/v glycerol and 2% SDS).Immediately before use, DTT (1%) was added to the first, and iodoacetamide (4%) to the second equilibration step.Then the IPGs were applied to 15%T, 2.6%C polyacrylamide gels without stacking gels.The gel format was 16 cm (high)616 cm (wide)61.0mm.Electrophoresis was performed in a Hoefer system for 19 h with voltage limited to 70 V per gel at room temperature.Proteins spots were visualized in analytical gels by silver staining (Amersham Pharmacia Biotech).Image analysis was performed using the program PDQuest software (Bio-Rad).

Contact angle measurements
Hydration solution (8 M urea, 2% CHAPS ) was used without Tergitol and with 0.1% to measure the contact angle on ceramic strip holders as solid phase.This latter was cleaned with a commercial cleaner, then carefully washed with water of surface quality (measured surface tension as 72 mN/m at 257C).The drop was allowed to grow very slowly until the radius of the three-phase contact line was above 4 mm to minimize the drop size effect.The operation was effected under a camera and contact angles were measured with a Krüss contact angle measuring system (G2).All the experiments were performed at room temperature (297C).

Results
Total proteins of muscle samples (30 mg) were resolved according to the basic protocol (see Section, Figs.1A  and 2A) or to the improved protocol (addition of 0.1% NP7 in the rehydration solution, Figs.1B and 2B).Focusing was performed with either a series of steps (Figs.1A  and 1B) or with a series of gradients (Figs.2A and 2B).In the absence of the wetting agent, NP7, point streaking occurs due to underfocusing at high salt concentrations (Figs.1A and 2A).Focusing time was not long enough to achieve steady-state focusing.Better results are obtained by using the wetting agent in the rehydration solution and there is a significant improvement in pattern quality (Figs.1B and 2B).The majority of proteins appears as clear, distinct, round spots and a true steady step is obtained, revealing a highly reproducible 2-D pattern (Figs.1B and 2B).Sample application at the anode did not influence the final result (samples entered the gel properly, without protein precipitation at the application point).Interestingly, it appears that the linear voltage Figure 1A and 2A.Typical 2-D protein pattern of muscle extract obtained in the absence of the nonionic agent, Tergitol NP7.The first dimension was an isoelectric focusing in immobiline gel ranging from pH 4-7 as indicated along the horizontal axis, and the second dimension is by molecular weight as determined by relative mobility in an SDS polyacrylamide gel shown on the vertical axis.Thirty mg of total protein was loaded onto the first dimension.Focusing was performed either through a series of voltage steps (Fig. 1A) or through a series of voltage gradients (Fig. 2A). Figure 1B and 2B.Typical 2-D protein patterns of muscle extract obtained in the presence of the nonionic agent, Tergitol NP7.The first dimension was an isoelectric focusing in immobiline gel ranging from pH 4-7 as indicated along the horizontal axis, and the second dimension is by molecular weight as determined by relative mobility in an SDS polyacrylamide gel shown on the vertical axis.The total protein of the sample loaded on the first dimension was 30 mg.Two different run programs were used.Focusing was performed through a series of voltage steps (Fig. 1B) or through a series of voltage gradients (Fig. 2B).
sweep run is more efficient than the voltage step (Fig. 2B).Note that there is no precipitation of the sample at any point on the gel and that resolution of high molecular weight proteins is better.
Together, the sample application and the addition of a wetting agent allow us to use lower protein quantity that the usual method of sample application in the whole rehydration solution (here 250 mL) and no wetting agent.As an argument, these optimized conditions were also tested with 120 mg of muscle extract loaded onto a narrow range of pH, i.e. 4-7 (Fig. 3, area A).As a rough estimate, three time more proteins are viewed.Until now, no poorly soluble protein was found during mass spectra identification, which seems to signify that the wetting agent does not enhance their solubility, as shown by example with special membrane protein adapted detergents [16].B).We have gained protein spots which can be evaluated roughly to an incraese of 300% of proteins.

Discussion
In the absence of Tergitol NP7, we have observed that whatever the running conditions used for IEF, during the first step (200 V), corresponding to the sample entry into the strip, the current rapidly raises toward high values, at least 50 mA, which is the maximum current setting.After this jump, the current decreases exponentially as proteins and other components migrate to their equilibrium position.The steady-state occurs some hours later, generally on the potential plateau, and as the current reaches a practically constant value less than 50 mA, the focusing begins as the current becomes constant.
During the formation of this peak current, the real potential developed in the electrolysis cell is the result of the applied voltage and a large voltage drop.The efficacy of sample entry into the strip is lowered.As the ionic composition increases (due to Tris, DTT, SDS, IPG buffer, proteins), the peak current increases together with its duration.As a consequence, a longer focusing time is required to reach steady-state.Straightforwardly, this peak current corresponds to a capacity current which arises from spatial ion distribution in the strip.When a step of potential is applied, the ions move towards the electrodes; as a consequence of this charge distribution, the total electric charge Q increases from zero to a constant value.The corresponding current is then ic = dQ/dt = C dV/dt (where C = capacity, V = potential.).ic depends on C and, according to the double layer theory [10], C depends on salt concentration.So, as the salt concentration increases, C increases with ic.Moreover, ic is proportional to the gradient dV/dt.A first solution consists to lower the total salt concentration and the V versus t rise.In our case, to overcome these difficulties, a wetting agent is added to the rehydration solution which in fact is a nonionic detergent, Tergitol NP7.As an experimental consequence, the capacity current is abolished, and a resistive current is established; with our experimental conditions, a constant 145 MO resistance is measured.Now, the potential profile, step or gradient, is really the fixed one.The role of Tergitol is to create a wetting film between the strip and the bottom of the strip holder so that the ionic conductance is distributed between the strip and the film.If the strip and the wetting film respectively has Z1 and Z2 impedances, the resulting impedance Z is: Z = Z1Z2/(Z11Z2), and if Z2 ,, Z1, Z * Z2.
The strip conductance is shunted and mainly the current passes through the wetting film.It is now not necessary to wait for the establishment of the current plateau, and the electrofocusing starts at the beginning of the step or gradient.An alternative technical solution may be the development of voltage generators allowing the maintenance of a fixed potential in each strip holder, as obtained with "potentiostats" in electrochemical apparatus.Also, the stability of a wetting film can be greatly improved by a rough strip holder surface.
Contact angle measurements give a proof of the reality of the wetting layer, as shown in Figs.4A, B and C. Hydration solution without Tergitol does not wet the strip holder material (ceramic) and a 677 contact angle is measured.(Fig. 4B).(The contact angle of pure water on the strip holder material (ceramic) is about 757 which means that this surface solid is as hydrophobic as the current surface polymer (Fig. 4A)).When 0.1% Tergitol NP7 is added to the hydration solution, the contact angle becomes near zero (Fig. 4C) and the wetting is said to be perfect; so, in this case a liquid drop spreads on the solid to form a wetting film.The reader who wants further information on wetting, should refer to the specialized papers [11][12][13][14][15].

Concluding remarks
In this study, we have shown that the addition of a wetting agent to the rehydration solution, markedly improves the quality of 2-D gels.Moreover, the best resolution is obtained and the sample entry is probably more efficient when focusing is performed through a series of voltage gradients.When a capacity peak current appears, many factors can affect the amount of time required for complete focusing and thus the reproducibility of analyses.The performances of IEF in the presence of a relatively high salt concentration may also be largely improved by the addition of a wetting agent which reduces the increase of capacity current as well as the conductivity of the sample, compared to the strip.Thus, this procedure may be helpful to resolve membrane protein samples which generally contain relatively high salt concentrations.

Figure 3 .
Figure 3.The optimized loading and focusing conditions tested with 120 mg of muscle extract loaded onto a narrow range IPG strip of pH range 4-7 (area A) compared with 30 mg loaded onto a narrow range IPG strip of pH range 4-7 (areaB).We have gained protein spots which can be evaluated roughly to an incraese of 300% of proteins.

Figure 4 .
Figure 4. Sessile drop on strip holder ceramic: A, pure water; B, 8 M urea, 2% CHAPS hydration solution; C, 8 M urea, 2% CHAPS, 0.1% NP7 hydration solution.In C, the liquid drop spreads on the surface to give a perfect wetting i.e., a wetting liquid layer.