Electrophoresis 1997. 18. 127-135 Two-dimensiunal electrophoresis of membrane proteins 127

CCline Adessi' Christine Miege* Catherine Albrieux' Thierry Rabilloud'

'Laboratoire de Chimie des ProtBines, 'Laboratoire de Physiologie Cellulaire VBgBtale, 'Laboratoire de BioenergCtique Cellulaire et Pathologique, CEA, Grenoble, France

Two-dimensional electrophoresis of membrane proteins: A current challenge for immobilized pH gradients

Membrane proteins were separated by high resolution two-dimensional (2-D) electrophoresis. On isoelectric focusing (IEF) with immobilized pH gradients severe protein losses in the resulting 2-D map were observed when compared with carrier ampholyte-based IEF. This has been noticed for two different bio- logical systems, namely the chloroplast envelope of spinach and the endocytic vesicles from Dictyostelium discoideum. The possible mechanisms of these losses on immobilized pH gradients are discussed?

1 Introduction

Since its introduction in the mid-seventies, high resolu- tion 2-D electrophoresis has been used for many types of proteins, including membrane proteins (e.g. [ 1-51). However, conventional isoelectric focusing, where the pH gradient is established with carrier ampholytes, is not devoid of problems. These include pH gradient instabil- ity with time, and irreproducibility in the long term, mainly due to the undefined chemical composition of carrier ampholytes. These drawbacks were eliminated by the use of immobilized pH gradients for the focusing step [6,7]. In this system, the pH gradient is formed by a small number of defined chemicals, covalently grafted to the polyacrylamide matrix. This allows true steady-state focusing, with greatly increased reproducibility. Addi- tional advantages of immobilized pH gradients include their high capacity [8], which allows preparative runs of microgram amounts of proteins for structural analysis [9, 101. Separation of membrane proteins has been shown to be feasible [ l l , 121 and 2-D electrophoresis, with ade- quate resolution of membrane proteins with immobi- lized pH gradients has been reported [13]. The quantita- tive aspects of separation, however, were not thoroughly investigated in the studies mentioned above, which dealt with purely analytical, mainly descriptive procedures. We have undertaken a study of membrane proteins for carto- graphic purposes on two widely different systems, the chloroplast envelope of spinach and the endocytic vesicles from Dictyostelium discoideum. The cartographic purposes implied that microgram amounts of proteins be obtained after the 2-D electrophoretic step, in order to obtain sequence information for polypeptide identifica- tion, requiring separation of high amounts of membrane proteins (several hundred micrograms). We report here that the use of immobilized pH gradients may result in severe protein loss, which was not experienced with car- rier ampholyte-based isoelectric focusing.

Correspondence: Dr. Thierry Rabilloud, DBMS, BECP, CEA-Grenoble, 17 rue des martyrs, F-38054 Grenoble Cedex 9, France (Tel: +33-4- 7688-3212; Fax: +33-4-7688-5155)

Nonstandard abbreviations: BisTris, N,N-bis(hydroxyethy1)-N,N,N-tris (hydroxymethy1)aminomethane; PDA, 1,4-bis(acryloyl)piperazine; RUBISCO, ribulose 1,s biphosphate carboxykinase

Keywords: Membrane proteins / Two-dimensional polyacrylamide gel electrophoresis / Immobilized pH gradients

2 Materials and methods

2.1 Chemicals and materials

Immobilines, Pharmalytes 3-10, gel cassettes, the gra- dient maker and the equipment for running the IPG gels (Multiphor IT and Dry-Strip kit) were from Pharmacia; carrier ampholytes, Resolytes 4-8, were from BDH. Second-dimensional SDS-gels were cast and run in a Bio-Rad Multi Cell. 1,4-Bis(acryloyl)piperazine (PDA) and protein assay reagent were from Bio-Rad, HEPES hemisodium salt from Sigma, and acrylamide from Amresco. All other chemicals, of analytical grade, were from Fluka, Gel-Bond PAG film was from FMC, and Gel Fix Covers from Serva.

2.2 Protein sample preparation

2.2.1 Dic@osteliurn vesicles

2.2.1.1 Colloidal iron particles

Magnetite-dextran, a colloidal suspension of iron oxide (y-Fe,O,) coated with Dextran T-40, was synthesized as described by Rodriguez-Paris et al. [14]. The magnetic solution was dialyzed for two days against cold water, followed by centrifugation to remove large aggregates; the unbound dextran was not removed. The solution was found to be stable at 10 mg of iron/mL at 4°C for at least two months.

2.2.1.2 Biological preparation of endocytic vesicles by a magnetic fractionation approach

D. discoideum strain AX2 (ATCC 24397) was grown in axenic medium at 21°C [15]. Magnetic purification of endocytic vesicles was essentially conducted as described by Rodriguez-Paris et al. 1141. Briefly, the cells (100 mL; 1 X lo7 cells per mL) were fed for 3 min with the col- loidal iron suspension (final concentration of 1 mg iron per mL). The endocytic activity was then stopped by addition of an equal volume of an ice-cold hypoosmotic buffer (5 mM glycine, 100 mM sucrose, pH 8.5, GS buffer, supplemented with 0.5 O/o bovine serum albumin). The cells were washed three times with the same buffer and broken with a cell cracker [16]. The postnuclear superna- tant was loaded onto a column filled with stainless steel

* Results presented in part at the international ICES and SFE mee- ting Electrophoresis '95, Paris, August 30-September 2, 1995

0 VCH Verlagsgesellschaft mbH, 69451 Weinheim, 1997 0173-0835/97/0101-0127 $10.00+.25/0

128 C . Adessi cf a / . Electruphormis 1997. 18, 127-135

Figure 1. 2-D electrophoresis of Dictyostelium endocytic vesicle proteins. Eighty pg of endocytic vesicle proteins, solubilized in SDS and urea, were loaded onto the focusing gels. Detection was carried out by silver staining. (A) IPG focusing (pH 4-8), sample appli- cation by reswelling. (R) IPG focusing (pH 4-8) sample application by loading at the cathode. (C) Carrier ampholyte focusing (gra- dient measured from pH 4.2 to 7.9), 12 000 Vh. (D) Carrier ampholyte focusing (gradient measured from pH 4.2 lo 7.5), 25 000 Vh. Arrows in (C) and (D): horizontal streaks attributed to dextran.

wool (Spontex, Nanterre, France) placed in a strong 40 min). The purified vesicles, corresponding to 100 pg magnetic field (0.15 Tesla). The column was washed with of total protein (measured by quantitative amino acid ice-cold GS buffer supplemented with 2 mM EDTA analysis), were solubilized at room temperature in 50 pL (GSE buffer), and then removed from the magnetic lysis buffer containing 1 O/o SDS, 20 r n M DTT, 25 mM Tris- field. The retained fraction was eluted with four volumes HCL, pH 8.5. Then the dextran-coated iron oxide was of GSE buffer and pelleted by centrifugation (15 000 g, pelleted by centrifugation (200 000 g, 15 min, Beckman

Electrophoresis 1997, 18. 127-135 Two-dimensional electrophoresis of membrane proteins 129

TLlOO centrifuge). Urea (72 mg) and a concentrated solu- tion, 46 yL, was added to the supernatant in order to obtain 150 yL of a final solution containing 20 mM DTT, 8 M urea, 4% CHAPS, 0.2% Triton X-100. The solution was supplemented with Resolytes 4-8 (2.4%) or with Pharmalytes 3-10 (0.4%) for carrier ampholyte gradients and for immobilized pH gradients, respectively.

2.2.2 Chloroplast envelopes

Chloroplasts were isolated from 3-4 kg of spinach (Spinaciu olerucea L.) leaves obtained from local mar- kets. Deveined leaf sections were cut into chilled medium (2.5 L/kg of leaves) containing 330 mM sucrose, 25 mM tetrasodium pyrophosphate, 0.1% bovine serum albumin; the pH was adjusted to 7.8 with HCl. The leaves were homogenized for 2-3 s in a 4 L Waring blender. All operations were carried out at 0-5°C. A crude chloroplast pellet was obtained as described by Douce and Joyard [17] and purified further by isopycnic centrifugation in Percoll gradients. Purified intact chlo- roplasts were then lysed in hypotonic medium as de- scribed by Douce and Joyard [17]. At this step of purifica- tion, protease inhibitors (1 mM PMSF, 1 mM benzami- dine, and 0.5 mM caproic acid) were added to prevent protein degradation. The stroma, envelope membranes, and thylakoids were purified from the lysate (swollen chloroplasts) by centrifugation through a step sucrose gradient as described by Douce and Joyard [17]. Enve- lope membranes were stored in liquid nitrogen, in 50 mM MOPS-NaOH, pH 7.8,l mM DTT [18], 1 mM ben- zamidine and 0.5 mM caproic acid. For 2-D electropho- retic analysis, the envelopes were thawed, collected by centrifugation (100 000 g, 1 h) and resuspended in lysis buffer (9 M urea, 4% CHAPS, 0.4% Triton X-100, 40 r n M DTT and 0.4 or 1.6% Pharmalyte 3-10).

2.2.3 Keratinocyte total extract

Transformed keratinocytes (line TR 146 [19]) were grown in DMEM, supplemented with 10% fetal calf serum. The cells were harvested by trypsinization, rinsed in PB S and lysed in ten volumes of lysis buffer (9 M urea, 4O/o CHAPS, 10 mM spermine base, 20 mM DTT). Extraction was carried out for 1 h at room temperature, and the nucleic acids were pelleted by ultracentrifugation (200 000 g 1 h). The resulting supernatant was supple- mented with carrier ampholytes (Pharmalytes 3-10, 0.4% final) and stored frozen at -20°C. Protein concen- trations were determined by a dye-binding assay [20].

2.3 IEF in immobilized pH gradients

2.3.1 Gel casting

The Irnmobiline concentrations used to generate the pH gradients (mean buffering power, 3 mequiv.1-l.pH-1) were calculated according to published recipes [2 11. The acrylamide percentage was either 4 or 4.5%T for sample application by loading, or 3.2-3.5 %T for sample applica- tion by rehydration [lo]. PDA was selected as a cross- linker (2.7%C), since it gives larger pores than Bis [22].

To cast the low-percentage gels, the previously described modifications [lo] were used. Linear gradients with or without plateaus were cast. Polymerization was carried out at 50°C for 1 h [23]. The gels were then washed with 0.1 M sodium ascorbate for 1 h [24], then with water for 3 X 20 min and finally with 2% glycerol for 30 min. The gels were then dried overnight at room temperature with a fan or underneath a chemical hood. The dried gels were protected with a Gel-Fix cover sheet (Serva) and stored dry at -20°C.

2.3.2 IPG strip rehydration and running

Four mm wide IPG strips were cut from the dry plate with a paper cutter. They were rehydrated in grooved chambers as previously described [lo]. The rehydration solution contained 8 M urea, 4% CHAPS, 0.4°/o-1.60/o Pharmalytes 3-10, 0.1 mg/mL Orange G , 10 mM DTT, 0.4% Triton X-100 and 0.1 O/o sodium taurodeoxycholate. If needed, the protein sample was added to this solution to a final volume of 400-500 yL. Once the chamber had been assembled, the solution was poured into the rehy- dration groove with the help of special micropipette tips for electrophoresis (Bio-Rad). The strip, with the Gel-Fix Cover peeled off, was then inserted into the groove. If needed, the groove was filled with pure rehydration solu- tion. Rehydration was allowed to proceed overnight to ensure maximal diffusion of the proteins within the strip. The first-dimensional strips were run on a Dry- Strip Kit according to the manufacturer's instructions with the previously described modifications [ 101. Instead of using a paper electrode lying transversely to the gels, the electrode strip paper was cut into ca. 1.5 cm long pieces. These were wetted with water and placed longitu- dinally on the gels with a 3-5 mm strip overlap. The electrodes were then placed distally from the gel, so that the paper between the electrode and the strip could act as a trap for ionic compounds. The entire set-up was covered with low viscosity silicon or paraffin oil. The samples were applied at one end of the gels using the sample cups for immobilized pH gradients (Pharmacia). Sample application was tested at the cathode and at the anode, with volumes up to 120 pL. IEF was carried out at 150 V for 30 min, 300 V for 2 h30, 700 V, 1500 V, and 2000 V, 30 min each, and finally 2800-2900 V to reach a total of 50-60 kVh.

2.3.3 Equilibration

After the IEF run, the oil was poured off and the strips were equilibrated while in place in the running setup. The first equilibration solution contained 30 Yo glycerol, 6 M urea, 2.5% SDS, 0.15 M BisTris-0.1 M HCI and 0.8% DTT. This was applied to the strips for 10 min. The second equilibration step was also carried out for 10 min in the same solution, with exception that DTT was replaced by 4% iodoacetarnide (IAM) [25]. The strips were then sealed at the top of the 1.5 mm thick second- dimensional gel (Bio-Rad vertical system) with the help of 1% low-melting agarose in 0.2% SDS, 0.15 M BisTris- 0.1 M HC1 buffer, supplemented with bromophenol blue as a tracking dye.

130 C . Adessi cf a / . Electrophoresis 1997, 18, 127-135

Figure 2. 2-D electrophoresis of chloroplast envelope proteins. Two hundred pg of chloroplast envelope proteins, solubilized in CHAPS and urea, were loaded onto the focusing gel. Detection by silver staining. (A) IPG focusing (pH 4-8), sample application by reswelling. (B) IPG focusing (pH 4-8), sample application by loading at the anode. (C) IPG focusing (pH 4-8), sample application by loading at the cathode. (D) Carrier ampholyte focusing (gradient measured from pH 4.1 to 8.1), 15 000 Vh. Major matching spots in all gels are indicated by various symbols (arrows, arrowheads, etc.) . This figure shows the massive overloading encountered in silver-stained gels with 200 pg of proteins in conventional carrier ampholyte focusing.

2.4 IEF in carrier ampholyte-based gradients: gel (4.S0/oT, 2.7% C-PDA) contained 9 M urea, 4% CHAPS, 0.4% Triton X-100, 2% Resolyte 4-8 and 0.4% Pharma- lyte 3-10. Polymerization was initiated by addition of 1 pL of TEMED and 4 pL of 10% ammonium persulfate per mL of the mixture, and allowed to proceed for 2 h at

casting, running and equilibration

Tube gels (3 mm diameter, 160 mm long) were cast in cut 1 mL disposable glass pipets (Corning). The gel

Electrophoresis 1991, 18, 127-135 Two-dimensional electrophoresis of membrane proteins 13 1

room temperature. The tubes were placed in a Chamber Model 175 (Bio-Rad). The sample was applied at the cathodic end. The anodic electrolyte was 15 mM phos- phoric acid and the cathodic electrolyte was 20 mM degassed sodium hydroxide. The gels were run at 250 V for 2 h, 500 V for 2 h and 1500 V for 15 h (total 24 kVh). After the run, the gels were removed from the running tube by water pressure. The first cm of the gel (anode) was dipped into a solution of pyronin Y (1%) for about 10 s before complete expulsion of the gel into the tube containing the equilibration buffer (2.5% SDS, 0.15 M BisTris - 0.1 M HCl, 2 mM tributylphosphine, 20% gly- cerol). The gels were equilibrated for 20 min, loaded onto the SDS gels and sealed in place with agarose in the stacking buffer (0.15 M BisTris - 0.1 M HCl), contai- ning 0.2% SDS. Measurement of the pH gradient was carried out by running a gel loaded with lysis buffer, with or without proteins, in parallel with gels loaded with the samples of interest. After the run, the gel was extruded from the tube and cut into 1 cm sections. Each section was incubated for 15 min in 1 mL of degassed 10 mM KCl, and the pH of the resulting solution was read on a standard pH meter. At analytical protein loa- dings (less than 200 pg) no significant interference due to the presence of proteins on the pH gradient was detected.

2.5 SDS-PAGE

The gels used in this study were continuous 10%T, 2.7%c gels using either Bis or PDA as a cross-linker. The gel buffer (final concentration) was 0.167 M Tris - 0.1 M HCI. A 5 mm wide stacking gel (4%T) was cast in the 0.15 M BisTris - 0.1 M HCl buffer. The upper electrode buffer (made fresh every time) was 0.1 M Tris, 0.1 M tau- rine, 0.1% SDS, while the lower electrode buffer was the standard Tris-glycine-SDS buffer (twice concentrated) and was kept in the electrophoresis cell for approxi- mately three months at 4°C. Electrophoresis was carried out at 25 V for 1 h and then at 35-40 mA/gel for 5-6 h or 15 mA/gel overnight, until the tracking dye reached the bottom of the gel.

2.6 Staining

Depending on the purpose of the gel (analytical or mic- ropreparative), different staining protocols were used. For analytical gels (25-200 pg of a complex sample loaded in the first dimension) tetrathionate-silver nitrate staining was used [26]. The gels (up to four per dish) were fixed for at least 3 X 30 min in 10% acetic acid and 30% ethanol. They were then sensitized overnight in a solution containing 0.5 M potassium acetate, 2.5 g/L potassium tetrathionate, 0.5 O/o glutaraldehyde and 20% ethanol. Next they were rinsed 6 X 20 min with water and impregnated with the silvering solution (12 mM silver nitrate, i.e. 0.2%, 600 pL/L 37% formaldehyde and 5 mM HEPES hemisodium salt) for 1-2 h. All steps were carried out in polyethylene food boxes with up to four gels per box. To avoid glove marks, the gels were kept in the same container from the first fixation to the end of the silvering. During solution changes, they were kept in place with a thin polycarbonate sheet (Bio-Rad separating sheets for multigel casting) while the con-

tainer was emptied and refilled. After the silvering step, each gel was taken out of the silvering bath, rinsed in water for 5-15 s and developed (one gel per container) in 3 O/o potassium carbonate containing 250-300 yL/L 37% formaldehyde and 125 pL/L 10% sodium thiosul- fate pentahydrate. When the desired result was obtained (typical development time 10-15 min) the development was stopped by placing the gels (up to four per dish) in 5% Tris-2.5% acetic for at least 30 min. The gels were then rinsed several times with water prior to drying. For micropreparative scaling, the gels were stained for 24 h by colloidal Coomassie Blue G [27], with the modifica- tions described by Anderson et al. [28].

3 Results

3.1 2-D electrophoresis of endocytic vesicles proteins

Preliminary solubilization tests carried out in these vesicles showed that SDS was mandatory to solubilize the proteins with good efficiency, and that iron removal was required to obtain good results, both in standard SDS-PAGE and in 2-D electrophoresis [29,30]. However, the patterns obtained by reswelling the IPG strip with the protein were rather disappointing (Fig. 1A). Some improvement was obtained by loading the sample at the cathodic end of the strip (Fig. lB), while loading at the anodic end was disastrous, probably because of carry-over of undissociated SDS-protein complexes into the anodic paper. The patterns were not improved by increasing the carrier ampholyte concentration in the IPG strip from 0.4 to 1.6%. Only few proteins were observed on these 2-D maps, while standard SDS-PAGE patterns revealed a host of proteins [29, 301. We therefore tested conventional 2-D electrophoresis with carrier ampholytes. Somewhat improved patterns were initially obtained (Fig. 1C) with standard focusing times (- 10 kVh), but prominent horizontal streaks (arrows) prevented sensitive detection. These streaks being attrib- uted to the presence of dextran in the sample (arising from the coating of the iron particles), we increased focusing to more than 20 kVh to remove the dextran bands electrophoretically. This improved the patterns considerably, which eventually revealed the expected high number of proteins (Fig. 1D). This necessity for pro- longed focusing seen in carrier ampholyte-based focu- sing is only due to the staining interference of dextran and not to a general slowing down of the focusing pro- cess. We therefore do not think that insufficient focusing is the cause of the poor results obtained in IPG.

3.2 2-D electrophoresis of chloroplast envelope proteins

To test whether this relative failure of IPGs was due to the nature of the vesicular proteins or to an interference with the extraneous compounds used to prepare the vesicles (iron, dextran, etc.), we tested the separation on chloroplast envelope proteins. In this case, the mem- brane can be directly solubilized in urea and detergent (CHAPS plus Triton X-loo), and the nonproteinaceous contaminants are limited to lipids and some natural dyes (residual chlorophyll). High quality patterns were

132 C. Adessi er al. Elecrrophoresis 1997, 18, 127- 135

obtained by reswelling the IPG strip with the protein extract (Fig. 2A) or by sample application at either side (Fig. 2B and C). Milligram amounts of proteins could be loaded for micropreparative purposes (not shown) but peptide microsequencing of the major spots did not reveal any of the previously identified membrane pro- teins of this type of extract (e.g. protein E24, a 24 kDa protein of pZ 4.6 [31, 321). Mainly contaminating soluble proteins, e.g. ribulose 1,5-biphosphate carboxy kinase (RUBISCO) fragments, ribosomal proteins or mem- brane-associated proteins (the spinach homolog of the pea membrane-associated protein described in [33]) were identified, indicating that many true membrane proteins were lost at some stage of the process. Again, the stain- ing intensity with silver was considerably less than expected from the protein loading (200 pg). This prompt- ed us to focus these proteins with carrier ampholytes (Fig. 2D). This overloaded pattern corresponded to the intensity expected for the protein load (200 pg). To check whether this result was due to an interference of IPGs with silver staining, additional gels, loaded with 400 pg, were run either with IPG or with carrier ampholyte IEF, and stained with colloidal Coomassie Blue. The results, shown on Fig. 3, clearly indicate that the poor detection sensitivity obtained with IPG is the result of severe pro- tein losses at the focusing step.

3.3 2-D electrophoresis of a whole cell extract

Finally, to check whether the poor performance of the IPG was due to a problem in the batches of gradients used for the above-mentioned analyses or to sample application, these strips were tested with a standard whole cell extract from keratinocytes, in parallel with carrier ampholyte-based focusing. The results, displayed in Fig. 4, clearly show what was initially expected, i.e. sharper resolution of IPGs (Fig. 4B), without any great losses compared to carrier ampholyte-based focusing (Fig. 4A). In addition, no detrimental effect from the re- swelling of the IPG strip with the protein extract was observed. On the contrary, horizontal streaking of the major cytoskeletal proteins was reduced by reswelling

Figure 3. 2-D electrophoresis of chloro- plast envelope proteins. Four hundred pg of chloroplast envelope proteins, solubi- lized in CHAPS and urea, were loaded onto the focusing gel. Detection by col- loidal Coomassie Blue staining. (.4) II’G focusing (pH 4-8), sample application by reswelling. Ladder at the right: molecular weight markers. (B) Carrier ampholyte focusing (gradient measured from pH 4.1 to 8.1), 15 000 Vh. Arrow: large subunit of RUBISCO.

the IPG strip with the protein extract (Fig. 4B-D). These results clearly assigned the problems encountered with the membrane proteins as being linked to the above class of proteins.

4 Discussion

In their early days, IPGs suffered from a number of problems, mainly linked to protein-matrix interactions, with e.g., massive sticking of some proteins at the appli- cation site [34]. Addition of carrier ampholytes to the matrix considerably enhanced the protein solubility [35], improving isoelectric focusing. Addition of high concen- trations of carrier ampholytes to IPG gels proved equally successful for membrane proteins [ l l , 121. This modifica- tion, along with improved equilibration procedures [36] allowed good 2-D patterns of membrane proteins to be obtained without signs of massive protein-matrix interac- tions [13]. However, we have shown in this report that even good patterns can mask severe problems clue to important protein losses in the 2-D process. Below we discuss where these problems may occur.

The first hypothesis would be an interaction, probably hydrophobic, between the protein and the matrix, pre- venting focusing, as in the early days of IPG [34]. From the strategy we have adopted, this seems unlikely. Indeed, if any strong interaction between a rather abun- dant protein and the matrix were to occur, sample appli- cation by rehydration would reveal a horizontal streak across part of the gel or the whole gel [lo]. Due to the high sensitivity of silver staining, even medium-abun- dance proteins would give rise to this type of streaking. This is, however, not observed in our 2-D patterns in which proteins simply appear underrepresented. This finding favors another hypothesis, one in which the inte- raction with the IPG matrix becomes prominent at or close to the isoelectric point. In this model the proteins focus correctly but become insoluble at their p l and are not transferred to the second-dimensional gel. Isoelec- tric insolubilization of proteins in IPGs may well be a more general phenomenon, not restricted to membrane

Elecrrophoresrs 1997. 18. 127-135 Two-dimensional electrophorcsis of membrane proteins 133

Figure 4. 2-D electrophoresis of total keratinocyte proteins. One hundred pg of total proteins, extracted by CHAPS and urea from the keratinocyte cell line TR 146, were loaded onto the focusing gel. Detection by silver staining. (A) Carrier ampholyte focusing (gradient measured from pH 4.1 to 7.9), 12 000 Vh. (B) IPG focusing (pH 4-8), sample application by loading at the cathode. (C) IPG focusing (pH 4-8) sample application by loading at the anode. (D) IPG focusing (pH 4-8) sample applica- tion by reswelling. Streaking in the area of the major cytoskeletal proteins induces the impression of a higher yield in (A) to (C) compared with (D). However, no spots can be found in (B) or (C) that are absent in (D) while spots present in (D) but not in (B) or in (C) can be found (arrowheads). This indicates that sample application by rehydration is probably not the best method for all spots, but is probably the best compromise.

proteins. For example, enzyme visualization in IPG gels is claimed to retain sharp bands even after long incuba- tions [12], indicating that the focused proteins interact

with the matrix, which prevents free diffusion as found in carrier ampholyte-based focusing. This interaction has been demonstrated [37], but it has been postulated that

134 C. Adessi P I nl Electrophoresis 19Y7, 18, 127-135

it occurs via salt formation. In reality, this may be more complex, involving both salt formation and hydrophobic interactions. While the former are easily broken in the buffers used to equilibrate the IPG gels in 2-D electro- phoresis, the latter are known to be much stronger and difficult to dissociate, even with detergents. It has been observed that addition of urea to the equilibration buffer is beneficial [36], and this has been attributed to decreas- ing diffusion. This benefit may rather be due to a de- crease in the hydrophobic interactions between the focused proteins and the matrix by the chaotrope. To alleviate these problems of poor solubility, we tried to improve protein solubility by adding more carrier ampholytes to the gels, as recommended earlier [ 13, 351, without any improvement. A prolonged equilibration time, or different temperatures of the sealing agarose for better protein solubilization with SDS also did not bring any improvement.

In conclusion, the strength of these hydrophobic interac- tions might well be a clue to the problems encountered in 2-D electrophoresis with IPGs. In most cases, the interactions between the focused proteins and the gels are sufficiently weak to be broken by the equilibration mixture, and adequate transfers from the IPG to the SDS gel can be achieved. However, in the case of more hydrophobic proteins, as exemplified by membrane pro- teins, the transfer is incomplete due to stronger interac- tions between the focused proteins and the IPG gel matrix. Attempts to demonstrate incomplete elution by silver staining of the IPG strips, before and after the second-aimensional SDS-electrophoresis, were unsuc- cessful because the soft IPG gels (3.2%T) did not with- stand the silver staining process and detached from the Gel-Bond support during fixation. The absence of ver- tical streaking indicates that elution requires a high SDS- concentrations, present in the salt-SDS moving boun- dary. Once this front has left the IPG strip, the remain- ing proteins are exposed to 0.1% SDS only, which seems inadequate for elution. As a result, the eluted protein enters the SDS gel, forming a regular spot, while the residual protein in the IPG strip is not transferred to the SDS gel, with potential formation of vertical streaks.

Efficacious protein transfer appears to depend on insolu- bilization on the IPG matrix, at the isoelectric point, and mobilization by the equilibration solution. The elution power of the equilibration solution (which depends on the concentration of the compounds present and the duration of the equilibration step) can be taken as con- stant. Insolubilization of the protein will depend both on the concentration and the nature of the protein. Conse- quently, elution will vary widely from one protein to an- other, and, for a given protein, will decrease when the amount increases. This explains why the phenomenon is so prominent for high protein loadings (e.g. Fig. 3) , but can remain unnoticed at low analytical loadings [13]. In addition, the former explains the results obtained for complex samples, such as vesicles or envelope fragments. In such samples, which contain both soluble proteins (e .g . the luminal proteins of the vesicles) and membrane- anchored proteins, the soluble proteins become overre- presented in the IPG 2-D pattern; this was observed on

extreme cases, if the sample contains a variety of soluble or weakly hydrophobic proteins, the patterns can appear good but poorly representative of the real protein com- position of the sample. This current status should, however, not be considered definitive proof of incompa- tibility between hydrophobic proteins and IPG gels. As the problems now seem to be identified, it is likely that further alterations in the interfacing solutions between the IPG and the SDS-dimension will bring a sol.ution.

We are deeply indebted to Mathilde VinCon and Jerome Garin .for microsequence analyses.

Received October 26, 1995

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