Experiments on bidirectional electrophoresis techniques in plant proteomics
Bidirectional gel electrophoresis and mass spectrometry for protein identification using fixed pH gradients (IPGs ) are currently the main techniques for proteomic analysis. Bidirectional electrophoresis can be used to separate very complex mixtures of proteins based on isoelectric point, molecular mass, solubility, and relative abundance and to obtain a complete protein profile. The source of this experiment is the "Guide to Plant Proteomics Experiments" [France] H. Tillmant, M. Zivi, C. Damerweil, V. Mitchen, eds.
Operation method
Bidirectional electrophoresis in plant proteomics
Materials and Instruments
Urea Lysis Solution IPG Dry Strip Hydration Solution IPG Strip Equilibration Solution SDS Gel Buffer Electrode Buffer Reservoir Acrylamide Methylenedioxybisacrylamide Solution Ammonium Persulfate Solution Agarose Solution Move 3.1 First direction: Isoelectric focusing in IPG adhesive strip (IPG-IEF) For more product details, please visit Aladdin Scientific website.
Isoelectric Focusing Instrument IPGphor Multi-Vertical SDS Electrophoresis Instrument
The first direction of bi-directional electrophoresis with IPG (IPG-Dalt), Isoelectric Focusing (IEF), is performed using individual, 3 mm wide, IPG adhesive strips attached to a GelBond PAG support membrane. Pre-made Immobiline dry glue sticks are available in 7 cm, 11 cm, 18 cm, and/or 24 cm lengths for virtually any desired pH range. For example, IPG 3-10 and IPG 3-11 for wide pH ranges, IPG 4-7 or IPG 6-9 for medium pH ranges, and IPG 4-5 or IPG 4.5-5.5 for narrow pH ranges (e.g., GE Healthcare, Bio-Rad, Sigma-Aldrich, Serva). Alternatively, lab-made IPG dry glue sticks can be used. For details of IPG instillation, interested readers may refer to the previously published method [21].
Prior to IEF, the dry IPG strips are hydrated and then placed on the cooling tray of a horizontal isoelectric focuser [13, 22]. Recently, IPCMEF has been simplified and accelerated by the use of an integral system called the IPGphor, which features hydration of individual IPG dry strips in a holding tank or sample loading at the same time as hydration, optional cup loading, and subsequent IEF at high voltage (8000 V), which does not require manipulation of the IPG dry strips after they have been placed in the ceramic holding tank.
When using IPG tape with a pH greater than 9, DTT depletion can cause the level of the alkaline end to drift. To eliminate drift, reducing agents such as DTT can be replaced with hydroxyethyl dithioate (HED) (DeStreak, GE Healthcare) in the hydration solution of the tape to stabilize the disulfide bonds in the cysteine. In addition to removing drift, the use of HED makes the spots clearer and improves reproducibility [23] . Furthermore, isoelectric focusing of extremely basic proteins with isoelectric points greater than 10, such as ribosomes and nucleoproteins, is greatly simplified by using IPGphor.
1. Hydration and Sampling of IPG Gel Strips
Prior to IEF, dry IPG adhesive strips must be hydrated to their original thickness of 0.5 mm in a hydration cassette or tray. dry IPG adhesive strips can be hydrated with a hydration solution that has the sample dissolved in it (hydration loading) [24] or hydrated with a hydration solution that does not contain the sample, and then loaded with a cup loading method. Although hydration sampling is more convenient, it is not recommended when the sample contains proteins with high molecular mass (< 100 kDa), extreme basicity, and/or extreme hydrophobicity. This is because these proteins do not readily enter the gel. The reason for this may be the hydrophobic interactions between the protein and the walls of the hydration disk or the limiting effect of the pore size of the gel matrix. The latter phenomenon is particularly noticeable if the sample volume significantly exceeds the volume expected to be reached after hydration of the IPG gel. High molecular mass proteins are more likely to remain in the excess hydration solution than to enter the IEF gel. Cross-contamination is also a problem, so hydration trays must be thoroughly cleaned between experiments. In conclusion, hydration upsampling is not as reliable as cup upsampling, especially in quantitative experiments.
For cup sampling, the dry IPG tape is still hydrated with the hydration solution. after the IPG tape is hydrated, the sample (20-100 μl) dissolved in lysis buffer is added to a disposable plastic or silicone cup on the surface of the IPG tape (see Note 3). The best results are obtained when the sample is added at the pH extremes, i.e., near the cathode or anode. In most cases it is better to sample near the anode than near the cathode. When using an alkaline pH gradient, such as IPG 6-12 or 9-12, all samples must be taken from the anode [15-17] .
The amount of protein sampled on a single IPG strip is determined by several factors. As a rule of thumb, the longer the separation distance (i.e., the longer the strip), the narrower the pH gradient range, and the less sensitive the protein assay, the more protein will be required. 50-100 μg is the recommended sample size for analytical (silver-stained) bi-directional electrophoresis gels of 20 cm X 20 cm, and as much as 1 mg ( or more) is recommended for micropreparation gels. When using IPG gels with a very narrow pH range, we strongly recommend using only pre-separated samples [ 25 ].
1 ) Hydration of dry IPG gels in a hydration tray
( 1 ) If sampling is done at the same time as hydration [24], dissolve cell lysate or tissue samples (5-10 mg protein/ml) directly into the appropriate amount of IPG dry gel strip hydration solution. For hydration of 180 mm long, 3 mm wide strips, 350 μl of the above solution can be pipetted into the grooves in the IPG dry strip hydration tray (Figure 13-2). If the IPG tape is longer or shorter, the volume of hydration solution should be converted accordingly (e.g., 450 μl for a 240 mm long dry IPG tape).
( 2 ) Remove the protective film from the surface of the IPG tape and place the IPG tape into the groove, adhesive side down, avoiding air bubbles. Then cover the IPG adhesive strip with IPG dry adhesive strip covering oil (to avoid drying out the adhesive strip during hydration). The tape should be able to move and not stick to the hydration tray before covering. After covering, hydrate the IPG tape at approximately 20°C overnight. High temperatures (> 37°C) risk carbamylation, while low temperatures (< 15°C) cause urea to crystallize in the IPG glue.
( 3 ) If cup sampling is used, dry IPG glue sticks are hydrated overnight in the hydration tray with no sample in the hydration solution as described in step 2 above.
2) Hydrate strips in the IPGphor holding tank and sample at the same time.
( 1 ) Solubilize the proteins with Sample Lysis Buffer (i.e., Urea/Thiourea Lysis Buffer) and dilute the extracts with IPG Dry Strip Hydration Buffer.
( 2 ) Place the desired number of IPGphor Holding Troughs (Figure 13-2) in the cooling tray/electrode area of the IPGphor.
( 3) Aspirate 350 μl of sample hydration buffer (when using 180 mm long IPG tape) into the bottom of the holding tank.
( 4 ) Remove the protective film from the IPG tape and slowly lower the IPG tape (adhesive side down) into the hydration solution. Avoid air bubbles. The tape should still be able to move and not stick to the hydration tray. Cover the IPG tape with 1 to 2 ml of IPG Dry Tape Covering Oil and close the plastic lid. Press down on the support block under the lid to ensure that the IPG tape maintains good contact with the electrode during hydration. Apply a low voltage (30-50 V) during hydration to allow better access of high molecular mass proteins into the glue [15, 28]. 
2. Perform IPG-IEF (Multiphor ll Unit) on a plate apparatus.
Hydrated IPG gels can be placed directly on the cooling plate of the IEF instrument if the following conditions are met: (1) If the run time does not exceed 12 h (which often occurs with wide or medium pH range IPGs, e.g., IPGs 3 to 10 or 4 to 7). (ii) If the pH gradient does not exceed pH 10.0. (iii) If a very small volume of sample (20 μl) is added by cup sampling only [3, 22]. Large volumes (up to 100 μl) are easier to sample using the cups included in the Immobiline Dry Strip Kit. When performing an IEF with the Dry Tape Kit, the IPG tape can be covered with silicone oil or dry tape covering oil. When the sample is extremely basic (pH > 10.0) proteins or micro-preparative electrophoresis with a narrow pH gradient (pH range < 1 unit) must be covered with a covering oil, etc. When the pH range is wide and the pH gradient does not exceed pH 10.0 (e.g., IPG 4-7 or 3-10), the Dry Strip Kit can be used without a covering oil.
( 1 ) Place the cooling tray into the Multiphor ll electrophoresis apparatus. Pipette 3~4 ml of kerosene or IPG Dry Strip Covering Oil onto the cooling tray, and then place the Immobiline Dry Strip Tray onto the cooling tray (Fig. 13-2). Avoid air bubbles between the glue strip tray and the cooling tray.
( 2 ) Connect the electrodes on the tape tray to the Multiphor II electrophoresis instrument.
( 3 ) Fill the tape tray with about 10 ml of dry IPG tape cover oil or silicone oil and place the corrugated Immobiline tape scale on top of the cover oil in the tape tray.
( 4 ) After the IPG Tape has been hydrated (see 13. 3.1 Section 1.1), remove the hydrated IPG Tape from the hydration tray with clean tweezers. Rinse the strip with deionized water, then place the strip between two layers of moistened filter paper and use the filter paper to absorb the excess covering oil from the strip for a few seconds. This prevents urea from crystallizing on the glue surface during the IEF process. Transfer the hydrated IPG tape (with the glue side up and the acid end aligned with the anode) into the tank and close to the scale. Arrange the IPG strips so that the edges of the strips facing the anode are neatly aligned.
( 5 ) Cut two IEF electrode strips (GE Healthcare) or strips of filter paper (e.g., MN440, Macherey Nagel, Germany) 2 mm thick, the length of which corresponds to the width of all the IPG adhesive strips aligned in the tape tray. The electrode strips were soaked in deionized water, excess liquid was removed with filter paper, and the moistened IEF electrode strips were placed near the cathode and anode of the neatly arranged adhesive strips.
( 6 ) Place the electrode on the IEF electrode strip and gently press down on the electrode.
( 7 ) If the sample has entered the strip while hydrated, cover the strip with approximately 80 ml of dry strip covering oil and go to step 12. If using the cup sampling method, continue to step 8.
( 8 ) Place the sample cup into the cup slot, but avoid contact with the surface of the tape. Also, make sure there is a few millimeters between the sample cup and the anode (or cathode, if using cathodic sampling).
( 9 ) Move the upper sample cups to a suitable position, one upper sample cup on each adhesive strip, and then gently press the upper sample cups downward. The sample cups should be in close contact with the adhesive strips but should not damage the surface of the adhesive strips.
( 10 ) After placing the sample cups, fill the tape tray with approximately 80 ml of dry tape cover oil to completely cover the IPG tape. If the covering oil leaks into the sample cup, suck out the covering oil, readjust the sample cup and check again for leaks. Add a few drops of Dry Strip Cover Oil to each sample cup. If a wide pH gradient of 3 to 10 is used for IEF, the addition of Cover Oil can be omitted.
( 11 ) Aspirate the sample and add to the bottom of the sample cup under the Cover Oil. Check again for leaks.
( 12 ) Close the lid of the isoelectric focusing box and start electrophoresis according to the parameters in Table 13-1. The voltage should be limited to 150 V, 300 V, and 600 V for the first few hours to allow better access of the sample into the gel, then electrophoresis to steady state at a maximum voltage of 3500 V (see Note 4). The current is limited to 0.05 mA/IPG strip. The optimal focusing temperature is 20°C [ 26] .
( 13 ) When the IEF is complete, remove the electrodes, sample cup tank, and IEF electrode strip from the tape tray. Use clean tweezers to remove the IPG tape from the tape tray. If the IPG strips are not to be used immediately for second direction electrophoresis and/or for further study, they can be stored at -70°C between two layers of plastic film for several months. 
3. Using the IPGphor Electrophoresis Apparatus for IPG-IEF
IPG-IEF in bi-directional electrophoresis can be simplified by using a total system called the IPGphor (GE Healthcare and more recently Bio-Rad has developed a similar system). The IPGphor consists of a Peltier element with precise temperature control (at 19.5-20.5°C) and a programmable power supply. The heart of the instrument is a thin trough made of alumina ceramic of various lengths (7 cm, 11 cm, 13 cm, 18 cm, or 24 cm), called a holding tank, in which IPG adhesive strips can be hydrated and sampled simultaneously, and then subjected to an IEF. Once the strips have been placed in the tank, the subsequent steps do not require any further manipulation of the strips (Fig. 13-2). The IPGphor is programmable and can store up to 10 different programs. The instrument supports delayed start, which allows the user to hydrate the strips with a sample-containing hydration solution in the holding tank in the afternoon, and then the IEF is automatically initiated in the evening and completed the next morning.
When separating proteins in the alkaline pH range (> pH 10.0), the cup sampling method of individually sampling different hydrated IPG strips yields much better separations than the hydration sampling method. Samples can be cupped using either the Special Cup Sampling ("Universal") IPGphor Holding Tank or the Combined Cup Sampling Holding Tank ("Combined") (Figure 13-2). Cup loading allows for up to 100 μl of sample (see Note 3). The gel bath platform is temperature adjustable and the gel bath can be connected to a power supply. In addition to ease of operation, another advantage of the IPGphor is the short focusing time. This is due to the high voltage (up to 8000 V) at which the IPGphor can be used for IEF.
Typical operating conditions for IEF with the IPGphor are shown in Table 13-2 and Table 13-3. As pointed out earlier, in order to allow the high molecular mass proteins to enter the polyacrylamide gel better, a low voltage (30-50 V) should be applied to both ends of the gel during hydration, otherwise it will cause difficulties in the hydration of the samples [15, 28]. The voltage should then be gradually increased to 8000 V. If the separation length of the IPG tape is < 11 cm, the voltage should be limited to 5000 V. The voltage should be increased to 8000 V. If the separation length is less than 11 cm, the voltage should be limited to 5000 V. For best results when separating samples with a high salt content or when using narrow pH intervals, a moistened piece of filter paper (4 mm x 4 mm) can be placed between the electrode and the IPG tape before the voltage is raised to 8000 V (see note 4). When the IEF is finished, store the IPG tape as described in Section 13.3.1, step 13. 
1) Hydration Sampling IEF
( 1 ) Place the desired number of holding slots on the cooling tray or on the electrode area of the IPGphor (Figure 13-2). Pipette an appropriate amount (e.g., 350 μl for a 180 mm long IPG strip) of the sample-containing IPG strip hydration solution into the holding tank, place the IPG strip adhesive side down into the hydration solution, and cover with IPG strip covering oil. The method is described in 13. 3.1 Section 1. 2).
( 2 ) Set up the IPGphor ( desired hydration time, volt-hours, voltage gradient).
( 3 ) After the IPG tape is hydrated (minimum 6 h required, usually overnight), start the IEF with the parameters listed in Table 13-2.
( 4 ) Upon completion of the IEF, IPG glues that are not immediately subjected to second direction electrophoresis are stored at -70°C between two layers of plastic film.
2) Cup Sample IEF
( 1 ) Hydrate dry IPG strips in a hydration tray with a sample-free hydration solution After the IPG strips have been hydrated, remove the hydrated IPG strips from the hydration tray or box with clean tweezers. Rinse the strip with deionized water, then place the strip between two layers of moistened filter paper and use the filter paper to absorb the excess covering oil from the strip for a few seconds. This will prevent the urea from crystallizing on the surface of the gel during the IEF process. See 13. 3.1 Section 2.
( 2 ) Place the desired number of cup sample holding slots on the electrode zone of the cooling tray or IPGphor, making sure that the tip (anode) of the holding slot is connected to the anode zone of the electrode zone. Instead of using individual glue holders, a combination of glue holders may be used.
( 3 ) Place the hydrated IPG adhesive strip into the cup holder (or composite holder) with the adhesive side up and the tip of the adhesive strip (acid end) aligned with the anode. Ensure that the cathode of the IPG adhesive strip is approximately 1.5 cm from the end of the tank and is connected to the electrode by the electrode wire.
( 4 ) Wet two electrode filter paper spacers (size: 4 mmX10 mm) with deionized water, absorb excess deionized water with filter paper, and place the wetted electrode filter paper spacers on the surface of the IPG tape between the anode and cathode electrodes and the IPG tape. If necessary (e.g. when the sample has a high salt content), the filter paper spacer can be replaced with a new one after a few hours.
( 5 ) Place the active electrode on the electrode filter paper spacer. Clamp the electrode so that it presses firmly against the electrode filter paper spacer.
( 6 ) Place the removable sample cup near the anode or cathode and gently press the cup against the surface of the IPG tape. The sample cup should be in close contact with the IPG tape without damaging the tape surface.
( 7 ) To ensure that the sample cup does not leak, add 100 μl of IPG cover oil to the cup. If leakage is detected, remove the cover oil and blot the cover oil with a piece of tissue paper, then reposition the cup. Check again for leakage. Blot out the covering oil before sampling.
( 8 ) Cover each strip with 2 to 4 ml of IPG Strip Covering Oil (it is recommended that silicone oil or kerosene not be used as a substitute for IPG Strip Covering Oil). In case the covering oil leaks into the sample cup, reposition the cup and blot the covering oil from the cup with a piece of cotton paper. Check again for leakage and add the sample (20-100 μl) to the sample cup.
( 9 ) Set up the instrument (hours of volts required, voltage gradient, temperature, etc.) and perform the IEF with the parameters recommended in Tables 13-2 and 13-3. omit the low voltage hydration step recommended in Table 13-2 for hydrated samples.
( 10 ) Upon completion of the IEF, proceed to equilibration or second direction IEF (SDS-PAGE) (see Section 13.3.3), or store IPG adhesive strips sandwiched between two layers of plastic film at -70°C for several months.
3.2 IPG Strip Equilibration
It is important to equilibrate the IPG adhesive strips so that the proteins separated in the strips interact adequately with the SDS prior to the second direction of separation (SDS-PAGE). Since the focused proteins are more tightly bound to the IPG gel than to the carrier ampholyte gel, a relatively long equilibration time (10-15 min), as well as urea and glycerol, are required to improve the transfer of proteins between the first and second directions. Among them, urea and glycerol can mitigate the effect of electroosmosis. The most commonly used procedure is to equilibrate IPG strips in a buffer originally proposed by Gorg et al [13] [ 50 mmol/L Tris-HCl (pH 8.8), 2% (m/V) SDS, 1% (m/V) DTT, 6 mol/L urea, and 30% (m/V) glycerol; Table 13-4] for 10-15 min, and then equilibrate the strips for 10-15 min in the buffer by replacing DTT in the buffer with urea and glycerol. The strips are then equilibrated in a buffer formed by replacing the DTT in the above buffer with 4% (m/V) iodoacetamide for a further 10-15 min. The latter step is used to alkylate the free DTT, which would otherwise migrate in the second direction to the SDS-PAGE gel, resulting in dot trailing. This phenomenon is observed after silver staining. More importantly, iodoacetamide alkylates sulfhydryl groups and prevents their redox action. We strongly recommend the use of this reduction/alkylation two-step procedure due to its ability to considerably simplify sample preparation for subsequent mass spectrometric identification (in-gel digestion of proteins). After equilibration, the IPG gel is placed on the surface of a second horizontal or vertical SDS-PAGE gel.
( 1 ) Dissolve 100 mg of DTT (Sigma-Aldrich) in 10 ml of equilibrium solution to prepare Equilibrium I. The equilibrium is then prepared in the same manner as the equilibrium solution.
a. Prepare 10 ml for each strip.
b. Place each focused strip into a test tube (250 mm long, 20 mm inner diameter) and add 10 ml of Equilibrant I to each test tube.
c. Seal the tubes with Pamfilm and shake them on a shaker for 15 min, then pour out the Equilibrant. A shorter equilibration time (10 min) may be used, but there is a risk that some of the proteins may not come out of the IPG tape when the sample goes into the SDS-PAGE gel. If this is the case, the IPG tape should be stained after it has been removed from the SDS gel to check that all the proteins have left the IPG tape.
( 2 ) Prepare Equilibrium II by dissolving 0.4 g of iodoacetamide (Sigma-Aldrich) into 10 ml of Equilibrium Solution.
a. Prepare 10 ml of each IPG strip.
b. Add 10 ml of Equilibrium II and 50 μl of bromophenol blue (Serva) solution as SDS-PAGE indicator to each strip and equilibrate for 15 min with gentle shaking again.
( 3 ) Pour out Equilibrium II and perform SDS-PAGE (see section 13.3.3). If performing SDS-PAGE in a horizontal electrophoresis tank (e.g., Muhiphor II ), briefly rinse the IPG strips with deionized water, then place the strips on one side of a piece of filter paper and wait a few minutes for excess equilibrium to be aspirated. If performing SDS-PAGE in a vertical electrophoresis tank (e.g., EttanDalt ), briefly rinse the IPG gel strip with electrode buffer. 
3.3 Second Direction: Multiplexed Vertical SDS-PAGE
SDS-PAGE can be performed on either horizontal or vertical systems [29] . Horizontal devices are suitable for precast gels (ExcelGel SDS; Amersham Biosciences/ GE Healthcare). Vertical systems, on the other hand, are used in electrophoresis where multiple gels are performed in parallel, especially in large-scale proteomic analyses. Such analyses often require several batches of second-direction SDS-PAGE electrophoresis to be performed simultaneously to achieve higher throughput and best reproducibility [31] .
1. Filling SDS gels
( 1 ) Filling plate (200 mm X 250 mm) consists of two 3 mm thick glass plates in the shape of a book. The two panes were connected by a hinge strip with two 1 mm thick edge strips between the panes. Fourteen panels were stacked vertically into the gluing mold of the Ettan Dah II. The hinge bar is stacked facing right and the panels are separated by plastic dividers (e.g. 0.05 mm thick polyester sheets).
( 2 ) Place the front plate of the gluing mold in place and screw on the nut (hand tight) (Figure 13-2).
( 3 ) Support a funnel about 30 cm above the top of the potting mold with a ring stand and connect it with a polyethylene tube (5 mm ID). The other end of the tube is connected to a metal connector on the side chamber on one side of the gluing mold.
( 4 ) Fill the side chamber with 100 ml of indicator.
( 5 ) Add TEMED and ammonia persulfate solution (Table 13-5) to the gel solution just prior to filling. When filling, pour the gel solution (830 ml) into the funnel. Avoid air bubbles in the tube. Do not fill the gel plate with acrylamide solution. This is due to the space (approximately 10 mm) required to secure the IPG strip to the top of the SDS gel with hot agarose.
( 6 ) When the solution has been filled, remove the tube from the side chamber interface. The level of the indicator in the side chamber will drop at this point.
( 7 ) Carefully add about 1 ml of Covering Solution to the top of each piece of adhesive to make the adhesive surface flat and smooth.
( 8 ) Allow the gels to polymerize at approximately 20°C for at least 3 h. Overnight polymerization is preferred for better reproducibility.
( 9 ) After polymerization of the gel, remove the front plate of the potting mold and carefully remove the gel plate from the mold. A razor blade may be used to separate the glue sheets. Remove the separators between the sheets.
( 10 ) Wash the glue boards with water to remove the acrylamide from the outer surface of the glue boards, and then drain the excess liquid from the top end of the glue. Since only 12 plates can be used at a time for electrophoresis, discard any undesirable plates, especially those of uneven thickness. This is usually the case for the outside of the plate.
( 11 ) If the polymerized gels are not to be used immediately, they can be wrapped in plastic and stored in the refrigerator (4°C). Store for up to 2 days. 
2. Multiplex SDS-PAGE using the Ettan Dalt II Vertical Electrophoresis Tank
( 1 ) Add 1875 ml of Electrode Buffer Reservoir and 5625 ml of deionized water to the lower tank of the Ettan Dalt II electrophoresis tank. Mix and turn on the cooler (25°C).
( 2 ) Place the DALT gel plate (with SDS gel inside) vertically in the gel plate rack in order to place the IPG tape.
( 3) Briefly rinse the equilibrated IPG Strips with Electrode Buffer (diluted 1:1 with water) and place the strips on top of the DALT Plate.
a. Using a thin spatula or ruler, push on the support film behind the IPG adhesive strip so that the strip enters the space between the two glass plates.
b. Add 2 ml of hot (75°C) agarose solution and continue to push the strip down onto the surface of the SDS adhesive until they are in close contact. avoid air bubbles between the IPG adhesive strip and the surface of the SDS adhesive.
c. If molecular mass standard proteins are to be added at the same time as electrophoresis, soak a piece of filter paper (2-4 mm2 ) with 5 μl of electrode buffer containing SDS standard proteins. After drying the filter paper, place it on the left or right side of the IPG adhesive strip.
d. The dried filter paper with the SDS Protein can be stored at -70°C in a microcentrifuge tube.
( 4 ) Allow the agarose to solidify for at least 5 min before placing the plates in the electrophoresis apparatus (see step 5). repeat the above steps for the remaining IPG film strips. Although it is not necessary to bury the strips in agarose, doing so ensures a tighter bond between the IPG glue and the top of the SDS gel.
( 5 ) Wet the outside of the gel plate by dipping it into the electrode buffer to make it easier to place in the electrophoresis tank. Insert the gel plate into the electrophoresis tank. If necessary, place an empty gel plate in an empty slot in the electrophoresis tank. Place the upper tank through the gel plate and add 2.5L of electrode buffer (1250 ml reservoir + 1250 ml deionized water) to it.
( 6 ) Close the safety cover of the electrophoresis tank and start the SDS-PAGE, starting with a run at 5 mA per gel (set up to 100 V) for about 2 h. Then run at 15 mA per gel (set up to 200 V) overnight for about 16 h, or higher current for a faster run (30 mA per gel for about 8 h).
( 7 ) Terminate electrophoresis when the bromophenol blue trace has migrated out of the lower end of the gel.
( 8 ) At the end of electrophoresis, carefully open the gel plate with a plastic spatula. Remove the agarose from the polyacrylamide gel with the spatula. Carefully peel the gel from the glass plate, carry it by its lower edge and place it in the box containing the fixative or staining solution.