To get the best performance from your PROTEAN II xi 3 Cell, familiarize yourself with the components by assembling and disassembling the cell before using it.
Central Cooling Core
The central cooling core provides the cooling capability which prevents thermal band distortion during electrophoretic separations. The cooling core can be connected to any circulating cooling source. The serpentine flow pattern assures even heat distribution over the entire gel area. An ethylene glycol:water (20:80) solution is recommended as coolant. Other coolants may damage the plastic during extended exposure. If a circulating bath is not available, the core can be connected to a tap water line, or simply filled with 1.8 liters of coolant and plugged to act as a heat sink during electrophoresis.
The central cooling core has a raised upper electrode which is housed in a protective casing, and the lower electrode is recessed to prevent accidental damage.
The sandwich clamps consist of a single screw mechanism which makes assembly, alignment, and disassembly of the gel sandwich an effortless task. The clamps exert an even pressure over the entire length of the glass plates, providing a leak-proof seal and preventing plate damage due to uneven pressure. Each pair of clamps consists of a left clamp and a right clamp. The sandwich clamps can accommodate up to two 1.5 mm thick gels.
The casting stand is separate from the PROTEAN II xi cell so that two gel sandwiches can be cast while others are being run. The one-piece molded unit has two casting slots and one alignment slot. Gel sandwiches are assembled, aligned, and cammed into the stand quickly, without the use of grease.
Double gels may also be cast. Two 1.5 mm (or thinner) gels may be cast in each sandwich, so that up to four 1.5 mm gels can be run at once.
Upper Buffer Chamber
The completed gel sandwich attaches to the central cooling core so that the outer plate of the sandwich forms the side of the upper buffer chamber. The inner plate is clamped against a rubber gasket on the central cooling core to provide a greaseless, leak-free seal or the upper buffer. Each sandwich forms one side of the cathode chamber. Tube gel adaptors also snap onto the central cooling core to form the upper buffer chamber walls (one adaptor per side). If only one gel is to be run, an upper buffer dam is attached to the core to form the complete upper buffer chamber. The upper buffer chamber will hold approximately 350 ml of buffer when full.
Lower Buffer Chamber
The lower buffer chamber of the PROTEAN II xi cell encloses the unit and provides stability during electrophoresis. The molded unit requires a minimum buffer volume of only 1.1 liters for 20 cm plates, while providing excellent heat exchange through the central cooling core.
Combined with the lower buffer chamber, the lid acts to fully enclose the PROTEAN II xi cell during electrophoresis, thus providing electrical insulation. The lid cannot be removed without disconnecting the electrical circuit. It can be placed on the lower chamber in only one alignment, so that the anode and cathode connections cannot be accidentally reversed. The lid also holds the coiled electrical leads when not in use.
Tube Gel Adaptor
The tube gel adaptor clamps onto the central cooling core in one easy motion and provides a leak-proof seal for the upper buffer chamber at voltages up to 1,000 V (especially useful for 2-D applications). The molded construction produces a lightweight, yet durable adaptor unit, which has a gel tube locator at the bottom to hold the tubes in a vertical orientation.
Each adaptor can hold up to 8 tubes (from 1.0 mm ID to 6 mm ID); 16 tube gels can be run at once using two tube adaptors.
Note: The upper buffer dam may not be used opposite a tube gel adaptor.
The alignment card greatly simplifies sandwich assembly by keeping the spacers upright during sandwich alignment. A sandwich is assembled by placing two spacers on top of the large outer plate. The alignment card is placed between the two spacers, and the shorter inner plate is then placed on top. Following attachment of the clamps, the sandwich assembly is transferred to the alignment slot of the casting stand for final adjustments.
Assembling the Glass Plate Sandwiches
1. Assembling Single Sandwiches
Note: Instructions for assembling 16 cm and 20 cm sandwiches are identical, except, of course, for the lengths of the components. To insure proper alignment, make sure all plates and spacers are clean and dry before assembly. The PROTEAN II xi plate washer/holder simplifies glass plate washing and also makes an ideal storage system for clean, dry glass plates. Each plate holder will accommodate up to 8 PROTEAN II xi plates or up to 18 Mini-PROTEAN®II plates.
- Assemble the gel sandwich on a clean surface. Lay the long rectangular plate down first, then place two spacers of equal thickness along the long edges of the rectangular plate. Next, place a short plate on top of the spacers so that it is flush with one end of the long plate.
- Locate both the right and left sandwich clamps, and loosen the single screw of each by turning counterclockwise. Place each clamp by the appropriate side of the glass plate stack, with the locating arrows facing up and toward the glass plates.
- Grasp the whole glass plate sandwich firmly with your right hand. With your left hand guide the left clamp onto the sandwich so that the long and short plates fit the appropriate notches in the clamp. Tighten the single screw enough to hold plates in place.
- Place the right clamp on the right side of the plates, and tighten the clamp screw.
- Level the casting stand on a flat surface with the alignment slot facing you. Check to see that gaskets are clean and free of residual acrylamide to insure a good seal. Place a flat, grey, silicone gasket in each casting slot.
Note: Always use the alignment slot to properly orient the gel sandwich. Failure to use this slot for alignment can result in casting leaks while pouring the gel or buffer leaks during the run.
- Place the assembled gel sandwich in the alignment slot of the casting stand. Loosen the clamp screws, and allow the plates and spacers to align at the surface of the alignment slot. Insert a PROTEAN II xi alignment card between the two glass plates to keep the spacers upright while additional alignment adjustments are made. As an alternative, the alignment card can be positioned between the glass plates when the sandwich is first assembled as in step 1.
- Simultaneously push inward on both clamps at the locating arrows, and tighten both clamp screws just enough to hold the sandwich in place. Pushing inward on both clamps at a point below the locating arrows will insure that the spacers and glass plates are flush against the sides of the clamps.
- Loosen one clamp screw slightly. While pushing down on the spacer with one finger, tighten the clamp screw finger-tight with the other hand (see photo). This will insure proper sealing when solution is poured. Tighten the other clamp screw in the same manner. It is important to visually inspect the sandwich while it is in the alignment slot to insure that there are no gaps between the glass plates and the surface of the alignment slot.
- Remove the alignment card. Pull the gel sandwich from the alignment slot. Check that the plates and spacers are flush at the bottom. If not, realign the gel sandwich as in steps 6-8.
Note: The easiest way to check for proper alignment is to run a fingernail across the contact area between the glass plates and spacer. If your fingernail catches or drops as you move from plate to spacer to plate, you must realign the sandwich before proceeding to step 10.
- The cams on the casting stand should be handle up and pulled out. Place the aligned sandwich into one of the casting slots with the longer plate facing you (and the arrows on the clamp facing away from you). When the sandwich is placed correctly, push the cams in, and turn them 180° so that the handles of the cams point downward. The sandwiches are now ready for gel casting.
2. Assembling Multiple or “Double-up” Gel Sandwiches
Up to four gels can be run at one time by doubling up gel sandwiches (i.e., 2 gels/side). Double gels are assembled, aligned, and cammed in a manner very similar to that described for single gels.
- Lay down a long rectangular plate, two spacers, and a notched inner plate.
- Place two more spacers on top of the notched inner plate. Place the short inner glass plate on top of this set of spacers to form the complete double sandwich
- Apply the sandwich clamps as described in Section 3.1, steps 3-4. Insert two alignment cards between each of the sandwiches to keep the spacers upright during sandwich alignment. Align, and then cam the whole assembly into the casting stand. The sandwiches are now ready for gel casting.
Casting the Gels
1. Casting Discontinuous (Laemmli) Gels)
Discontinuous gels consist of a resolving or separating (lower) gel and a stacking (upper) gel. The stacking gel acts to concentrate large sample volumes, resulting in better band resolution than is possible using the same volumes on a gel without a stack. Molecules are then completely separated in the resolving gel. The most popular discontinuous buffer system is that of Laemmli. (.)
- Prepare the monomer solution by combining all reagents except ammonium persulfate (APS) and TEMED (see ). De-aerate the solution under vacuum for at least 15 minutes.
- Place a comb completely into the assembled gel sandwich. With a marker pen, place a mark on the glass plate 1-2 cm below the teeth of the comb. This will be the level to which the separating gel is poured. Remove the comb.
- Add APS and TEMED to the deaerated monomer solution, and pour the solution to the mark, using a glass pipet and bulb. The easiest way to pour is to flow the solution down the middle of the outside plate of the gel sandwich. Another way to pour is to flow the solution down the side of one of the spacers. An alternative method is to use a syringe and Tygon tubing to load the solution from near the bottom of the sandwich. In all cases, pour the solution smoothly to prevent it from mixing with air.
- Immediately overlay the monomer solution with water, water-saturated isobutanol, or t-amyl alcohol. The advantage of using isobutanol or t-amyl alcohol is that the overlay solution can be applied rapidly with a Pasteur pipet and bulb because very little mixing will occur. If water is used to overlay, it must be done using a needle and syringe, using a steady, even rate of delivery to prevent mixing.
- Allow the gel to polymerize for 45 minutes to 1 hour. Rinse off the overlay solution completely with distilled water. This is especially important with alcohol overlays. Do not allow alcohols to remain on the gels more than 1 hour, or dehydration of the top of the gel will occur.
Note: It is sometimes convenient to cast the separating portion of a discontinuous gel the afternoon before casting the stacking gel and running the gel. If the stacking gel is to be cast the following day, place approximately 5 ml of 1:4 diluted stock solution B (see ) on top of each separating gel after rinsing with deionized water. This will prevent dehydration of the separating gel during overnight storage at room temperature.
- Prepare the stacking gel monomer solution. Combine all reagents except APS and TEMED, and deaerate under vacuum at least 15 minutes.
- Dry the area above the separating gel completely with filter paper before pouring the stacking gel.
- Place a comb in the gel sandwich, and tilt it so that the teeth are at a slight (~10°) angle. This will prevent air from being trapped under the comb teeth while pouring the monomer solutions.
- Add APS and TEMED to the degassed monomer solution, and pour the solution down the spacer nearest the upturned side of the comb. Pour until all the teeth have been covered by solution. Then properly align the comb in the sandwich, and add monomer to fill completely.
Generally, an overlay solution is not necessary for polymerization when a comb is in place.
- Allow the gel to polymerize 30-45 minutes. Remove the comb by pulling it straight up slowly and gently.
- Rinse the wells completely with distilled water. The gels are now ready to be attached to the central cooling core, the sample loaded, and the gels run.
2. Casting Continuous Gels
Continuous gels are ones in which the entire gel is of one composition. This type of gel is often used in non-denaturing (native) buffer systems.
- Prepare the monomer solution. Combine all reagents except APS and TEMED. Degas under vacuum for at least 15 minutes.
- Place a comb in the glass sandwich so that the teeth are tilted at approximately a 10° angle.
- Add APS and TEMED to the degassed monomer solution, and use a pipet and bulb to pour the solution down the spacer nearest to the upturned side of the comb. Pour until the bottoms of all the teeth are covered. Then adjust the comb to its proper position. Add monomer solution to fill the sandwich completely. No overlay solution is necessary.
- Let the gel polymerize for 45 minutes to 1 hour. The gel is now ready to load and run. Remove the comb, and rinse thoroughly with distilled water.
3. Casting Gradient Gels
Polyacrylamide concentration gradients, made with an external gradient former (Model 385, catalog number 165-2000), can be introduced into the PROTEAN II xi gel sandwich assembly either from the bottom or from the top. A peristaltic pump is required for introduction from the bottom. Introduction from the top can be done by gravity flow or with a peristaltic pump. Follow the gradient former instructions for formulating the gradient. If gradients are pumped into the gel sandwich from the bottom, the low density solution (low percent monomer) must enter first. If the gradient enters from the top, the high density solution (high percent monomer) enters first.
Pouring Gradient Gels from the Bottom
- After assembling the gel sandwich as described in Section 4, attach the gradient former tubing to a gradient pouring needle (catalog number 165-2007).
- Cam the gel sandwich to the casting stand, turn the casting stand on its side, locate the bottom filling ports directly under the space in the sandwich, and insert the bottom-fill needle through the rubber gasket, so that it protrudes about 2 mm into the sandwich. Make sure the needle opening faces one of the glass plates.
- Stand the casting stand up on a level surface, add APS and TEMED to the monomer solutions, and begin pumping the gradient monomer solution.
- Once the gradient is poured (this should take no more than 10 minutes from the time the initiators are added to the first monomer solution), withdraw the needle from the gasket, and immediately clean the gradient former, tubing, and needle by pumping distilled water through them.
- Overlay the monomer solution (see Section 4.1), or insert the comb (see Section 4.2), depending on whether the gel is discontinuous or continuous, respectively.
Pouring Gradient Gels by Top-Filling
- Attach the gradient maker tubing to a needle (catalog number 165-1943), a long cannula, or a piece of tubing that will fit between the glass plates of the gel sandwich.
- Insert the needle, cannula, or tubing as far as possible into the sandwich, and center it.
- Add APS and TEMED to the monomer solution, and begin pumping the gradient. As the monomer level in the sandwich approaches the tip of the needle, withdraw the needle slowly, so that it always stays above the monomer level.
- Immediately rinse any remaining monomer out of the needle by pumping water through the gradient former and out the needle.
- Overlay the gel, or insert the comb, as outlined in Sections 4.1 and 4.2, respectively.
4. Casting Agarose Gels
Agarose gels in vertical apparatus may slide down between the glass plates. To prevent this, one of the two plates used to form the agarose gel sandwich should be a frosted inner plate (catalog number 165-1825 for 16 cm gels and catalog number 165-1826 for 20 cm gels).
- Assemble the glass plate sandwich as outlined in Section 3, using one frosted plate and one regular clear plate. The frosted plate should be the shorter inner plate (the plate on the inside during running). This will allow visualization of the tracking dye during the run. Make sure that the frosted side of the plate is on the inside of the gel sandwich.
- Place the sandwich assembly in a warm air incubator (50 °C) for 5-10 minutes before casting the gel. This will prevent premature gelling of the agarose.
- Cam the warm assembly to the casting stand.
- Immediately pour the molten (55 to 60 °C) agarose. One convenient method of pouring is to tilt the sandwich assembly and pour agarose directly down the long rectangular glass plate.
- Insert the prewarmed comb carefully.
- Allow the agarose to cool to ambient temperature before use.
- Remove the comb very slowly to avoid tearing the gel.
Note: There are some special tricks to properly remove a comb from a vertical agarose gel. The most important point is to introduce water or buffer under the comb while it is being removed. To introduce buffer or water, use a squirt bottle or a needle and syringe to force fluid under the teeth of the comb while it is slowly removed from the gel. Another option is to insert the comb only partway into the gel. This can easily be done with the aid of comb conversion screws (catalog number 165-1859). The three standard screws on the comb are replaced with the three large head comb conversion screws, with the protruding head of the conversion screws resting on top of the longer outer glass plate. The well depth of the comb is limited to 10 mm instead of the standard 25 mm well depth.
Assembling the Upper Buffer Chamber
- Lay the central cooling core down flat on a lab bench. Make sure the core caps are in place on the cooling core ports.
- Seat the white U-shaped gasket onto the core with the flat (non-stepped) side down.
Note: To help insure a good upper buffer seal with your gaskets for the PROTEAN II xi cell, lubricate the entire front of the gaskets (the shaded portion) with water or upper buffer prior to attaching the gel sandwich to the central cooling core. This will allow the glass plate sandwich to slide onto the gasket properly.
- After the gel is cast and the comb is removed (if applicable), release the gel sandwich from the casting stand by turning the cams to the “up” position and pulling them outward. Pull the gel sandwich straight out of the stand.
- With the short glass plate facing the cooling core (and the locating decal on the clamps facing the core), position the gel sandwich so that the grooves in the upper portion of the clamps are fitted onto the locating pins on the central cooling core.* The gel sandwich should be positioned at an angle of 20° with the cooling core. Keeping this angle to a minimum and lubricating the gasket will prevent distortion of the gasket while the sandwich slides into place.
*Note: The locating pins on the central cooling core must be tightly secured in place to insure optimal pressure during operation. If these pins are vibrated loose during transport or repeated use, the decrease in pressure on the clamp can be enough to allow the cell to leak. If the pins are loose, they can be gently tightened using a coin or screwdriver.
- With your fingers below the latch on the cooling core and your thumbs resting on the clamps, gently push the gel sandwich onto the cooling core with one simple motion. The upper edge of the short inner glass plate should be butted against the notches of the U-shaped gasket, and the tabs of each clamp should be held securely against the latch assemblies on both sides of the cooling core.
- Turn the central cooling core to its other side, and repeat steps 1 through 5 to attach the second gel sandwich to it.
Note: When the gel sandwich has been properly installed, the shorter inside glass plate will be forced against the notch in the U-shaped gasket to create a leak-proof seal. Always inspect the contact between the gasket and glass plate to make sure the glass plate is butted against the notch in the gasket and is not resting on top of or below this notch. Improper installation of the gel sandwich can result in buffer leakage during the run. As a standard procedure, pour buffer into the upper buffer compartment, and check for buffer leaks prior to a run. In addition, we recommend marking the level of the upper buffer on the glass plates prior to electrophoresis. Checking this level after 1-2 hours will show whether a slow leak is occurring. This is especially important when the electrophoresis cell is being used for overnight experiments.
Use of the Buffer Dam
If only one gel is to be run, the acrylic buffer dam must be attached to the cooling core on the other side to form the complete upper buffer chamber. Position the acrylic plate between two clamps by matching the notches on the clamps to the notches on the acrylic plate (as in Section 3.1, steps 2-3), and then slide the dam up each clamp as far as possible. No further alignment is necessary. The acrylic buffer dam fits both 16 and 20 cm clamps.
Sample loading can be done in one of three ways. The most common method is to load liquid samples into wells formed by casting a gel with a well-forming comb. The second method uses the entire gel surface as a single well for liquid samples. The third method involves placing a tube gel or gel strip over the entire gel surface, as in 2-D procedures.
Loading the Sample Wells
- Prepare 1.5 liters of electrode buffer. Set aside 350 ml for the upper buffer chamber.
- Pour 325-350 ml of electrode buffer into the upper buffer chamber. At this point, check the integrity of the upper buffer seal. If the buffer appears to be leaking, remove the gel sandwich assemblies, re-lubricate the gasket, and then re-attach the gel sandwich assemblies.
- Place the remainder of the electrode buffer into the lower buffer chamber. Lower the central cooling core into the lower buffer chamber at a slight angle to prevent air entrapment under the gel sandwich. A few, isolated bubbles under the gel will not affect the run. With 16 cm plates, it is necessary to dilute the lower buffer with distilled water to a level of 1 cm above the bottom of the gel plates. Be sure to mix the lower buffer well with a stir bar on a magnetic stirrer.
Note: Dilution of the lower buffer by up to 1:2 with distilled H2O will have no detrimental effect on resolution. Dilution of the upper buffer is not recommended.
- Load the samples into the wells under the electrode buffer with a Hamilton syringe. Insert the syringe to about 1-2 mm from the well bottom before delivery. Disposable pieces of plastic tubing may be attached to the syringe to eliminate the need for rinsing the syringe between samples.
Note: The sample buffer must contain either 10% sucrose or glycerol in order to underlay the sample in the well without mixing.
- An easier method of sample loading is to use an Eppendorf-like pipettor and disposable tips. To accomplish this successfully, use the optional beveled short plate (catalog number 165-1827 for 16 cm and 165-1828 for 20 cm) so that you can insert the pipet tip further into the well before sample delivery. This will prevent inter-well mixing of samples. Or, use the Bio-Rad Prot-Elec tips (catalog number 223-9915 or 223-9917), which are designed for loading samples into wells.
Loading a Single Sample Per Gel
In this procedure, a gel is cast without using a comb, forming a flat gel surface. This gel is cast with an overlay solution. This type of sample application can be used for preparative purposes, but it is most often used in blotting applications. After electrophoresis, the sample is transferred electrophoretically to a membrane, which then can be cut into several identical strips for analysis. Follow the instructions for casting the separating portion of a discontinuous gel (Section 4.1), except pour the gel nearly to the top of the sandwich. Allow just enough room for sample loading. (A stacking gel may also be added to this type of gel.)
- Prepare electrode buffer, and add to lower reservoir as in Section 6.1. Place the central cooling core in the lower chamber, and add electrode buffer to the upper reservoir chamber.
- The sample may be loaded with an Eppendorf-type pipettor, with a needle and syringe, or with a Hamilton syringe. Start at one end of the gel, and deliver the sample gently and evenly over the entire length of the gel. Layer the sample as closely as possible (1-2 mm) to the surface of the gel.
Gels as Samples
All two-dimensional techniques involve this variation of sample loading. Either a cylindrical gel or a strip of a slab gel may be placed on top of a slab gel for separation into a second dimension.
Running the Gel
- Place the lid on top of the lower buffer chamber to enclose the PROTEAN II xi cell fully. Note that the lid can be placed only in one orientation, so that the anode and cathode connections cannot be reversed.
- Attach the electrical leads to a suitable power supply, with the proper polarity (this connection could accidently be reversed).
- Apply power to the PROTEAN II xi cell and begin electrophoresis. As a safety precaution, always set voltage, current, and power limits when possible. The table below gives specific running conditions.
Tare recommended power conditions for optimal resolution with minimal distortion.
We recommend that gels be run under constant current conditions with an appropriate power supply, such as the PowerPac 3000 or Model 1000/500 power supply.
|Gel Thickness||Current Conditions (mA per gel)|
|Stacking Gel||Separating Gel|
Run time is between 4 and 5 hours, depending on length of gel. Under constant current conditions, voltage will gradually increase during the run. As a safety precaution, always set voltage, current, and power limits when possible.
- [bibliplug last_name=”Laemmli” year=”1970″]
- The BIORAD PROTEAN II xi cell Instruction Manual