Bio-Rad PROTEAN II XL Cell User Manual

PROTEAN

II xi Cell

PROTEAN II xi 2-D Cell

Instruction Manual

For Technical Service, call your local Bio-Rad office or in the U.S. call 1-800-424-6723

Note

To insure best performance from the PROTEAN® II xi cell, become fully acquainted with these operating instructions before using the cell to separate samples. Bio-Rad recommends that you first read these instructions carefully. Then assemble and disassemble the cell completely without casting a gel. After these preliminary steps, you should be ready to cast and run a gel.

Bio-Rad also recommends that all PROTEAN II xi cell components and accessories be cleaned with a suitable laboratory cleaner (such as Bio-Rad Cleaning Concentrate, catalog #161-0722) and rinsed thoroughly with distilled water, before use.

Model

Catalog No.

Date of Delivery

Warrant Period

Serial No.

Warranty

Bio-Rad Laboratories warrants the PROTEAN II xi cell against defects in materials and workmanship for 1 year. If any defects occur in the instrument during this warranty period, Bio-Rad Laboratories will repair or replace the defective parts free. The following defects, however, are specifically excluded:

1. Defects caused by improper operation.

2. Repair or modification done by anyone other than Bio-Rad Laboratories or an authorized agent.

3. Use of fittings or other spare parts supplied by anyone other than Bio-Rad Laboratories

4. Damage caused by accident or misuse.

5. Damage caused by disaster.

6. Corrosion due to use of improper solvent or sample.

7. This warranty does not apply to parts listed below:

• Platinum wire

• Glass plates

For any inquiry or request for repair service, contact Bio-Rad Laboratories after confirming the model and serial number of your instrument.

Nonidet is a trademark of Shell International Petroleum Company. Parafilm is a trademark of American National Can Company. Triton is a trademark of Dow Chemical Company. Tygon is a trademark of Norton Company.

Table of Contents

Section 1 General Information ............................................................................... 1

1.1 Introduction .............................................................................................. 1

1.2 Specifications ........................................................................................... 1

1.3 Safety ...................................................................................................... 2

Section 2 Description of Major Parts ..................................................................... 3

2.1 Central Cooling Core ................................................................................ 3

2.2 Sandwich Clamps .................................................................................... 3

2.3 Casting Stand .......................................................................................... 3

2.4 Upper Buffer Chamber ............................................................................. 4

2.5 Lower Buffer Chamber ............................................................................. 4

2.6 Lid ........................................................................................................... 4

2.7 Tube Gel Adaptor ..................................................................................... 4

2.8 Alignment Card ........................................................................................ 4

Section 3 Assembling the Glass Plate Sandwiches.............................................. 4

3.1 Assembling Single Sandwiches ................................................................ 4

3.2 Assembling Multiple or “Double-up” Gel Sandwiches ............................... 8

Section 4 Casting the Gels ..................................................................................... 9

4.1 Casting Discontinuous (Laemmli) Gels ...................................................... 9

4.2 Casting Continuous Gels .......................................................................... 11

4.3 Casting Gradient Gels .............................................................................. 11

4.4 Casting Agarose Gels ............................................................................... 13

Section 5 Assembling the Upper Buffer Chamber ................................................ 15

5.1 Assembly ................................................................................................. 15

5.2 Use of the Buffer Dam .............................................................................. 17

Section 6 Loading the Samples ............................................................................. 18

6.1 Loading of Sample Wells .......................................................................... 18

6.2 Loading a Single Sample Per Gel ............................................................. 18

6.3 Gels as Samples ...................................................................................... 18

Section 7 Running the Gel...................................................................................... 19

Section 8 Set-up Options ....................................................................................... 19

8.1 Buffer Recirculation .................................................................................. 19

8.2 Cooling Options ....................................................................................... 20

Section 9 Removing the Gels ................................................................................. 21

Section 10 Two-Dimensional Electrophoresis ......................................................... 22

10.1 Sequence of Steps for 2-D Protocol ......................................................... 22

10.2 Protocol for IEF First Dimension................................................................ 22

Section 11 Maintenance of Equipment ................................................................... 28

Section 12 Troubleshooting Guide -

Section 13 Equipment and Accessories .................................................................. 31

13.1 PROTEAN II xi Cell - Slab Configurations .................................................. 31

13.2 Accessories ............................................................................................. 31

13.3 PROTEAN II xi Cell - 2-D Configuration .................................................... 34

13.4 Accessories ............................................................................................. 34

13.5 Electrophoresis Chemicals ....................................................................... 35

13.6 Power Supplies ........................................................................................ 36

Section 14 Appendix ................................................................................................ 37

14.1 Reagents and Gel Preparation for SDS-PAGE Slab Gels .......................... 37

14.2 Separating Gel Preparation ...................................................................... 38

14.3 Stacking Gel Preparation .......................................................................... 38

14.4 Running Conditions .................................................................................. 39

14.5 Comparison of Coomassie Blue and Silver Staining.................................. 39

14.6 2-D Stock Solutions ................................................................................. 39

14.7 Running Conditions .................................................................................. 40

Section 15 Bibliography ........................................................................................... 41

15.1 General ................................................................................................... 41

15.2 Native Gel Systems ................................................................................. 41

15.3 SDS Gel Systems .................................................................................... 41

15.4 Urea Gel Systems ................................................................................... 42

15.5 Two-Dimensional IEF / SDS-PAGE Gel Systems ...................................... 42

15.5 References .............................................................................................. 42

PAGE, SDS-PAGE, 2-D IEF/SDS-PAGE

........... 29

Section 1 General Information

1.1 Introduction

The PROTEAN When used with the various combs, spacers, and accessories available, the PROTEAN II xi cell is suitable for most common electrophoretic techniques, including SDS electrophoresis, two-dimensional (2-D) electrophoresis, native electrophoresis, and agarose gel electrophoresis. The PROTEAN II xi cell can run up to 4 slab gels or 16 tube gels simultaneously. The basic unit accommodates gels 16 or 20 cm long. The 20 cm gels offer increased resolution capability, which is especially useful in 2-D applications.

The central cooling core of the PROTEAN II xi cell assures even heat distribution over the entire gel length, permitting excellent resolution with minimal band distortion. Only 1.5 liters of buffer are required. The raised electrode position insures safe operation even with extended overnight runs.

The unique single-screw sandwich clamps allow rapid assembly of the gel sandwiches, while providing even pressure distribution along the entire gel length. This even pressure minimizes the risk of breaking the glass plates, a common problem with multi-screw clamps. The PROTEAN II xi alignment card helps keep spacers upright during sandwich alignment. The combination of the clamps, alignment card, and the casting stand permits assembly and casting of gels in minutes, with little effort. After casting, the completed gel sandwich attaches to the central cooling core with a single motion.

The PROTEAN II xi cell is the instrument of choice for 2-D electrophoresis. With the optional tube adaptors, one can run the first dimension IEF tube gel, and then overlay this gel onto the second dimension SDS slab gel. Thus, the complete 2-D procedure can be done with one dedicated instrument.

II xi cell is a vertical slab electrophoresis instrument which combines versatility with practicality.

1.2 Specifications

Construction:

Cooling core and tube gel adaptor molded polysulfone

Lid and lower buffer chamber molded polycarbonate

Clamps, casting glass and Polytetraﬂuoroethylene (PTFE)-filled stand, and cams molded poly-carbonate

Electrical leads flexible, coiled Electrodes platinum, 0.010 inch diameter

(0.254 mm)

Shipping weight 11 kg (24 lb, 3 oz) Overall size 26 cm (L) x 19 cm (W) x 30 cm (H) Gel size 16 x 16 cm slab or 16 x 20 cm slab

1 to 6 mm diameter tube gels Glass plate sizes 16 cm cell: 16 x 20 cm (inner plate)

18.3 x 20 cm (outer plate)

Cooling core, maximum flow rate 2 liter/min

Maximum coolant temperature Do not exceed 50°C Voltage limit 1,000 volts DC

Note: PROTEAN II xi cell components are not compatible with ethanolamine, ethylene diamine, chlorinated

hydrocarbons (e.g., chloroform), aromatic hydrocarbons (e.g., toluene, benzene), or acetone. Use of such organic solvents voids all warranties. Cyanoacrylate and other adhesives will also attack the cell components. Contact your local Bio-Rad office for compatibility information before using any adhesive or organic solvent with this cell.

1.3 Safety

Power to the PROTEAN II xi cell and PROTEAN II xi 2-D cell is to be supplied by an external DC voltage power supply. This power supply must be ground isolated in such a way that the DC voltage output floats with respect to ground. All of Bio-Rad's power supplies meet this important safety requirement. Regardless of which power supply is used, the maximum specified operating parameters for these cells are:

1000 VDC maximum voltage limit 80 Watts maximum power limit 50 °C maximum ambient temperature limit

Current to the cell, provided from the external power supply, enters the unit through the lid assembly, providing a safety interlock to the user. Current to the cell is broken when the lid is removed. Do not attempt to circumvent this safety interlock, and always turn the power

supply off before removing the lid, or when working with the cell in any way.

Important

This Bio-Rad instrument is designed and certified to meet IEC1010-1* safety standards. Certified products are safe to use when operated in accordance with the instruction manual. This instrument should not be modified or altered in any way. Alteration of this instrument will:

• Void the manufacturer's warranty

• Void the IEC1010-1 safety certification

• Create a potential safety hazard

Bio-Rad is not responsible for any injury or damage caused by the use of this instrument for purposes other than for which it is intended or by modifications of the instrument not performed by Bio-Rad or an authorized agent.

*IEC 1010-1 is an internationally accepted electrical safety standard for laboratory instruments.

Section 2 Description of Major Parts

Fig. 1. PROTEAN® II xi 2-D cell including tube adaptor. 1. Lower buffer chamber, 2. Lid with electrical leads in place,

3. Cooling core with glass plate sandwich attached and core caps in place, 4. Casting stand with glass plate sandwich in alignment slot, 5. Tube gel adaptor, 6. Sandwich clamps, 7. Buffer dam, 8. Alignment card.

2.1 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.

2.2 Sandwich Clamps

The unique PROTEAN II xi 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.

2.3 Casting Stand

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.

2.4 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 for 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.

2.5 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.

2.6 Lid

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.

2.7 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.

2.8 Alignment Card

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.

Section 3 Assembling the Glass Plate Sandwiches

3.1 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.

1. 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.

2. 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.

3. 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.

4. Place the right clamp on the right side of the plates, and tighten the clamp screw.

5. 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.

6. 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.

7. 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.

8. 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.

9. 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.

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.

3.2 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.

Note: In order to run four gels instead of two, it is necessary to order two notched inner glass plates and a set of four spacers of equal length.

1. Lay down a long rectangular plate, two spacers, and a notched inner plate.

2. 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.

3. 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.

Section 4 Casting the Gels

4.1 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. This formulation is included in the Appendix.

1. Prepare the monomer solution by combining all reagents except ammonium persulfate (APS) and TEMED (see Section 14.1 for formulations). Deaerate the solution under vacuum for at least 15 minutes.

2. 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.

3. 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.

4. 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.

5. 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 Section 14.1) on top of each separating gel after rinsing with deionized water. This will prevent dehydration of the separating gel during overnight storage a room temperature.

6. Prepare the stacking gel monomer solution. Combine all reagents except APS and TEMED, and deaerate under vacuum at least 15 minutes.

7. Dry the area above the separating gel completely with filter paper before pouring the stacking gel.

8. 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.

9. 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.

10. Allow the gel to polymerize 30-45 minutes. Remove the comb by pulling it straight up slowly and gently

11. 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.

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