The BIORAD Mini-PROTEAN 3 cell

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In our laboratories, we use two types of vertical gel electrophoresis equipment, of which the BIORAD Mini-PROTEAN^® 3 Cell is depicted above (the BIORAD PROTEAN^® II xi Cell is ).

Before you attempt any experiment using this equipment, you should familiarise yourself with its component parts, both by studying this on-line material and by actually examining and handling its components in the laboratory…

0. The BIORAD Mini-PROTEAN 3 cell components

To get the best performance from your Mini-PROTEAN 3 cell, familiarize yourself with the components by assembling and disassembling the cell before using it.

0.1. Gel casting components

The Casting Stand secures the Gel Cassette Assembly during gel casting. It contains pressure levers that seal the Gel Cassette Assembly against the casting gaskets.

The Casting Frame when placed on the benchtop, evenly aligns and secures the Spacer Plate and the Short Plate together to form the gel cassette sandwich prior to casting.

One Casting Frame, a Spacer Plate, and a Short Plate form one Gel Cassette Assembly.

A Spacer Plate and Short Plate with polymerized gel form a Gel Cassette Sandwich after casting.

The Spacer Plate is the taller glass plate with gel spacers permanently bonded. Spacer Plates are available in 0.5 mm, 0.75 mm, 1.0 mm, and 1.5 mm thicknesses, which are marked directly on each Spacer Plate.

The Short Plate is the shorter, flat glass plate that combines with the Spacer Plate to form the gel cassette sandwich.

A selection of molded combs is available. These are used to form wells in the polymerised gel.

0.2. Electrophoresis components

The Mini Tank and Lid combine to fully enclose the inner chamber during electrophoresis. The lid cannot be removed without disrupting the electrical circuit.

The molded, one-piece buffer dam is used when running only one gel.

The Electrode Assembly holds the Gel Cassette Sandwich. It houses the sealing gasket, the upper and lower electrodes and the connecting banana plugs. The anode (lower electrode) banana plug is identified with a red marker and the cathode (upper electrode) banana plug with a black marker.

The Clamping Frame holds the Electrode Assembly and Gel Cassette Sandwich in place. Its pressure plates and closure cams seal the Gel Cassette Sandwich against U-shaped gaskets on the Electrode Assembly to form the inner buffer chamber.

The Electrode Assembly, two Gel Cassette Sandwiches or one gel cassette sandwich and a buffer dam, and the Clamping Frame form the Inner Chamber.

Another diagram of the whole cell:

To perform a complete electrophoresis run using this apparatus, the following steps have to be carried out:

1. Set Up and Basic Operation.
2. Assembly of the Electrophoresis Module.
3. Sample Loading.
4. The electrophoresis run.

1. Set Up and Basic Operation

Gel Cassette Sandwich Preparation (for Hand Cast Gels)

Glass Cassette and Casting Stand Assembly

Gel Casting

Discontinuous Polyacrylamide Gels

1. Place a comb completely into the assembled gel cassette. Mark the glass plate 1 cm below the comb teeth. This is the level to which the resolving gel is poured. Remove the comb.
2. Prepare the resolving gel monomer solution by combining all reagents except APS and TEMED (See the .) Degas the solution under vacuum for at least 15 minutes. Do not use a sink water aspirator.
3. Add APS and TEMED to the degassed monomer solution and pour to the mark using a glass or disposable plastic pipette. Pour the solution smoothly to prevent it from mixing with air.
4. Immediately overlay the monomer solution with water or t-amyl alcohol.
   Note: If water is used, add it slowly and evenly to prevent mixing.
   Do not overlay with butanol or isobutanol
5. Allow the gel to polymerize for 45 minutes to 1 hour. Rinse the gel surface completely with distilled water. Do not leave the alcohol overlay on the gel for more than 1 hour because it will dehydrate the top of the gel.
   Note: At this point the resolving gel can be stored at room temperature overnight. Add 5 ml of a 1:4 dilution of 1.5 M Tris-HCI, pH 8.8 buffer (for Laemmli System) to the resolving gel to keep it hydrated. If using another buffer system, add 5 ml 1 x resolving gel buffer to the resolving gel surface for storage.
6. Prepare the stacking gel monomer solution. Combine all reagents except APS and TEMED. Degas under vacuum for at least 15 minutes.
7. Before casting the stacking gel, insert a piece of filter paper to dry the area in between the glass plates above the resolving gel. Take care not to touch the surface of the gel.
8. Add APS and TEMED to the degassed stacking gel monomer solution and pour the solution between the glass plates. Continue to pour until the top of the short plate is reached.
9. Insert the desired comb between the spacers starting at the top of the Spacer Plate, making sure that the tabs at the ends of each comb are guided between the spacers. It is easiest to insert the combs starting at an angle and insert well 1 first, then 2, 3, and so on until the combs is completely inserted. Seat the comb in the gel cassette by aligning the comb ridge with the top of the Short Plate.
10. Allow the stacking gel to polymerize for 30-45 minutes.
11. Gently remove the comb and rinse the wells thoroughly with distilled water or running buffer.
12. Rinse the Casting Frame(s) and Stand with distilled, deionized water after use.

1. Prepare the monomer solution by combining all reagents except the APS and the TEMED. Degas under vacuum for 15 minutes (see ).
2. Add APS and TEMED to the degassed monomer solution and pour the solution between the glass plates. Continue to pour until the top of the Short Plate is reached.
3. Insert the desired comb between the spacers starting at the top of the Spacer Plate, making sure that the tabs at the ends of each comb are guided between the spacers. It is easiest to insert the combs starting at an angle and insert well 1 first, then 2, 3, and so on until the combs is completely inserted. Seat the comb in the gel cassette by aligning the comb ridge with the top of the Short Plate.
4. Allow the gel to polymerize for 45 minutes to 1 hour.
5. Gently remove the comb and rinse the wells thoroughly with distilled water or running buffer.
6. Rinse the Casting Frame(s) and Stand with distilled, deionized water after use.

2. Electrophoresis Module Assembly

3. Sample Loading

1. Load the samples into the wells with a Hamilton syringe or a pipette using gel loading tips.
2. If using Bio-Rad’s patented sample loading guide, place it between the two gels in the Electrode Assembly. Sample loading guides are available for 9, 10, 12 and 15 well formats.
   
3. Use the Sample Loading Guide to locate the sample wells. Insert the syringe or pipette tip into the slots of the guide to fill the corresponding wells.
   Note: Load samples slowly to allow them to settle evenly on the bottom of the well. Be careful not to puncture the bottom of the well with the syringe or pipette tip.

4. Electrophoresis run

4.1. Mini Tank Assembly

Place the lid on the Mini Tank. Make sure to align the colour coded banana plugs and jacks. The correct orientation is made by matching the jacks on the lid with the banana plugs on the electrode assembly. A stop on the lid prevents incorrect orientation.

4.2. Power Conditions

Insert the electrical leads into a suitable power supply with the proper polarity.

Apply power to the Mini-PROTEAN 3 cell and begin electrophoresis; 200 volts constant is recommended for SDS-PAGE and most native gel applications. Run time is approximately 35 minutes at 200 volts for SDS-PAGE.

4.3. Gel Removal

After electrophoresis is complete, turn off the power supply and disconnect the electrical leads.

Remove the tank lid and carefully lift out the Inner Chamber Assembly. Pour off and discard the running buffer.

Note: Always pour off the buffer before opening the cams to avoid spilling the buffer.

Open the cams of the Clamping Frame. Pull the Electrode Assembly out of the Clamping Frame and remove the Gel Cassette Sandwiches.

Remove the gels from the Gel Cassette Sandwich by gently separating the two plates of the gel cassette. The green, wedgeshaped, plastic Gel Releaser may be used to help pry the glass plates apart.

Run the sharp edge of the Gel Releaser or a razor blade along each spacer to separate the gel from the spacer. Remove the gel by floating it off the glass plate by inverting the gel and plate under fixative or transfer solution, agitating gently until the gel separates from the plate.

Rinse the Mini-PROTEAN 3 cell electrode assembly, Clamping Frame and Mini Tank with distilled, deionized water after use.

5. References

1. BIORAD Mini-PROTEAN® 3 Cell Instruction Manual.
2. BIORAD Mini-PROTEAN® 3 Cell Assembly Guide