Bio-Rad Trans-Blot Plus Cell User Manual

Trans-Blot®Plus

Electrophoretic

Transfer Cell

Instruction Manual

Catalog Number

170-3990

For Technical Service Call Your Local Bio-Rad Office or in the U.S. Call 1-800-4BIORAD (1-800-424-6723)

Table of Contents

Page

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

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

1.2 Specifications ................................................................................................2

1.3 Safety.............................................................................................................3

Section 2 Set Up and Basic Operation of the Trans-Blot Plus Cell .............5

2.1 Components ..................................................................................................5

2.2 Additional Components .................................................................................6

2.3 Setting up the Tank and Cooling System .....................................................6

2.4 Assembling the Gel Sandwich and Cassette................................................8

2.5 Transfer .........................................................................................................9

2.6 Draining the Tank ........................................................................................10

2.7 Running Acidic Transfers ............................................................................10

Section 3 Transfer Conditions .......................................................................11

3.1 General Guidelines and Running Conditions .............................................11

3.2 Notes on Electrophoretic Transfer Conditions............................................15

3.3 Buffer Formulations .....................................................................................17

Section 4 Strategies for Optimizing Electrophoretic Transfer ...................21

4.1 Optimizing Protein Transfer ........................................................................21

4.2 Optmizing DNA and RNA Transfer .............................................................23

Section 5 Choice of Blotting Membranes.....................................................23

5.1 Protein Blotting Membranes........................................................................23

5.2 DNA and RNA Blotting Membranes............................................................24

Section 6 Troubleshooting .............................................................................25

Section 7 Maintenance....................................................................................28

Section 8 Product Information.......................................................................29

Section 9 References ......................................................................................30

Section 10 Warranty..........................................................................................31

Section 1 General Information

1.1 Introduction

The Trans-Blot Plus cell is an electrophoretic transfer cell designed for use with large format gels, such as those used with the PROTEAN Plus Dodeca cell, and for high-throughput blotting applications with smaller format gels, such as those used with the Criterion Dodeca cell. The Trans-Blot Plus cell is supplied with three gel holder cassettes, each with an effective blotting area of 26.5 x 28 cm. A pair of plate electrodes- a platinum-coated titanium anode and a stainless steel cathodemay be positioned 4 cm, 7 cm, or 10 cm apart for electrophoretic transfers of one, two, and three gel-containing cassettes, respectively. This variable placement ensures a minimum distance between electrodes, which increases the field strength and efficiency of transfer. Cooling, which is achieved with the Super cooling coil and a refrigerated recirculating water bath, is required for high-intensity transfers and is recommended for longer, overnight transfers. The Trans-Blot Plus tank is designed to simultaneously accommodate the two plate electrode cards, three gel holder cassettes and the Super cooling coil.

1.2 Specifications

Trans-Blot Plus cell tank

Overall dimensions 39.4 cm x 17.27 cm x 30 cm Material Acrylic Buffer requirement 12 liters Buffer capacity 14 liters

Electrodes

Electrode plate dimension 22.86 cm x 24.45 cm Electrode card dimension 36.2 cm x 28.26 cm Material Support card Molded Polycarbonate

Anode plate Platinum coated titanium Cathode plate Stainless steel

Distance anode to cathode Adjustable to 4 cm, 7 cm or 10 cm

Cassettes

Cassette dimension 28 x 30.7 cm Material Molded Polyphthalamide (PPA) Blotting area 26.5 x 28 cm Gel capacity per cassette 1 PROTEAN Plus, PROTEAN II XL, or

PROTEAN II xi gel; 4 Criterion gels or 9 ReadyGel gels

Gel/cassette assembly tray

Material Molded PETG Overall dimensions 42 x 42 x 6.3 cm

1.3 Safety

Power to the Trans-Blot Plus cell is 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 the cell are:

300 VDC Maximum voltage limit

300 Watts Maximum power limit

40°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. Always turn the power supply off before removing the lid, or when working with the cell in any way.

The Trans-Blot Plus is certified to meet EN61010-1* safety standard for safety of laboratory equipment. Certified products are safe to use when operated in accordance with the instruction manual. This safety certification does not extend to other equipment or accessories not EN61010-1 certified, even when connected to the Trans-Blot Plus.

This instrument should not be modified or altered in any way. Alteration of this instrument will void the manufacturer’s warranty, void the EN61010-1 safety certification and create a potential safety hazard for the user.

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.

*EN61010-1 is an internationally accepted electrical safety standard for laboratory instruments.

Trans-Blot Plus Cell Assembly of Parts

Fig. 1.

Super Cooling Coil

Cathode Plate (Black)

Gel Holder Cassettes (3)

Lid

Anode Plate (Red)

Buffer Tan

r a

l o

l u

l l

Handles

Drain Port

Section 2 Set up and Basic operation

2.1 Components

Buffer Tank and Lid

The buffer tank and lid combine to fully enclose the inner chamber during electrophoresis. The lid cannot be removed without disrupting the electrical circuit. Handles on both sides of the tank facilitate transport. Guide marks on the front and back of the tank identify appropriate fill levels for transfer buffer. On the inside, the tank has 5 separate slots for variable placement of the electrode cards and gel holder cassettes and a designated space for the cooling coil. Multiple ports on the lid allow three different connection points for the cathode (black) electrode card.

Gel Holder Cassettes

Each gel holder cassette has an overall effective blotting area of 26.5 x 28 cm. The gel/membrane sandwich is placed into the cassette between two fiber pads, which are also included. The gel holder cassette design includes three separate clamps that ensure even pressure across the gel and membrane sandwich. A detachable hinge mechanism prevents gel sandwiches from slipping during assembly.

Super Cooling Coil

Coolant from a refrigerated circulator (see Additional Components) passes through the Super cooling coil to cool the transfer buffer during high intensity or prolonged runs.

Electrode Cards

The Trans-Blot Plus cell is supplied with a pair of plate electrodes- a platinum-coated titanium anode and a stainless steel cathode. Both electrode cards are removable. The anode plate (red) must be placed into the slot that is nearest the front face of the buffer tank. The placement of the cathode plate (black) is variable; the cathode plate may be positioned in either the third, fourth or fifth slot (4 cm, 7 cm, or 10 cm away from the anode) for electrophoretic transfers of one, two, and three gel-containing cassettes, respectively. The electrode cards are held in place in the tank with the nylon screws (provided).

Roller

The roller is used to ensure proper contact between gel and membrane and to remove trapped bubbles during sandwich assembly.

Stir bar

A 3" x ½" stir bar is included with the Trans-Blot Plus cell and should be used during every electrophoretic run to maintain uniform conductivity and temperature during transfer.

Drain Port and Quick-Connect Fitting

The drain port and quick-connect fitting on the side of the Trans-Blot Plus cell facilitate draining the buffer from the tank after transfer. Remove at least half of the transfer buffer volume prior to moving the tank.

2.2 Additional Components

Magnetic stir plate

A magnetic stir plate with a surface area that is sufficient to accommodate the Trans-Blot Plus cell is required for constant stirring of transfer buffer during electrophoresis. Recommended stir plates include the PC-610 from Corning, the Cimarec 3 from Thermolyne and the Vel A from Cole Parmer.

Refrigerated circulator

The Trans-Blot Plus cell requires a refrigerated circulator to work with the Super cooling coil for optimal results. The recommended minimum cooling capacity of the refrigerated circulator is 300W at 20°C and minimum pump flow rate is 4 L/min. Recommended chillers include the Model RTE-7 from Thermo NESLAB and the Model WKL 26 from Thermo Haake.

Tubing

Tubing with a 3/8 " internal diameter is required to connect both ends of the Super cooling coil to the refrigerated circulator and for draining the tank using the quick-release fitting and drain port.

Gel/ Cassette Assembly Tray

The optional gel/ cassette assembly tray is large enough to accommodate a gel holder cassette and the buffer required for sandwich assembly and may be purchased separately (see Product Information).

2.3 Setting up the tank and cooling system

1. Prepare the transfer buffer. See Section 3.3 for buffer formulations. Generally,

15 liters of transfer buffer will suffice for electrophoresis, gel equilibration, and

sandwich assembly.

2. Position the anode (red) electrode card into the tank, in the slot that is nearest

the front face of the tank (see Figures 1 and 2). This is the only position

possible for the anode plate since it is the only position that will provide a

connection between the plate electrode and the anode leads in the lid.

Tighten the electrode card in place with the white nylon screw.

3. Position the cathode (black) electrode card into the third, fourth or fifth slot from

the front (4 cm, 7 cm, or 10 cm away from the anode card), depending on

whether you will be transferring one, two, or three gel-containing cassettes,

respectively (see Figure 2). Tighten the electrode card in place with the white

nylon screw.

Fig. 2.

4. If necessary, adjust the position of the cathode leads (black) on the lid so that

they correspond to the position of the cathode electrode plate (black) within the

tank.

5. Position the Trans-Blot Plus cell on a magnetic stir plate.

6. Add transfer buffer to the appropriate fill line. Choose the fill line that is

appropriate for the number of gel holder cassettes that you are using.

Note: In order to avoid overflow and electrical hazards, do not fill the tank beyond the indicated buffer level lines marked on the tank.

7. Place the stir bar in the tank.

8. Start cooling. Cooling is required for high intensity field conditions and is

recommended for prolonged, unsupervised runs. Place the Super cooling coil

into its designated slot at the rear face of the tank. Connect the Super cooling

coil to the refrigerated circulator according to the manufacturer's instructions.

Avoid restrictive fittings, internal diameter reductions and excessive extensions

of tubing. Turn on power to the stir plate.

Note: To test that the cooling system is functioning properly before the first transfer run is initiated, place the Trans-Blot Plus cell on the magnetic stirrer and pour in 11 L of buffer (or water). With no power applied to the tank, the buffer should cool by 5°C in approximately 20 min.

Super Cooling Coil

Cathode Plate (Black)

Gel Holder Cassette (1)

Anode Plate (Red)

Super Cooling Coil

Cathode Plate (Black)

Gel Holder Cassettes (2)

Anode Plate (Red)

Super Cooling Coil

Cathode Plate (Black)

Gel Holder Cassettes (3)

Anode Plate (Red)

One Gel Holder Cassette Loaded

Two Gel Holder Cassettes Loaded

Three Gel Holder Cassettes Loaded

SLOT

Cathode Plate (Black)

Super Cooling Coil

MAX LEVEL

MIN LEVEL

Trans-Blot Plus Cell

1 CASSETTE

Gel Holder Cassettes (2)

Anode Plate (Red)

2.4 Assembling the Gel Sandwich and Cassette Assembly

Each gel sandwich will contain the gel and membrane sandwiched between two pieces of blot absorbent filter paper.

To prevent contamination, always wear gloves when handling the gels, membranes and filter paper to prevent contamination.

An optional gel/ cassette assembly tray is available for the Trans-Blot Plus cell (see Product Information). This tray is large enough to accommodate the gel holder cassette during sandwich assembly. The lid of the tray can be used for soaking membranes.

Fig. 3.

1. For each gel, cut one piece of membrane and two pieces of filter paper to the

dimensions of the gel. Pre-cut membranes and filter papers are available (see

Product Information).

2. Equilibrate gels and membranes by soaking them in transfer buffer for 15 minutes.

3. Pour ~ 3 liters of transfer buffer into a tray for assembly of the cassettes.

4. Place the black cassette plate into the tray with the clamps in their fully extended

position.

5. Place a fiber pad on the black cassette plate, making sure it is thoroughly wet.

6. Place a piece of filter paper on top of the fiber pad. Make sure there is enough

buffer to thoroughly wet the filter paper.

7. Carefully place the equilibrated gel on top of the filter paper.

Fiber Pad

Filter Paper

Membrane

Gel

Filter Paper

Gel Holder Cassette

(Red Plate)

Clamp

Hinge

Fiber Pad

Gel Holder Cassette (Black Plate)

Note: Extra care is required when handling large gels, first align one side of the gel with the side of the filter paper and slowly lower the rest of the gel.

8. Carefully place the pre-soaked membrane on top of the gel. Make sure the

membrane is properly positioned as it touches the gel. To avoid ghost prints or

artifacts, do not move the membrane after it is positioned. Use the roller to

remove any air bubbles and to ensure proper contact between the gel and

membrane.

9. Wet a second piece of filter paper in transfer buffer and place it on top of the

membrane.

10.Soak a fiber pad in transfer buffer and place it on top of the filter paper.

11. Place the hinge of the upper cassette plate (red) into the hinge mechanism of

the lower plate, and lower the upper cassette plate on top of the gel sandwich.

Make sure that the gel sandwich is aligned below the rim of the hinge so that

the cassette will close properly.

12. Working with one side at a time, apply firm pressure to the area adjacent to a

clamp and slide the clamp in.

13. Once the cassette is closed and locked, insert it into the tank with the hinge

side up. Make sure the red cassette plate faces the red electrode plate (see

Figure 4).

Fig. 4.

2.5 Beginning Transfer

1. Once all the cassettes are in place, check that the buffer level is between the

maximum and minimum levels indicated on the tank.

2. Turn on the stir plate and check that the stirring and cooling are working properly.

3. Place the lid on the tank.

MAX LEVEL

MIN LEVEL

Trans-Blot Plus Cell

1 CASSETTE

2 CASSETTES

3 CASSETTES

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