Bio-Rad Trans-Blot Cell User Manual

Trans-Blot

Electrophoretic

Transfer Cell

Instruction

Manual

Catalog Numbers 170-3910, 170-3925, 170-3926, 170-3939, 170-3945, 170-3946,

170-3950, 170-3951

170-3953 and 170-3954

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

Note

1. Assembly & Disassembly

To insure best performance from the Trans-Blot electrophoretic transfer cell, become fully acquainted with these operating instructions before using the cell to transfer samples. Bio-Rad recommends that you first read these instructions carefully. Then assemble and disassemble the cell completely. After these preliminary steps, you should be ready to transfer a sample.

2. Wash Cell Before Use

Bio-Rad also recommends that all Trans-Blot electrophoretic transfer cell components and accessories be cleaned with a suitable laboratory cleaner (such as Bio-Rad Cleaning Concentrate, catalog number 161-0722) and rinsed thoroughly with distilled water, before use.

Warranty

Bio-Rad Laboratories warrants the Trans-Blot electrophoretic transfer 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.

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

Model

Catalog Number

Date of Delivery

Warranty Period

Serial Number

Invoice Number

Purchase Order Number

Table of Contents

Section 1 Introduction..................................................................................................1

1.1 Specifications .............................................................................................................1

1.2 Safety Instructions......................................................................................................2

Section 2 Trans-Blot Cell Assembly............................................................................4

2.1 Assembly of the Unit for Standard Transfers............................................................4

2.2 High Intensity Field Option .......................................................................................7

2.3 Acidic Transfers .........................................................................................................8

Section 3 Transfer Conditions .....................................................................................8

3.1 General Guide for Transfer Buffers and Running Conditions ..................................8

3.2 Notes on Electrophoretic Transfer Conditions ..........................................................9

3.3 Buffer Formulation...................................................................................................11

Section 4 Strategies for Optimizing Electro-Elution ..............................................12

4.1 Optimizing Protein Transfer ....................................................................................12

4.2 Optimizing DNA and RNA Transfer.......................................................................13

Section 5 Choice of Blotting Membranes.................................................................14

5.1 Protein Blotting Membranes ....................................................................................14

5.2 DNA and RNA Blotting Membrane........................................................................14

Section 6 Troubleshooting Guide ..............................................................................15

Section 7 Product Information..................................................................................22

Section 8 References ...................................................................................................22

Section 1 Introduction

Blotting was first performed by Southern1in 1975 with the transfer of DNA from agarose gels to nitrocellulose membranes. Blotting has subsequently been applied to RNA

2-4

and pro-

tein

5,6

from both agarose and polyacrylamide gels. To overcome the inefficiencies observed in various capillary transfers, electric current was used to elute protein from polyacrylamide gels, as first described by Towbin7et al. in 1979. Since that time, electrophoretic transfer has been applied to DNA and RNA.

8-14

Numerous publications have dealt with the topic of pro-

tein electrophoretic transfer technique.

15-26

There have also been several reviews summariz-

ing the literature on electrophoretic blotting.

27-33

The Trans-Blot cell is available with standard electrode cards or with plate electrodes, which consist of a platinum-coated titanium anode and a stainless steel cathode. The TransBlot cell allows electrophoretic transfers to be performed in the standard configuration, with either set of electrodes positioned 8 cm apart, using up to three gel holders positioned between the electrodes. Removable electrodes can be positioned 4 cm apart with one gel holder between. This allows generation of a high intensity electrical field, when used in combination with the PowerPac 200 Power Supply. An optional Super Cooling Coil is available for temperature control during routine blots and high power transfers.

1.1 Specifications

Construction

Trans-Blot tank Molded polysulfone Electrode cards Cathode 18” Anode 25” Anode plate electrode Platinum-coated titanium Cathode plate electrode Stainless steel Gel holder Acrylic

Overall dimensions 24 x 18 x 9.5 cm

Gel holder dimensions 21x17cm

Buffer capacity 3L

Transfer capacity 3 gel holders

Cleaning Use mild soap and warm water to clean the elec-

trodes, cassettes, and buffer tank. Use special care when cleaning the electrode cards or plate electrodes. Avoid stretching or breaking the platinum wires. Avoid scratching or marring the platinum plate. Do not use abrasives or strong detergents. The cathode plate (stainless steel) can be cleaned with a mild abrasive to remove salt that may be deposited during normal operation. Rinse the fiber pads under hot water and then in distilled, deionized water.

Chemical compatibility The Trans-Blot cell components are not compati-

ble with chlorinated hydrocarbons (e.g., chloroform), aromatic hydrocarbons (e.g., toluene, benzene), or acetone. Use of organic solvents

voids all warranties.

1.2 Safety Instructions

Power to the Trans-Blot 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

200 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 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 IEC 1010-1* 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.

Trans-Blot Cell Description & Assembly of Parts

Lid

Fiber pad

Filter paper

Membrane

Gel Filter paper Fiber pad

Gel holder cassette

Super cooling coil

Plate cathode

Buffer tank

* Wire electrode

cards not shown

Plate anode

Section 2 Trans-Blot Cell Assembly

2.1 Preparation for Blotting

1. Prepare the transfer buffer. (See Section 3.3 for buffer formulation. Using buffer chilled to

4 °C will improve heat dissipation.)

2. Cut the membrane and the filter paper to the dimensions of the gel. Always wear gloves

when handling membranes to prevent contamination. Equilibrate the gel and soak the

membrane, filter paper, and fiber pads in transfer buffer (15 min—1 hour depending on

gel thickness).

3. Prepare the gel sandwich.

Place the cassette, with the gray side down, on a clean surface.

Place one pre-wetted fiber pad on the gray side of the cassette.

Place a sheet of filter paper on the fiber pad.

Place the equilibrated gel on the filter paper.*

Place the pre-wetted membrane on the gel.*

Complete the sandwich by adding a piece of filter paper on the membrane.*

* Removing any air bubbles which may have formed is very important for good results. Use a glass tube to gently roll air bubbles out.

Add the last fiber pad.

Fiber pad

Filter paper

Membrane

Gel

Filter paper

Fiber pad

Cathode (-) side

Anode (+) side

4. Close the cassette firmly, being careful not to move the gel and filter paper sandwich.

Lock the cassette closed with the white latch.

5. Place the cassette in tank. Repeat for any other cassettes you may want to run. Completely

fill the tank with buffer.

6. If running high intensity or high voltage with plate electrodes, add the super cooling coil

in any available slot.

Note: For high intensity runs (4 cm inter-electrode distance) only one cassette can be

placed in the tank. The Super Cooling Coil must be used. It is placed outside the electrodes.

See Section 2.2, high intensity field option. Two cassettes can be run with the Super

Cooling Coil in the standard field with the electrodes 8 cm apart, and three cassettes can

be run at low voltage without the super cooling coil.

7. Addition of a standard stir bar will help to maintain even buffer temperature and ion dis-

tribution in the tank. Set the speed as fast as possible to keep ion distribution even.

8. Put on the lid, plug the cables into the power supply, and run the blot. Refer to Section 3

for run times and voltage setting with various buffers.

9. Upon completion of the run, disassemble the blotting sandwich and remove the mem-

brane for development. Clean the cell, fiber pads, and cassettes with laboratory detergent

and rinse well with deionized water.

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