BIO RAD Trans-Blot SD Instruction Manual
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BIO RAD Trans-Blot SD Instruction Manual

BIO RAD Trans-Blot SD Instruction Manual

Semi-dry electrophoretic transfer cell
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Trans-Blot
SD
Semi-Dry
Electrophoretic
Transfer Cell
Instruction
Manual
Catalog Number
170-3940
For Technical Service Call Your Local Bio-Rad Office or in the U.S. Call 1-800-4BIORAD (1-800-424-6723)

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  • Page 1 ® Trans-Blot Semi-Dry Electrophoretic Transfer Cell Instruction Manual Catalog Number 170-3940 For Technical Service Call Your Local Bio-Rad Office or in the U.S. Call 1-800-4BIORAD (1-800-424-6723)
  • Page 2 Note To insure the best performance from the Trans-Blot SD semi-dry 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 without transferring sample. After these preliminary steps, you should be ready to transfer a sample.

  • Page 3: Table Of Contents

    Chemical Reagents..................... 4 Section 3 Safety Instructions ..................5 Section 4 Trans-Blot SD Assembly ................6 Preparation for Blotting ..................... 6 Assembly of the Unit for Standard Transfers............7 Assembly of the Unit for Acidic Transfers ............... 10 Section 5 Buffer Formulation ..................

  • Page 4: Introduction

    DNA and RNA blotting. For blotting PCR fragments, plasmid and vector DNA, and RNA with the SD cell, use the Trans-Blot SD DNA blotting kit. DNA or RNA can be blotted from agarose gel to ®...

  • Page 5: Equipment And Reagents

    Section 2 Equipment and Reagents 2.1 Equipment and Accessories Catalog Number Product Description 170-3940 Trans-Blot SD Electrophoretic Transfer Cell Replacement Parts 170-3942 Trans-Blot SD Anode, platinum 170-3947 Trans-Blot SD Cathode, stainless steel DNA Blotting Kit 170-3957 Trans-Blot SD DNA/RNA Blotting Kit...
  • Page 6 Catalog Number Product Description Nitrocellulose Membrane (0.2 micron) Recommended uses 162-0112 Roll, 33 cm x 3 m, 1 Transfer of proteins 162-0146 Sheets, 7 x 8.4, 10 (smaller pore size retaining more low 162-0147 Sheets, 13.5 x 16.5 cm, 10 molecular weight pro- teins - PVDF is even DNA/RNA Blotting Accessories...

  • Page 7: Related Instruments

    2.2 Related Instruments Catalog Number Product Description Blotting Equipment 170-3910 Trans-Blot Electrophoretic Transfer Cell 170-3946 Trans-Blot Electrophoretic Transfer Cell, with plate electrodes 170-3945 Trans-Blot Plate Electrode Pair ® 170-3930 Mini Trans-Blot Electrophoretic Transfer Cell 170-3970 Western Processor ® 170-6545 Bio-Dot Microfiltration Apparatus 170-6542 Bio-Dot SF Microfiltration Apparatus...

  • Page 8: Safety Instructions

    Catalog Number Product Description Total Protein Detection Kits 170-6512 Biotin-Blot Protein Detection Kit 170-6517 Enhanced Colloidal Gold Total Protein Detection Kit Blotting Standards 161-0372 Precision Prestained Standards, 10–250 kD, 500 µl 161-0380 Precision Streptactin-HRP conjugate 161-0381 Precision Streptactin-AP conjugate 161-0305 Prestained SDS-PAGE Standards, Low range 161-0309 Prestained SDS-PAGE Standards, High range...

  • Page 9: Trans-Blot Sd Assembly

    Joule heat can be generated rapidly during semi-dry blotting. Transferring longer than 2 hours can damage the unit. 5. Power supply requirements. The Trans-Blot SD cell should only be used with the microprocessor-controlled Model 200/2.0 power supply (catalog numbers 165-4761 and 165-4762), or the Model 1000/500 power supply (catalog numbers 165-4710 and 165-4711).

  • Page 10: Assembly Of The Unit For Standard Transfers

    Low molecular weight macromolecules ( 10,000 daltons) may diffuse out of gels more readily. One can allow adequate gel pre-equilibration by changing the pre-equilibration buffer several times during a relatively short pre-equilibration period. This will help to limit diffusion of low molecular weight macromolecules while providing efficient salt reduction. 3.
  • Page 11 2. Place a pre-soaked sheet of extra thick filter paper onto the platinum anode. Roll a pipet or test tube over the surface of the filter paper (like a rolling pin) to exclude all air bubbles. If thick or thin filter paper is used, repeat with one or two more sheets of buffer- soaked filter paper.
  • Page 12 Trans-Blot SD cell. In this situation, the gel sandwich and electrodes will be exposed to excessive heat. This may result in a safety hazard. It is advisable to monitor resistance, power, and current during the run.

  • Page 13: Assembly Of The Unit For Acidic Transfers

    Section 5 Buffer Formulation The following buffers are recommended for use with the Trans-Blot SD cell. For protein transfers, the single buffer system of Bjerrum and Schafer-Nielsen provides more efficient elution than the original isotachophoretic system of Khyse-Andersen, which requires the use of three different buffers.

  • Page 14: Examples Of Specific Protocols

    Note: Some pH electrodes will not perform a proper measurement for the pH of Tris buffers. If the pH of the buffer is not correct, check the electrode to be sure it is designed to function with Tris buffers. If the pH electrode works properly with Tris buffers, and the pH is below 9.0, remake the buffer.

  • Page 15: Dna Blotting (For Acrylamide Gels With Dna 250 Bp To ~1 Kb)

    (For agarose gels with DNA up to 23 kb, RNA up to 3.5 kb) Refer to the Trans-Blot SD DNA blotting kit instruction manual for transfer protocol and conditions. DNA or RNA cannot be blotted from agarose gels without the use of the Trans- Blot SD DNA blotting kit.

  • Page 16: Troubleshooting Guide

    Nitrocellulose membranes have been used extensively for protein binding and detec- 7,19-22 ® tion. They can easily be stained for total protein by a dye stain (Amido Black, Coomassie blue, Ponceau S, Fast Green FCF, etc. ), or the more sensitive Colloidal Gold Total Protein Stain, and also allow either RIA, FIA, or EIA.

  • Page 17: Poor Binding To Nitrocellulose Membrane

    6. Methanol in the transfer buffer is restricting elution of proteins from the gel. Elimination of methanol results in increased transfer efficiency, but it also diminishes binding to nitrocellulose. Use PVDF. 7. Protein is precipitating in the gel. Try using SDS in the transfer buffer. SDS can increase transfer efficiency, but can also reduce binding efficiency to nitrocellulose and affect reactivity of some proteins with antibodies.

  • Page 18: Poor Detection Sensitivity Or No Reactivity

    8.4 Poor Detection Sensitivity or No Reactivity 1. Consult detection kit manual. 2. Antigen binding is incomplete. See Troubleshooting Sections 8.1–8.3. 3. Antibody reaction times are insufficient. Increase reaction times. 4. Sample load is insufficient. Increase the protein concentration applied to the gel. 5.
  • Page 19 Bio-Rad Laboratories Life Science Website www.bio-rad.com Bio-Rad Laboratories Main Office 2000 Alfred Nobel Drive, Hercules, CA 94547, Ph. (510) 741-1000, Fx. (510)741-5800 Also in: Australia Ph. 02 9914 2800, Fx. 02 9914 2889 Austria Ph. (01) 877 89 01, Fx. (01) 876 56 29 Belgium Ph. 09-385 55 11, Fx. 09-385 65 54 Group Canada Ph.

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