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.
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.
Table of Contents Page Section 1 Introduction .................................................................................................. 1 1.1 Specifications ............................................................................................................. 1 Section 2 Equipment and Reagents ............................................................................ 2 2.1 2.2 2.3 Equipment and Accessories ............................................................................
Section 1 Introduction Blotting was first performed by Southern1 in 1975 with the transfer of DNA from agarose gels to nitrocellulose membranes. Blotting has subsequently been applied to RNA2-4 and protein5,6 from both agarose and polyacrylamide gels. Membrane materials have been expanded to include PVDF for improved protein binding capacity.
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 170-3947 Trans-Blot SD Anode, platinum Trans-Blot SD Cathode, stainless steel DNA Blotting Kit 170-3957 Trans-Blot SD DNA/RNA Blotting Kit Power Supply 165-4761 165-4762 Model 200/2.0 Constant Voltage Power Supply, 100/120 V, 50/60 Hz Model 200/2.
Catalog Number Product Description Nitrocellulose Membrane (0.2 micron) 162-0112 Roll, 33 cm x 3 m, 1 162-0146 Sheets, 7 x 8.4, 10 162-0147 Sheets, 13.5 x 16.5 cm, 10 DNA/RNA Blotting Accessories (Blot paper) 170-3958 Extra Thick Blot Paper, 10 x 15 cm, 30 170-3959 Extra Thick Blot Paper, 15 x 15 cm, 30 170-3960 Extra Thick Blot Paper, 15 x 20 cm, 30 Zeta-Probe Membrane 162-0159 Roll, 30 cm x 3.
2.
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 161-0307 Biotinylated SDS-PAGE Standards Kit, Low range, HRP 161-0308 Biot
4. Lengthy transfer times are not recommended. Do not leave this instrument unattended. 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). Do not use the Model 250/2.
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. Cut the membrane to the dimensions of the gel.
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 buffersoaked filter paper. 3. Place the pre-wetted blotting media on top of the filter paper. Roll out all air bubbles. 4. Carefully place the equilibrated gel on top of the transfer membrane, aligning the gel on the center of the membrane.
7. Carefully place the cathode onto the stack. Press to engage the latches with the guide posts without disturbing the filter paper stack. 8. Place the safety cover on the unit. Plug the unit into the power supply. Normal transfer polarity is cathode to anode, i.e., red wire to red outlet and black wire to black outlet on the power supply. Caution: Do not reverse polarity. This will result in damage to the stainless steel cathode. 9. Turn on the power supply.
4.3 Assembly of the Unit for Acidic Transfers If an acidic transfer buffer is used, the transfer direction will be from the anode to the cathode. 1. Remove the safety cover and the stainless steel cathode assembly. 2. Place a pre-soaked sheet of extra thick filter paper onto the platinum anode. Roll out all air bubbles. If thin filter paper is used, repeat with two more sheets of buffer-soaked filter paper. If thick filter paper is used, repeat with one more sheet of buffer soaked filter paper. 3.
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. 2. SDS may be added to Buffer 1 to increase protein elution from the gel: 48 mM Tris, 39 mM glycine, (20% methanol), 1.3 mM SDS (0.0375%), pH 9.2 Dissolve 5.82 g Tris and 2.93 g glycine, and 0.
6.2 DNA Blotting (For acrylamide gels with DNA 250 bp to ~1 kb) Electrophoresis Run on a Polyacrylamide Gel 1. Prepare the stock electrophoresis 5x TBE buffer (Section 5). Dilute the stock to 1x. 2. Mix 10–15 µl of the sample with 5 µl of 5x dye buffer, heat to 65 °C for 5 min and load on a gel. 3. A 5% PAGE gel can separate DNAs from about 250 to 1,000 bp. 4. Run the gel in 1x TBE buffer at 100 V for 1–2 hours. Standard Blot to Zeta-Probe 1. From the 5x TBE electrophoretic buffer, dilute the stock to 0.
Nitrocellulose membranes have been used extensively for protein binding and detection.7,19-22 They can easily be stained for total protein by a dye stain (Amido Black, Coomassie® blue, Ponceau S, Fast Green FCF, etc.22), or the more sensitive Colloidal Gold Total Protein Stain, and also allow either RIA, FIA, or EIA.7 Nitrocellulose has a high binding capacity of 80–100 µg/cm2. Nonspecific protein binding sites are easily and rapidly blocked, avoiding subsequent background problems.
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. B. Swirls or missing patterns on blot; diffuse transfers 1.
8.4 Poor Detection Sensitivity or No Reactivity 1. 2. 3. 4. 5. Consult detection kit manual. Antigen binding is incomplete. See Troubleshooting Sections 8.1–8.3. Antibody reaction times are insufficient. Increase reaction times. Sample load is insufficient. Increase the protein concentration applied to the gel. Antigen may require specific temperature regulation during transfer to prevent denaturation. Use the Trans-Blot cell with the super cooling coil to transfer heat-sensitive proteins. 6.
Bio-Rad Laboratories Life Science Group Bulletin 0000 US/EG 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 Canada Ph. (905) 712-2771, Fx. (905) 712-2990 China Ph. 86-10-62051850/51, Fx. 86-10-62051876 Denmark Ph. 45 39 17 99 47, Fx. 45 39 27 16 98 Finland Ph.
