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

(1-800424-6723)

Table of Contents


Section 1 Introduction

1.1 Specifications


Section 2 (removed) Equipment and Reagents
2.1 Equip ment and Accessories
2.2Related Instruments

2.3 Chemical Reagents


Section 3 Safety Instructions

Section 4 Trans-Blot SD Assembly

4.1 Preparation for Blotting
4.2 Assembly of the Unit for Standard Transfers
4.3 Assembly of the Unit for Acidic Transfers


Section 5 Buffer Formulation

Section 6 Examples of Specific Protocols

6.1 SDS-Protein Blotting
6.2 DNA Blotting (For acrylamide gels with DNA 250 bp to 1 kb)
6.3 DNA RNA Blotting (For agarose gels with DNA up to 23 kb,

RNAupto3.Skb)

Section 7 Properties of Protein Blotting Niedia

Section 8 Troubleshooting Guide

8.1 Poor Transfer
8.2 Poor Binding to Nitrocellulose Membrane
8.3 High Background After Incubation with Antibody Probes; Nonspecific

or Nonquantitative Detection

8.4 Poor Detection Sensitivity or No Reactiviry


Section 9 References


Section 1
Introduction

1.1Specifications

Construction:

Trans-Blot SD body Molded polycarbonate
Anode Platinum-coated titanium
Cathode Stainless steel
Anode platform Precision machined acrylic

Overall size:37 cm x 24 cm x 11 cm
Maximum gel size: 25cm x 18.5cm
Cleaning:Do not immerse the unit in liquid. Use special care when cleaning the anode plate to avoid scratching or marring the platinum. Do not use abrasives or strong detergents. The cathode plate (stainless steel) can be cleaned with a mild abrasive to remove sait that may deposit during norma operation. The entire unit can also be periodically disassembled and cleaned with water to remove salt deposits.

Chemical compatibility:The semi-dry blotter components are not compatible with chlorinated hydrocarbons (e.g., chloroform), aromatic hydrocarbons (e.g., toluene, benzene), or acetone. Use of organic solvents voids all warranties.


Section 2 .... Dull catalogue numbers!

Section 3

Safety Instructions

Read the entire manual before beginning electrophoretic transfers.

5.Power supply requirements. The Trans-Blot SD c~l should only be used with the micro processor-controlled Model 200/2.0 power supply (catalog numbers 1654761 and 165- 4762), or the Model 1000/500 power supply (catalog numbers 165-4710 and 165-4711). Do not use the Model 250/2.5 power supply with this apparatus. The low voltage, high current operating conditions of the Trans-Blot SD cell are not compatible with the Model 250/2.5 power supply, and will cause the power supply to blow a fuse.
6.Do not operate this instrument in ambient temperatures exceeding 50 0C. Important

This Bio-Rad instrument is designed and certified to meet IEC l010-l* safety standards.Certified products are safe to use when operated in accordance with the instructtion manual. This instrument should not be modified in any way. Alteration of this instrument will:
°Void the manufacturer's warranty
Void the IEC1O1O-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.


1O10-I is an intemarionaily accepted electical satety standard for laboratory instruments.
Section 4
Tans-Blot SD Assembly

4.1Preparation for Blotting
1.

Prepare the transfer buffer. See Section 5 for buffer formulation.

Note:Buffer preparation is extremely important. Do not adjust transfer buffer pH by addition of acid or base unless specifically indicated in the instructions. Improperly prepared buffer will cause excess heat generation and safety hazards. Use only high quality, reagent grade methanol. Contaminated methanol can result in increased transfer buffer conductivity, as well as poor transfer of macromolecules.

2.Following electrophoresis, equilibrate the gels in transfer buffer. Equilibration facilitates the removal of electrophoresis buffer salts and detergents. If the salts are not removed, they will increase the conductivity of the transfer buffer and the amount of heat generated during the transfer. Also, low percentage gels (<12% acrylarnide) will shrink in methanol-containing buffers. Equilibration allows the gel to adjust to its final size prior to electrophoretic transfer. The length of time required for equilibration is dependent on the gel thickness. For example, 15 minutes for a 0.75 mm SDS-PAGE gel. Low molecular weight macromolecules (<10000 Daltons) may diffuse out of gels more readily. One can allow adequate gel pre-equllibration by changing the pre-equilibration buffer several times during a relatively short pre -equilibration period. This will help to limit diffasion of low molecular weight macromolecules while providing efficient salt reduction.

3.Cut the membrane to the dimensions of the gel. Wet the membrane by slowly sliding it at a 450 angle into transfer buffer and allowing it to soak for 15-30 minutes. Complete wetting of the membrane is important to insure proper binding. Abrupt wetting can lead to entrapment of air bubbles in the matrix. These air bubbles can block transfer of molecules. To avoid membrane contamination, always use forceps or wear gloves when handling membranes.
4.Cut filter paper to the dimensions of the gel. Two pieces of thick filter paper (or six pieces of thin filter paper) per gel are needed for each gel/membrane sandwich. Completely saturate the filter paper by soaking in transfer buffer. If more than one full-size gel is to be transferred at one time, cut a piece of dialysis membrane with the appropriate molecular weight cutoff to the dimensions of the gel. Completely wet die dialysis membrane in transfer 'uuffer. Spectr/Por dialysis membrane is recommended for this use.


4.2 Assembly of the Unit for Standard Transfers
Wear gloves for this procedure to avoid contamination of membranes.

1.Remove the safety cover and the stalniess steel cathode assembly.

2.Place a pre-soaked sheet of 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 thin filter paper is used, repeat with two more sheets of buffer-soaked 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. Transfer will be incomplete if any portion of the gel is outside the blotting media. Roll out all air bubbles.


5.

Place another sheet of pre-soaked filter paper on top of the gel, carefully removing air bubbles from between the gel and filter paper. If thin filter paper is used. place three sheets on top of the gel. and remove bubbles from between each layer.


6.If more than one full-size gel is to be transferred. place a sheet of pre-soaked dialysis membrane on top of the filter paper stack. Repeat the procedure from step 2. Up to four mini gels can be transferred at the same time by placing them side-by-side on the anode platform.

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. Transfer minl gels for 15-30 minutes at 10-15 V. Large gels can be transferred for 30 minutes to 1 hour at 15-25 V. do not exceed 25 V with this instrument. A current limit (3 mA/cm2 for large gels; 5.5 mA/cm2 for mini gels) is recommended to prevent excessive heating during the run. Under the strong fields developed by this apparatus, transfers may not always be quantitative. A certain quantity of protein may be transferred thiough the membrane and onto the filter paper below.
The Model 200/2.0 power suppiy is capable of a 200 waat output. This means that unless a current limit is set, uncontrolled conductivity changes may result in full power being delivered to the 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. Refer to the Model 200/2.0 Instruction Manual for setting current limits and run times, and monitoring these parameters.
10.Following transfer, turn the power supply off, and disconnect the unit from the power supply. Remove the safety cover and the cathode assembly discard the filter paper (and dialysis membrane, if used). The transfer efficiency can be monitored by staining the gel with Coomassie blue R-250 protein stain or with Bio-Rad's Silver Stain Kit. Alternatively, prestained molecular weight standards can be used, or a portion of the membrane can be stained for total protein with colloidal gold. Biotin Blot Total Protein Stain, or an anionic dye such as Amido Black. Zeta-Probe membrane can be stained with the Biotin-Blot Total Protein Stain.

4.3Assembly of the Unit for Acidic Transfers

If an acidic transfer buffer is used, the transfer direction will be from the anode to the cathode.

4.Place the pre-wetted blotting media on top of the gel. Roll out all air bubbles.

5.Place another sheet of pre-soaked ifiter paper on top of the blotting membrane. carefully removing all air bubbles. If thin ifiter paper is used, place three sheets on top of the membrane.

6.If more than one gel is to be transferred, place a sheet of pre-soaked dialysis membrane on top of the filter paper stack. Repeat the procedure from step 2.
7.Carefully place the cathode assembly onto the stack. Press to engage the latches with the guide posts, without disturbing the filter paper stack.
S.Place the safety cover on the unit. Plug the unit into the power supply, red wire to red outlet and black wire to black outlet.
Caution:Do not reverse polarity. This will damage the stainless steel cathode.
9.Turn on the power supply. Transfer mini gels for 15-30 minutes at 10-15 V. Large gels can be transferred for 30 minutes to 1 hour at 15-25 V. Do not exceed 25 V with this instrument. A current limit (3 mA/cm2 for large gels; 5.5 mA/cm2 for mini gels) is recommended to prevent excessive heating during the run.

Section 5
Buffer Formulation

48 mM Tris, 39 mM glycine, (20% methanol) pH 9.2

DO NOT ADD ACID OR BASE TO ADJUST pH. The buffer will range from pH 9.0 to 9.4, depending on the quality of the Tris, glycine, ddH2O, and methanol. Methanol should be analytical reagent grade, because metallic contaminants in low grade methanol will plate on the electrodes.
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 eludon 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 0.0375 g SDS or 3.75 ml of 10% SDS in ddH2O (add 200 ml of methanol); adjust the volume to 1 liter with ddH2O.
DO NOT ADD ACID OR BASE TO ADJUST pH.
3.Towbin transfer buffer for SDS-proteins using nltrocellulose (with methanol) or Zeta- Probe membrane (without methanol):7

25 mM Tris, 192 mM glycine (20% methanol), pH 8.3
Dissolve 3.03 g Tris and 14.4 g glycine in dd H2O (add 200 ml of methanol); adjust volume to 1 liter with dd H2O.
DO NOT ADD ACID OR BASE TO ADJUST pH.
4.Dunn carbonate transfer buffer for SDS-proteins using nitrocellulose (with methanol) or Zeta-Probe membrane (without methanol):17

l0mM NaCHO3, 3 mM Na2CO3 (20% methanol), pH 9.9
Dissolve 0.84 g NaHCO3 and 0.318 g Na2C03 (anhydrous) in dd H2O (add 200 ml of methanol); adjust volume to 1 liter with dd H2O.
DO NOT ADD ACID OR BASE TO ADJUST pH.
5.DNA transfer buffer for use with Zeta-Probe membrane:18

5x TBE stock solution (0.5 M Tris. 0.5 M boric acid, 10 mM EDTA in dd H2O; adjust volume to 1 liter with dd H2O. Dilute to 0.5x TBE with dd H2O for the working solution.
DO NOT ADD ACID OR BASE TO ADJUST pH.
6.5x dye buffer (20% Ficoll, 20 mM EDTA, 1% SDS, 0.2% bromophenol blue)


Section 6
Examples of Specific Protocols

Note: In order to determine the optimum conditions for a particular sample. a time course of transfer should be performed. Since many factors affect transfer, e.g., molecular weight, p1, porosity of the gel, it may not be necessary to transfer for the full time or to use high field intensity transfer conditions. Final transfer conditions for any protein should be determined empirically.
6.1SDS-Protein Blotting
Standard Blot to Nitrocellulose
1.Equilibrate the gel in 500 ml of Towbin buffer (Section 5) for 15 minutes.
2.Pre-chill buffer prior to transfer.
3.Assemble the sandwich as described in Section 4.2.
4.Refer to Section 4.2, step 9 for transfer conditions with either large or small gels.

6.2DNA 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 lx.
2.Mix 10-15 ul of the sample with 5 ul of 5x dye buffer, heat to 65 0C 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 lx ThE buffer at 100 V for 1-2 hours.

Standard Blot to Zeta-Probe
1.From the 5x TBE electrophoretic buffer, dilute the stock to 0.5x (Section 5) and pre-chill 1 L of the buffer.
2.Equilibrate the gel, extra thick blot paper, and Zeta-Probe membrane in 0.5x TBE buffer for at least 15 minutes.

Note: Zeta-Probe membrane will bind non-denatured nucleic acids. Therefore, denaturing is not mandatory before transferring. If non-denatured nucleic acids are transferred, the blotted Zeta-Probe membrane must be treated with NaOH prior to hybridization. Refer to the Zeta-Probe membrane instruction manual.
3.Assemble the sandwich as described in Section 4.2.
4.Run the transfer at 400 mA for 1 hour (voltage should not exceed 25 volts).
5.After transfer, separate the membrane from the gel, and rinse the membrane briefly in 0.5x TBE buffer.
6.Fix the DNA to the membrane by placing the membrane on several pieces of blot paper saturated with 0.4 N NaOH for 10 minutes.
7.Rinse the membrane in 2 x SSC for 10 minutes and bake at 80 0C for 1 hour (this is optional if probing immediately). The membrane is now ready for hybridization. Refer to the hybridization procedure in the Zeta-Probe blotting membrane instruction manual.
6.3DNA RNA Blotting
(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.

Section 7
Properties of Protein Blotting Media

Section 8
Troubleshooting Guide

8.1Poor Transfer
A.Molecules remain in the gel matrix (as detected by Coomassie blue or silver staining the gel).
1.Gel percentage is too high. Reduce %T (total monomer) or %C (crosslinker). A 5% C (with bis as the crosslinker) will produce the smallest pore size gel. Decreasing from this concentration will increase pore size and increase transfer efficiency.
2.Transfer time is too short. Increase time of transfer.
3.Charge to mass ratio is incorrect. Proteins near their isoelectric point at the pH of the buffer will transfer poorly. Try a more basic or acidic transfer buffer to increase protein mobility.
4.Protein is precipitating in the gel. Try using SDS in the transfer buffer. SDS can increase transfer efficiency, butcan also reduce binding efficiency to nitrocellulose and affect reactivity of some proteins with antibodies.

5.Power supply circuit tripped. Check the fuse.
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. Therefore, use Zeta-Probe membrane instead of nitrocellulose when methanol is not added to the transfer buffer.

7.Filter paper is too dry; insufficient buffer soaking the filter paper. Buffer is depleted early in the transfer. The filter paper should be fully saturated with buffer prior to transfer. Increase the number of sheets of filter paper, or use thicker Filter paper.

B.Swirls or missing patterns on blot; diffuse transfers.


1. Contact between blot membrane and gel is poor. Air bubbles or excess moisture remain between the blot and gel. Use a test tube or pipet to roll over the membrane carefully in both directions until excess moisture and air bubbles are removed from between gel and membrane and complete contact is established. Use thicker filter paper in the gel/membrane sandwich. Make sure that there are no air bubbles trapped between the ifiter paper and the gel.

2.The gel is not completely equilibrated in transfer buffer. Gel must be properly washed in transfer buffer to avoid shrinking or swelling during transfer. Increase time or number of washes.


3.If multiple gels are being transferred simultaneously, cross-contamination may be occurring. Use a smaller size pore dialysis membrane to separate gel/membrane sandwiches. Use the Trans-Blot cell or mini Trans-Blot cell for multiple transfers.

4.Power conditions are too high. Reduce the voltage. Check the buffer conductivity; improperly prepared buffer will result in excessive power delivered to the cell.

8.2 Poor Binding to Nitrocellulose Membrane.
1.Proteins separated by SDS-PAGE require 20% methanol in the transfer buffer for optimal protein binding. Make sure the buffer contains the proper amount of methanol.

2.Mixed ester celluloses bind proteins poorly. Use Bio-Rad's pure nitrocellulose.

3.Proteins may be transferring through the nitrocellulose, driven by the high field strength of the plate electrodes. Use Zeta-Probe membrane (higher binding capacity) or 0.2 micron nitrocellulose (smailer pore size). Transfer using the Trans-Blot cell or the Mini Trans-Blot cell with standard platinum wire electrodes.
4.Protein >15,000 daltons may show diminished binding to 0.45 micron nitrocellulose, or may be washed from the membrane during assays. Use Zeta-Probe membrane or 0.2 micron nitrocellulose. To increase stability of binding, proteins can be cross-linked to nitrocellulose with glutaraldehyde.22


5.Proteins can be removed from nitrocellulose by SDS, NP-40, and several other detergents. Use Tween-20 detergent in wash and antibody incubation steps. Reduce or eliminate detergents from buffers. Try gluteraldehyde fixation.

6.SDS in the transfer buffer will reduce binding efficiency of proteins. Use 20% methanol iri the transfer buffer and equilibrate the gel in methanol buffer prior to transfer.


8.3High Background After Incubation with Antibody Probes; Nonspecific or Nonquantitative Detection.

For a complete troubleshooting guide to Immun-Blot assays, consult the Immun-Blot assay kit manual or the Zeta-Probe instruction manual.


1.Blocking conditions are inappropriate. Be sure the blocker is pure protein. Increase the concentration or blocking time as necessary. Zeta-Probe membranes require more extensive blocking than nitrocellulose. e.g., with 5% non-fat dry milk (BLOTTO).3 Hemoglobin reacts with horseradish peroxidase: BSA may contain contaminants that react with lectins. etc. Match the blocker (0 the detection system.

2.Monoclonal antibodies may react nonspecifically (or not at all, see Troubleshooting Section W) with SDS denatured proteins. Compare binding of other monoclonals or polyclonal antibodies. ~ot native proteins.

3.Second antibody is impure. Use Bio-Rad's affinity purified blotting grade antibody conjugates.

4.Primary or second antibody is too concentrated. Dilute the antibodies appropriately.

5.First or second antibody is contaminated with nonspecific or species cross-reactive IgG. Use purified IgG first antibody fraction and affinity purified blotting grade second antibody.

6.Washes are insufficient. Increase the number and/or duration of washes. Include progressively stronger detergents in washes, e.g., SDS>NP-40>Tween-20. Also, include Tween-20 detergent in antibody buffers to reduce non-specific binding.

7.Reaction time in the substrate is excessive. Remove the blot from the substrate reaction when the signai~to-noise level is acceptable; do not over-develop.

8.4Poor Detection SensitiviW or No Reactivity.

1.Antigen binding is incomplete. See Troubleshooting Sections 8.1 - 8.3.

2.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.

3.Monoclonal antibodies might not recognize a denatured antigen. Assess binding of other monoclonals or polyclonal antibodies. Blot native proteins.

4.Enzyme conjugate or substrate inactivated. First or second antibody is inactive or non- saturating. Test enzyme, antibody, and substrate separately for activity. Increase concentration of first or second antibody. Eliminate detergents from reactions and washes. Avoid sodium azide when working with horseradish peroxidase, as it is a potent inhibitor of the enzyme.

5.For autoradiographs, exposure time is insufficient. Increase time to increase signal-to- noise ratio.

6.Antibody reaction times are insufficient. Increase reaction times.

7.Sample load is insufficient. Increase the protein concentration applied to the gel.

Section 9 References

*spectr/por is a trademark of Spectrum Medkai tndus'ries. Coomas sic is a trademark of I C I Organics. Inc.
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