Protein Electrophoresis and Western Blotting Education Center
Premixed transfer buffers are a convenient way to ensure high-quality, consistent gel transfer. We offer premixed western blot transfer buffers that are optimized for each gel chemistry. Use the tabs below to see the recommended transfer buffers and transfer conditions for the gel chemistry being used for western blot wet or semi-dry transfer. Find the suggested transfer buffer recipes for wet transfer below.
Recommended transfer buffers for Bis-Tris and Tris-Acetate Systems
| Protein sample | Gel chemistry | Transfer buffer |
|---|---|---|
| Broad-range MW (6-400 kDa) | Bolt Bis-Tris Plus | Bolt Bis-Tris transfer buffer Reduced samples: Antioxidant |
| Broad-range MW (6-400 kDa) | NuPAGE Bis-Tris | NuPAGE Bis-Tris transfer buffer Reduced samples: Antioxidant |
| High MW (40-500 kDa) | NuPAGE Tris-Acetate |
Transfer conditions using the Mini Bolt Module (Mini Gel Tank)
| Gel type | Membrane | Transfer buffer | Transfer conditions | Expected current* | Run time |
|---|---|---|---|---|---|
| Bolt Bis-Tris Plus | Nitrocellulose | Bis-Tris transfer buffer with 10% methanol and antioxidant for reduced samples | 10 V constant | Start: 160 mA End: 60 mA | 1 hr |
| PVDF | 20 V constant | Start: 340 mA End: 130 mA | 1 hr | ||
| NuPAGE Bis-Tris | Nitrocellulose | 10 V constant | Start: 160 mA End: 60 mA | 1 hr | |
| PVDF | 20 V constant | Start: 390 mA End: 130 mA | 1 hr | ||
| NuPAGE Tris-Acetate | Nitrocellulose | 10 V constant | Start: 150 mA End: 50 mA | 1 hr | |
| PVDF | 20 V constant | Start: 380 mA End: 130 mA | 1 hr |
* Current readings represent values when running a single gel, and can vary depending upon the power supply being used.
Transfer conditions using the XCell II Blot Module (SureLock Tank)
| Gel type | Membrane | Transfer buffer* | Transfer conditions | Expected current** | Run time |
|---|---|---|---|---|---|
| Bolt Bis-Tris Plus and NuPAGE Bis-Tris | Nitrocellulose or PVDF | Bis-Tris transfer buffer with 10% methanol and antioxidant for reduced samples | 30 V constant | Start: 170 mA End: 100 mA | 1 hr |
| NuPAGE Tris-Acetate | Nitrocellulose or PVDF | Bis-Tris transfer buffer with 10% methanol and antioxidant for reduced samples | 30 V constant | Start: 220 mA End: 180 mA | 1 hr |
* Transfer buffer with 10% methanol provides optimal transfer for a single gel in the blot module. If you are transferring 2 gels, increase the methanol content to 20% to ensure efficient transfer.
** Current readings represent values when running a single gel, and can vary depending upon the power supply being used.
Bolt Bis-Tris Plus System
NuPAGE System
Recommended transfer buffers for Tris-Glycine and Tricine Systems
| Protein sample | Gel chemistry | Transfer buffer |
|---|---|---|
| Broad-range MW (6-400 kDa) | Novex Tris-Glycine | Tris-Glycine transfer buffer |
| Low MW (2.5-40 kDa) | Novex Tricine |
Transfer conditions using the Mini Bolt Module (Mini Gel Tank)
| Gel type | Membrane | Transfer buffer | Transfer conditions | Expected current* | Run time |
|---|---|---|---|---|---|
| Novex Tris-Glycine | Nitrocellulose | Tris Glycine Transfer Buffer with 20% methanol | 10 V constant | Start: 70 mA End: 50 mA | 1 hr |
| PVDF | 20 V constant | Start: 160 mA End: 100 mA | 1 hr | ||
| Novex Tricine | Nitrocellulose | 10 V constant | Start: 70 mA End: 60 mA | 1 hr | |
| PVDF | 20 V constant | Start: 180 mA End: 150 mA | 1 hr |
* Current readings represent values when running a single gel, and can vary depending upon the power supply being used.
Transfer conditions using the XCell II Blot Module (SureLock Tank)
| Gel type | Membrane | Transfer buffer | Transfer conditions | Expected current* | Run time |
|---|---|---|---|---|---|
| Novex Tris-Glycine and Novex Tricine | Nitrocellulose or PVDF | Tris Glycine Transfer Buffer with 20% methanol. 1X Transfer Buffer should be pH 8.3 before addition of SDS or methanol. Do not adjust the pH. | 25 V constant | Start: 100 mA | 1-2 hr |
* Current readings represent values when running a single gel, and can vary depending upon the power supply being used.
Tris-Glycine and Tricine Systems
Recommended transfer buffers for Semi-dry Transfer
| Gel type | Transfer buffer |
|---|---|
| All gel chemistries | Power Blotter 1-Step Transfer Buffer (5X) Pierce 1-Step Transfer Buffer (1X) |
1-Step Transfer buffer is a high ionic strength formulation which allows for 5-minute to 12-minute protein transfer when used with compatible semi-dry blotting systems. 1-Step Transfer Buffer is compatible with Power Blotter and other protein semi-dry transfer devices, when they are paired with a suitable high-current power supply. Such devices should provide constant high current (1.3 to 5.0 amps) to rapidly transfer proteins via the high ionic strength conditions supplied by the transfer buffer.
Transfer conditions using the Power Blotter System
| Gel Size | Surface area (cm2) | Constant current (A) | Voltage limit (V) | Recommended transfer time (min)* | ||
|---|---|---|---|---|---|---|
| Low MW | Mixed MW | High MW | ||||
| 1 mini gel | ~60 | 1.3 | 25 | 5 | 7 | 10 |
| 2 mini gels or 1 midi gel | ~120 | 2.5 | 25 | 5 | 7 | 10 |
| 3 mini gels | ~180 | 3.8 | 25 | 5 | 7 | 10 |
| 4 mini gels or 2 midi gels | ~240 | 5.0 | 25 | 5 | 7 | 10 |
Ordering information
Transfer buffer formulations
The following transfer buffer recipes are provided to allow preparation of buffers from scratch. The pH listed for each buffer is for the 1X solution. Do not use acid or base to adjust the pH.
25X Tris-Glycine transfer buffer
- Dissolve the following reagents in 400 mL ultrapure water
Reagent Amount Concentration (1X) Tris Base 18.2 g 12 mM Glycine 90 g 96 mM - Mix well and adjust volume to 500 mL with ultrapure water.
- Before western transfer, dilute to 1X with water.
- Buffer is stable for 6 months at room temperature.
20X Bis-Tris Transfer buffer
- Dissolve the following reagents in 100 mL ultrapure water
Reagent Amount Concentration (1X) Bicine 10.2 g 25 mM Bis-Tris (free base) 13.1 g 25 mM EDTA 0.75 g 1 mL - Mix well and adjust volume to 125 mL with ultrapure water.
- Before western transfer, dilute to 1X with water.
- Buffer is stable for 6 months when stored at 4°C.
Recommended transfer buffers for Bis-Tris and Tris-Acetate Systems
| Protein sample | Gel chemistry | Transfer buffer |
|---|---|---|
| Broad-range MW (6-400 kDa) | Bolt Bis-Tris Plus | Bolt Bis-Tris transfer buffer Reduced samples: Antioxidant |
| Broad-range MW (6-400 kDa) | NuPAGE Bis-Tris | NuPAGE Bis-Tris transfer buffer Reduced samples: Antioxidant |
| High MW (40-500 kDa) | NuPAGE Tris-Acetate |
Transfer conditions using the Mini Bolt Module (Mini Gel Tank)
| Gel type | Membrane | Transfer buffer | Transfer conditions | Expected current* | Run time |
|---|---|---|---|---|---|
| Bolt Bis-Tris Plus | Nitrocellulose | Bis-Tris transfer buffer with 10% methanol and antioxidant for reduced samples | 10 V constant | Start: 160 mA End: 60 mA | 1 hr |
| PVDF | 20 V constant | Start: 340 mA End: 130 mA | 1 hr | ||
| NuPAGE Bis-Tris | Nitrocellulose | 10 V constant | Start: 160 mA End: 60 mA | 1 hr | |
| PVDF | 20 V constant | Start: 390 mA End: 130 mA | 1 hr | ||
| NuPAGE Tris-Acetate | Nitrocellulose | 10 V constant | Start: 150 mA End: 50 mA | 1 hr | |
| PVDF | 20 V constant | Start: 380 mA End: 130 mA | 1 hr |
* Current readings represent values when running a single gel, and can vary depending upon the power supply being used.
Transfer conditions using the XCell II Blot Module (SureLock Tank)
| Gel type | Membrane | Transfer buffer* | Transfer conditions | Expected current** | Run time |
|---|---|---|---|---|---|
| Bolt Bis-Tris Plus and NuPAGE Bis-Tris | Nitrocellulose or PVDF | Bis-Tris transfer buffer with 10% methanol and antioxidant for reduced samples | 30 V constant | Start: 170 mA End: 100 mA | 1 hr |
| NuPAGE Tris-Acetate | Nitrocellulose or PVDF | Bis-Tris transfer buffer with 10% methanol and antioxidant for reduced samples | 30 V constant | Start: 220 mA End: 180 mA | 1 hr |
* Transfer buffer with 10% methanol provides optimal transfer for a single gel in the blot module. If you are transferring 2 gels, increase the methanol content to 20% to ensure efficient transfer.
** Current readings represent values when running a single gel, and can vary depending upon the power supply being used.
Bolt Bis-Tris Plus System
NuPAGE System
Recommended transfer buffers for Tris-Glycine and Tricine Systems
| Protein sample | Gel chemistry | Transfer buffer |
|---|---|---|
| Broad-range MW (6-400 kDa) | Novex Tris-Glycine | Tris-Glycine transfer buffer |
| Low MW (2.5-40 kDa) | Novex Tricine |
Transfer conditions using the Mini Bolt Module (Mini Gel Tank)
| Gel type | Membrane | Transfer buffer | Transfer conditions | Expected current* | Run time |
|---|---|---|---|---|---|
| Novex Tris-Glycine | Nitrocellulose | Tris Glycine Transfer Buffer with 20% methanol | 10 V constant | Start: 70 mA End: 50 mA | 1 hr |
| PVDF | 20 V constant | Start: 160 mA End: 100 mA | 1 hr | ||
| Novex Tricine | Nitrocellulose | 10 V constant | Start: 70 mA End: 60 mA | 1 hr | |
| PVDF | 20 V constant | Start: 180 mA End: 150 mA | 1 hr |
* Current readings represent values when running a single gel, and can vary depending upon the power supply being used.
Transfer conditions using the XCell II Blot Module (SureLock Tank)
| Gel type | Membrane | Transfer buffer | Transfer conditions | Expected current* | Run time |
|---|---|---|---|---|---|
| Novex Tris-Glycine and Novex Tricine | Nitrocellulose or PVDF | Tris Glycine Transfer Buffer with 20% methanol. 1X Transfer Buffer should be pH 8.3 before addition of SDS or methanol. Do not adjust the pH. | 25 V constant | Start: 100 mA | 1-2 hr |
* Current readings represent values when running a single gel, and can vary depending upon the power supply being used.
Tris-Glycine and Tricine Systems
Recommended transfer buffers for Semi-dry Transfer
| Gel type | Transfer buffer |
|---|---|
| All gel chemistries | Power Blotter 1-Step Transfer Buffer (5X) Pierce 1-Step Transfer Buffer (1X) |
1-Step Transfer buffer is a high ionic strength formulation which allows for 5-minute to 12-minute protein transfer when used with compatible semi-dry blotting systems. 1-Step Transfer Buffer is compatible with Power Blotter and other protein semi-dry transfer devices, when they are paired with a suitable high-current power supply. Such devices should provide constant high current (1.3 to 5.0 amps) to rapidly transfer proteins via the high ionic strength conditions supplied by the transfer buffer.
Transfer conditions using the Power Blotter System
| Gel Size | Surface area (cm2) | Constant current (A) | Voltage limit (V) | Recommended transfer time (min)* | ||
|---|---|---|---|---|---|---|
| Low MW | Mixed MW | High MW | ||||
| 1 mini gel | ~60 | 1.3 | 25 | 5 | 7 | 10 |
| 2 mini gels or 1 midi gel | ~120 | 2.5 | 25 | 5 | 7 | 10 |
| 3 mini gels | ~180 | 3.8 | 25 | 5 | 7 | 10 |
| 4 mini gels or 2 midi gels | ~240 | 5.0 | 25 | 5 | 7 | 10 |
Ordering information
Transfer buffer formulations
The following transfer buffer recipes are provided to allow preparation of buffers from scratch. The pH listed for each buffer is for the 1X solution. Do not use acid or base to adjust the pH.
25X Tris-Glycine transfer buffer
- Dissolve the following reagents in 400 mL ultrapure water
Reagent Amount Concentration (1X) Tris Base 18.2 g 12 mM Glycine 90 g 96 mM - Mix well and adjust volume to 500 mL with ultrapure water.
- Before western transfer, dilute to 1X with water.
- Buffer is stable for 6 months at room temperature.
20X Bis-Tris Transfer buffer
- Dissolve the following reagents in 100 mL ultrapure water
Reagent Amount Concentration (1X) Bicine 10.2 g 25 mM Bis-Tris (free base) 13.1 g 25 mM EDTA 0.75 g 1 mL - Mix well and adjust volume to 125 mL with ultrapure water.
- Before western transfer, dilute to 1X with water.
- Buffer is stable for 6 months when stored at 4°C.
Download: Western Blotting Overview Brochure
Explore: Western Blot Protein Ladders
Download: HMW Protein Transfer Application Note
For Research Use Only. Not for use in diagnostic procedures.