Transfer Buffers for Western Blotting

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.

Transfer membranesTransfer systems

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 transfer buffer
Reduced samples: Antioxidant
Broad-range MW (6-400 kDa) NuPAGE Bis-Tris NuPAGE 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

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
* For gels thicker than 1 mm or for homemade gel formations, add 2 minutes of transfer time.


Ordering information

Transfer buffer formulations

The following tranfer 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

  1. 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
  2. Mix well and adjust volume to 500 mL with ultrapure water.
  3. Before western transfer, dilute to 1X with water.
  4. Buffer is stable for 6 months at room temperature.


20X Bis-Tris Transfer buffer

  1. 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
  2. Mix well and adjust volume to 125 mL with ultrapure water.
  3. Before western transfer, dilute to 1X with water.
  4. 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 transfer buffer
Reduced samples: Antioxidant
Broad-range MW (6-400 kDa) NuPAGE Bis-Tris NuPAGE 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

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
* For gels thicker than 1 mm or for homemade gel formations, add 2 minutes of transfer time.


Ordering information

Transfer buffer formulations

The following tranfer 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

  1. 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
  2. Mix well and adjust volume to 500 mL with ultrapure water.
  3. Before western transfer, dilute to 1X with water.
  4. Buffer is stable for 6 months at room temperature.


20X Bis-Tris Transfer buffer

  1. 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
  2. Mix well and adjust volume to 125 mL with ultrapure water.
  3. Before western transfer, dilute to 1X with water.
  4. Buffer is stable for 6 months when stored at 4°C.

Resources

Download the Power Blotter brochure

Download HMW protein transfer application note

Download free protein transfer technical handbook