Protein Electrophoresis and Western Blotting Education Center
One of the key steps in the western blot workflow is the transfer of proteins from the polyacrylamide gel after electrophoresis to the nitrocellulose or polyvinylidene difluoride (PVDF) membrane so that specific proteins can be detected using immune-detection techniques.
We have developed electrophoretic transfer systems for wet, semi-dry, and dry blotting methods. Use the table below to select the appropriate transfer method for your western blotting needs.
Which western blot transfer system is right for you?
| Wet transfer | Semi-dry transfer | Dry transfer | ||
|---|---|---|---|---|
 Mini Blot Module |  XCell II Blot Module |  SureLock Tandem Midi Blot Module |  Power Blotter Systems |  iBlot 3 Western Blot Transfer System |
| Capacity: 1 mini gel per blot module; 1–2 blot modules per tank | Capacity: up to 2 mini blots | Capacity: 1 midi gel per blot module; 1–2 blot modules per tank | Capacity: 1–4 mini or 1–2 midi gels | Capacity: 1–4 mini or 1–2 midi gels |
| Transfer time: 60 min | Transfer time: 60–120 min | Transfer time: 30 min | Transfer time: 5–10 min | Transfer time: as few as 3 min |
| Blotting area: 9 x 9 cm | Blotting area: 9 x 9 cm | Blotting area: 9.2 x 14.4 cm | Blotting area: 10 x 18 cm or 21 x 22.5 cm | Blotting area: 8.5 x 13.5 cm |
| Blotting area: Transfer buffer volume: 220 mL per blot module | Transfer buffer volume: 200 mL | Transfer buffer volume: 300 mL per blot module | Transfer buffer volume: Pre-cut membranes & filters: 50–100 mL; Pre-assembled Select transfer stacks: buffer not required | Transfer buffer volume: Buffer not required |
| Power supply: External | Power supply: External | Power supply: External | Power supply: Internal | Power supply: Internal |
| Required equipment: Mini Gel Tank: capacity for up to 2 Mini Blot Modules | Required equipment: XCell SureLock Mini-Cell: capacity for up to 2 blot modules | Required equipment: SureLock Tandem Midi Gel Tank: capacity for up to 2 blot modules | ––– | ––– |
| Learn more | Learn more | Learn more | Learn more | Learn more |
Comparison of western blot transfer methods
 |  |  | |
| Wet transfer | Semi-dry transfer | Dry transfer | |
|---|---|---|---|
| Setup | Hands-on, requires preparing transfer buffer ~15–20 min | ~5-10 min with Select stacks, ~10–15 min for pre-cut membranes and filters | ~5 min |
| Cleanup | Extensive clean-up after each use including hazardous methanol waste disposal | Light clean-up required after each use | Very minimal with extended used |
| Transfer time | 30–120 min | 7–10 min | as few as 3 min |
| Throughput | +++ | +++ | +++ |
| Performance | +++ | ++ | +++ |
| Ease of use | ++ | +++ | +++ |
| Transfer buffer requirements | Requires methanol | Methanol-free transfer buffers or no buffer required with pre-assembled stacks | No buffer required |
| Special considerations | Cooling may be required for longer transfers | Multiple systems can be utilized including Towbin buffers | Requires pre-assembled transfer stacks |
Comparison of wet, semi-dry and dry transfer methods
Efficient and reliable protein transfer from the gel to the blotting membrane is the cornerstone of a successful western detection experiment. Accuracy of results is dependent on the transfer efficiency of the western blotting method. Traditional wet transfer offers high efficiency, but at a cost of time and effort. Semi-dry blotting provides convenience and time savings, with flexibility to use multiple types of buffer systems or pre-assembled stacks that are buffer free. However, semi-dry transfer can have a lower efficiency of transfer of large molecular weight proteins (>300 kDa). Dry electroblotting offers both high quality transfer combined with speed as well as convenience since added buffers are not required for dry electroblotting.
Three major ways to transfer proteins from SDS-PAGE or native gels to nitrocellulose, PVDF or nylon membranes:
Wet tank electrotransfer
When performing a wet transfer, the gel is first equilibrated in transfer buffer. The gel is then placed in the “transfer sandwich” (filter paper-gel-membrane-filter paper), cushioned by pads and pressed together by a support grid. The supported gel sandwich is placed vertically in a tank between stainless steel/platinum wire electrodes and filled with transfer buffer.
Multiple gels may be electrotransferred in the standard field option, which is performed either at constant current (0.1 to 1 A) or voltage (5 to 30 V) from as little as 1 hour to overnight. Transfers are typically performed with an ice pack and at 4°C to mitigate the heat produced. A high field option exists for a single gel, which may bring transfer time down to as little as 30 minutes, but it requires the use of high voltage (up to 200 V) or high current (up to 1.6 A) and a cooling system to dissipate the tremendous heat produced.
Transfer efficiencies of 80–100% are achievable for proteins between 14–116 kDa. The transfer efficiency improves with increased transfer time. With increasing time, however, there is a risk of over-transfer (blew through) of the proteins through the membrane, especially for lower molecular weight (<30 kDa) proteins when using membranes with a larger pore size (0.45 µm).
Tank transfer apparatus for western blotting. Schematic showing the assembly of a typical western blot apparatus with the position of the position of the gel, transfer membrane, and direction of protein in relation to the electrode position.
Semi-dry electrotransfer
For semi-dry protein transfer, the transfer sandwich is placed horizontally between two plate electrodes in a semi-dry transfer apparatus. For this semi-dry transfer, it is very important that the gel is pre-equilibrated in transfer buffer. To maximize the current passing through the gel instead of around the gel, the amount of buffer available during transfer is limited to that contained in the sandwich, so it is helpful if the extra-thick filter paper (~3 mm thickness) and membrane are also sufficiently soaked in buffer. Likewise, it is key that the filter paper sheets and membrane are cut to the size of the gel.
One to four gels may be rapidly electroblotted to membranes. Methanol may be included in the transfer buffer, but other organic solvents, including aromatic hydrocarbons, chlorinated hydrocarbons and acetone, should not be used to avoid damage to the semi-dry blotter. Electrotransfer is performed either at constant current (0.1 up to ~0.4 A) or voltage (10 to 25 V) for 10 to 60 minutes. Methanol-free transfer buffers are recommended to reduce transfer time to 7 to 10 minutes. Transfer efficiencies of 60 to 80% may be achievable for proteins between 14 and 116 kDa, longer transfer times are required to transfer higher molecular weight proteins.
Semi-dry electroblotting transfer. The Invitrogen Power Blotter is designed specifically for rapid semi-dry transfer of 10–300-kDa proteins from polyacrylamide gels to nitrocellulose or PVDF membranes in 5 to 10 minutes. The Power Blotter features an integrated power supply optimized to enable consistent, high-efficiency protein transfer when used with commonly used precast or homemade gels (SDS-PAGE) and nitrocellulose or PVDF membranes.
Dry electrotransfer
Dry electroblotting methods use a specialized transfer sandwich containing innovative components that eliminate use of traditional transfer buffers. A unique gel matrix (transfer stack) that incorporates buffer is used instead of buffer tanks or soaked filter papers. The high ionic density in the gel matrix enables rapid protein transfer. During blotting, the copper anode does not generate oxygen gas as a result of water electrolysis, reducing blot distortion. Conventional protein transfer techniques, including wet and semi-dry, use inert electrodes that generate oxygen. Typically, transfer time is reduced by the shortened distance between electrodes, high field strength and high current.
Dry electroblotting transfer. The Invitrogen iBlot 3 Western Blot Transfer System provides fast western transfer without the need for buffers. This system efficiently blots proteins from acrylamide gels in as few as three minutes, and is compatible with both PVDF and nitrocellulose membranes. The iBlot 3 System has performance comparable to traditional wet transfer methods in a fraction of the time.
Which western blot transfer system is right for you?
| Wet transfer | Semi-dry transfer | Dry transfer | ||
|---|---|---|---|---|
Mini Blot Module | XCell II Blot Module | SureLock Tandem Midi Blot Module | Power Blotter Systems | iBlot 3 Western Blot Transfer System |
| Capacity: 1 mini gel per blot module; 1–2 blot modules per tank | Capacity: up to 2 mini blots | Capacity: 1 midi gel per blot module; 1–2 blot modules per tank | Capacity: 1–4 mini or 1–2 midi gels | Capacity: 1–4 mini or 1–2 midi gels |
| Transfer time: 60 min | Transfer time: 60–120 min | Transfer time: 30 min | Transfer time: 5–10 min | Transfer time: as few as 3 min |
| Blotting area: 9 x 9 cm | Blotting area: 9 x 9 cm | Blotting area: 9.2 x 14.4 cm | Blotting area: 10 x 18 cm or 21 x 22.5 cm | Blotting area: 8.5 x 13.5 cm |
| Blotting area: Transfer buffer volume: 220 mL per blot module | Transfer buffer volume: 200 mL | Transfer buffer volume: 300 mL per blot module | Transfer buffer volume: Pre-cut membranes & filters: 50–100 mL; Pre-assembled Select transfer stacks: buffer not required | Transfer buffer volume: Buffer not required |
| Power supply: External | Power supply: External | Power supply: External | Power supply: Internal | Power supply: Internal |
| Required equipment: Mini Gel Tank: capacity for up to 2 Mini Blot Modules | Required equipment: XCell SureLock Mini-Cell: capacity for up to 2 blot modules | Required equipment: SureLock Tandem Midi Gel Tank: capacity for up to 2 blot modules | ––– | ––– |
| Learn more | Learn more | Learn more | Learn more | Learn more |
Comparison of western blot transfer methods
| | | |
| Wet transfer | Semi-dry transfer | Dry transfer | |
|---|---|---|---|
| Setup | Hands-on, requires preparing transfer buffer ~15–20 min | ~5-10 min with Select stacks, ~10–15 min for pre-cut membranes and filters | ~5 min |
| Cleanup | Extensive clean-up after each use including hazardous methanol waste disposal | Light clean-up required after each use | Very minimal with extended used |
| Transfer time | 30–120 min | 7–10 min | as few as 3 min |
| Throughput | +++ | +++ | +++ |
| Performance | +++ | ++ | +++ |
| Ease of use | ++ | +++ | +++ |
| Transfer buffer requirements | Requires methanol | Methanol-free transfer buffers or no buffer required with pre-assembled stacks | No buffer required |
| Special considerations | Cooling may be required for longer transfers | Multiple systems can be utilized including Towbin buffers | Requires pre-assembled transfer stacks |
Comparison of wet, semi-dry and dry transfer methods
Efficient and reliable protein transfer from the gel to the blotting membrane is the cornerstone of a successful western detection experiment. Accuracy of results is dependent on the transfer efficiency of the western blotting method. Traditional wet transfer offers high efficiency, but at a cost of time and effort. Semi-dry blotting provides convenience and time savings, with flexibility to use multiple types of buffer systems or pre-assembled stacks that are buffer free. However, semi-dry transfer can have a lower efficiency of transfer of large molecular weight proteins (>300 kDa). Dry electroblotting offers both high quality transfer combined with speed as well as convenience since added buffers are not required for dry electroblotting.
Three major ways to transfer proteins from SDS-PAGE or native gels to nitrocellulose, PVDF or nylon membranes:
Wet tank electrotransfer
When performing a wet transfer, the gel is first equilibrated in transfer buffer. The gel is then placed in the “transfer sandwich” (filter paper-gel-membrane-filter paper), cushioned by pads and pressed together by a support grid. The supported gel sandwich is placed vertically in a tank between stainless steel/platinum wire electrodes and filled with transfer buffer.
Multiple gels may be electrotransferred in the standard field option, which is performed either at constant current (0.1 to 1 A) or voltage (5 to 30 V) from as little as 1 hour to overnight. Transfers are typically performed with an ice pack and at 4°C to mitigate the heat produced. A high field option exists for a single gel, which may bring transfer time down to as little as 30 minutes, but it requires the use of high voltage (up to 200 V) or high current (up to 1.6 A) and a cooling system to dissipate the tremendous heat produced.
Transfer efficiencies of 80–100% are achievable for proteins between 14–116 kDa. The transfer efficiency improves with increased transfer time. With increasing time, however, there is a risk of over-transfer (blew through) of the proteins through the membrane, especially for lower molecular weight (<30 kDa) proteins when using membranes with a larger pore size (0.45 µm).
Tank transfer apparatus for western blotting. Schematic showing the assembly of a typical western blot apparatus with the position of the position of the gel, transfer membrane, and direction of protein in relation to the electrode position.
Semi-dry electrotransfer
For semi-dry protein transfer, the transfer sandwich is placed horizontally between two plate electrodes in a semi-dry transfer apparatus. For this semi-dry transfer, it is very important that the gel is pre-equilibrated in transfer buffer. To maximize the current passing through the gel instead of around the gel, the amount of buffer available during transfer is limited to that contained in the sandwich, so it is helpful if the extra-thick filter paper (~3 mm thickness) and membrane are also sufficiently soaked in buffer. Likewise, it is key that the filter paper sheets and membrane are cut to the size of the gel.
One to four gels may be rapidly electroblotted to membranes. Methanol may be included in the transfer buffer, but other organic solvents, including aromatic hydrocarbons, chlorinated hydrocarbons and acetone, should not be used to avoid damage to the semi-dry blotter. Electrotransfer is performed either at constant current (0.1 up to ~0.4 A) or voltage (10 to 25 V) for 10 to 60 minutes. Methanol-free transfer buffers are recommended to reduce transfer time to 7 to 10 minutes. Transfer efficiencies of 60 to 80% may be achievable for proteins between 14 and 116 kDa, longer transfer times are required to transfer higher molecular weight proteins.
Semi-dry electroblotting transfer. The Invitrogen Power Blotter is designed specifically for rapid semi-dry transfer of 10–300-kDa proteins from polyacrylamide gels to nitrocellulose or PVDF membranes in 5 to 10 minutes. The Power Blotter features an integrated power supply optimized to enable consistent, high-efficiency protein transfer when used with commonly used precast or homemade gels (SDS-PAGE) and nitrocellulose or PVDF membranes.
Dry electrotransfer
Dry electroblotting methods use a specialized transfer sandwich containing innovative components that eliminate use of traditional transfer buffers. A unique gel matrix (transfer stack) that incorporates buffer is used instead of buffer tanks or soaked filter papers. The high ionic density in the gel matrix enables rapid protein transfer. During blotting, the copper anode does not generate oxygen gas as a result of water electrolysis, reducing blot distortion. Conventional protein transfer techniques, including wet and semi-dry, use inert electrodes that generate oxygen. Typically, transfer time is reduced by the shortened distance between electrodes, high field strength and high current.
Dry electroblotting transfer. The Invitrogen iBlot 3 Western Blot Transfer System provides fast western transfer without the need for buffers. This system efficiently blots proteins from acrylamide gels in as few as three minutes, and is compatible with both PVDF and nitrocellulose membranes. The iBlot 3 System has performance comparable to traditional wet transfer methods in a fraction of the time.
Resources
Download: Western Blotting Overview Brochure
Explore: Western Blot Protein Ladders
Explore: Chemiluminescent Detection Reagents
For Research Use Only. Not for use in diagnostic procedures.