Western Blot Protein Transfer | Thermo Fisher Scientific

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.

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Choosing the right western blot protein transfer system
Comparison of western blot transfer methods
Comparison of wet, semi-dry, and dry protein transfer methods
Related western blot transfer products
Frequently asked questions (FAQs)
Western blot transfer resources

Choosing the right western blot protein transfer system

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
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 protein 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 helps provide convenience and efficiency, 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.

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).

Click image to enlarge

Wet tank electrotransfer. 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.

schematic for semi-dry electroblotting transfer

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.

schematic of dry electroblotting transfer

Dry electroblotting transfer. The Invitrogen iBlot 3 Western Blot Transfer System helps provide 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.

Frequently asked questions (FAQs)

How long does it take to transfer western blots?

Transfer time depends on the method used: wet transfer typically takes 30–120 minutes, semi-dry transfer takes 7–10 minutes, and dry transfer can be completed in as few as 3 minutes.

What type of power supply is required for western transfers?

Wet and semi-dry transfer systems require an external power supply, while dry transfer systems like the iBlot 3 Western Blot System have a built-in power source for convenience.

What is the main difference between wet transfer and semi-dry transfer?

Wet transfer uses large volumes of buffer and is ideal for high-molecular-weight proteins, while semi-dry transfer uses minimal buffer and offers faster transfer times for most routine proteins.