4 Reasons to Switch Your Western Blot Transfer Method from Wet to Dry Blotting

by Halina Zakowicz, PhD, and Caleb Shearrow, MS

Dry blotting improves speed, convenience, and ease-of-use compared to wet transfers

Western blotting is a cornerstone of protein research, allowing researchers to identify and quantify specific proteins in a complex mixture. A key stage in western blotting is the transfer of proteins from the polyacrylamide gel to a membrane. There are three common transfer methods used in this process: wet, semi-dry, and dry.

In this guide, we’ll cover the basics of each and discuss four reasons to choose dry over semi-dry or wet blotting in terms of productivity, reliability, ease-of-use, and performance.

What is dry blotting?
Advantages of dry blotting
Learning resources

What is dry blotting, and how does it differ from other protein transfer methods?

In a dry blot transfer, the transfer stack (consisting of a gel, membrane, and filter papers) is placed between single-use copper electrodes and specialized gel matrices, which replace transfer buffer as the ion-carrying medium. This solid ion reservoir, as opposed to liquid transfer buffer, is what gives dry blotting its name.

In a wet transfer, the gel and membrane are assembled in a transfer cassette and submerged in transfer buffer. The transfer is performed in a wet environment for a longer period, often overnight.

Dry transfer systems like the Invitrogen ™ iBlot™ 3 Western Blot Transfer System offer substantial speed advantages over wet tank setups, completing the transfer in as few as 3-8 minutes. Additionally, dry transfer eliminates the need for sandwich assembly, buffer preparation, and tedious clean-up steps common in wet and semi-dry methods.

Figure 1. Schematic of iBlot ™ 3 Transfer stack showing current flow. The complete iBlot 3 Transfer Stack consists of two single-use copper electrodes sandwiching a nitrocellulose or PVDF membrane.

Advantages of Dry Blotting vs. Wet Blotting for Protein Transfer

1. Compared to wet transfer, dry blotting achieves better or equivalent transfer efficiency.

Semi-dry transfer systems have often fallen short of delivering the same transfer efficiency as wet tank transfer. This has led to a misconception among researchers that wet tank transfer offers superior transfer efficiency when compared to other transfer methods. However, the iBlot ™ 3 system utilizes dry transfer to consistently achieve equivalent or better transfer efficiency than wet transfer methods across a broad range of molecular weights. Figure 2 presents a comparison between dry transfer and wet transfer for detecting both the high molecular weight (HMW) protein, mTOR (289 kDa) and a smaller protein target, Ku80 (86 kDa) in HEK293 cells. With all other variables (blocking, primary and secondary antibody incubations, and washes) kept constant, the iBlot ™ 3 system’s dry transfer demonstrated superior transfer efficiency, as evidenced by a significantly higher chemiluminescence signal.

Figure 3. Chemiluminescence detection of mTOR and Ku80 in a dilution series of HEK293 cell lysates. Title: Comparison of dry (A) and wet (B) blot transfers of mTOR, a large 289 kDa protein, and Ku80, an 86 kDa smaller sized protein. The top blots (A) were performed using the iBlot3 dry blot system. The bottom blots (B) were performed using traditional wet transfer.

Figure 2. Comparison of dry (A) and wet (B) blot transfers using chemiluminescent detection of mTOR and Ku80 in a dilution series of HEK293 cell lysates. mTOR: Samples were separated using NuPAGE™ 3–8% Tris Acetate Mini Protein Gels. (A) Membrane image for dry transfer with the iBlot ™ 3 Western Blot Transfer System (8 min, 30 V, no cooling). (B). Membrane image for wet transfer (1 hr, 100 V). Ku80: samples were separated using Novex WedgeWell 4–20% Tris-Glycine Mini Protein Gels. (A) Membrane image for dry transfer with the iBlot 3 Western Blot Transfer System (6 min, 25 V, low cooling). (B) Membrane image for wet transfer (1 hr, 100 V).

2. Dry blotting is faster.

One major drawback of wet transfer is the extensive amount of time it takes to prepare the reagents, complete the transfer, and clean-up which typically ranges from 1.5 – 2 hours to overnight. Dry blotting saves time from the get-go by eliminating the preparation steps of buffer formulation, stack creation, and equipment cleaning after transfer.

In the case of the iBlot™ 3 system, a short inter-electrode distance combined with a high electric field strength and current reduce transfer time to as few as three minutes.

Overall, dry blotting enables more experiments to be completed in a day by reducing hands-on time by up to 90% (Fig. 3).

Figure 3. Comparison of Transfer Methods. When compared to wet tank and semi-dry transfer methods, dry transfer takes significantly less time to perform from start to finish. The quick setup is enabled by optimized pre-programmed transfer methods and pre-assembled transfer stacks which incorporate transfer buffer via built-in gel matrices. Rapid protein transfer is achieved on iBlot™ 3 in as few as 3 min. Since the transfer is completely contained within the transfer stack tray, clean up time is drastically reduced. Simply dispose of the tray and wipe down the blotting surface.

3. Dry blotting can reduce variability.

Transfer time is an often-overlooked source of variability in wet tank transfer. The misconception that longer transfer times result in better transfer efficiency is prevalent among researchers but often has the opposite effect. Compared to dry blotting, proteins transferred in a wet tank are more susceptible to membrane blow–through, resulting in reduced transfer efficiency.  Some proteins can also display weaker binding to the membrane in a wet transfer process, which results in a percentage of the bound target protein disassociating from the membrane during immunoprobing steps (Fig. 4).

Figure 4. Chemiluminescence detection of Ku80 in a dilution series of a HEK293 cell lysate at increasing transfer times. Samples were separated using Invitrogen™ Novex™ WedgeWell™ 4–20% Tris-Glycine Mini Protein Gels. (A) Membrane images for transfer times of 1, 2, and 16 hr. using traditional wet transfer methods (1 or 2 hr, 100 V at room temperature; 16 hr, 30 V at 4°C).

Wire electrodes, which generate O₂gas from water electrolysis, are another source of variability and distortions in banding patterns. These electrodes can corrode if not thoroughly cleaned, leading to inconsistent voltage and electrical current, both of which contribute to higher blot-to-blot variability. In contrast, the iBlot™ 3 dry blotting system utilizes single-use copper electrodes that remove these two sources of variability.

Because it is a single-use system, each transfer is performed using a fresh electrode. The copper electrodes do not generate oxygen gas, this prevents corrosion and results in reduced blot distortion.

iBlot™ 3 takes blot-to-blot reproducibility one step further by allowing the user the ability to better control temperature. Historically, researchers have only been able to control temperature by performing their protein transfer in a cold room. Now, iBlot™ 3 offers four different cooling settings that have been shown to improve blot-to-blot consistency (Fig 5).

Figure 5. The iBlot ™ 3 device delivers consistent results. A serial dilution of Thermo Scientific HEK293 lysate (lysed in Thermo Scientific RIPA buffer) was loaded onto four Invitrogen Bolt 4–12% Bis Tris plus gels. Proteins were transferred using the iBlot 3 Western Blot Transfer Device and iBlot ™ 3 Transfer Stacks, Midi, NC using the broad range (30–250 kDa) preprogrammed transfer method (25V, 6 min, low cooling).

4. Dry blotting is a simpler workflow with less clean-up.

New researchers often find protein transfer to be one of the most laborious western blotting steps. This is no surprise if they are using wet transfer, which requires users to accurately formulate transfer buffer, assemble a multi-component transfer stack, and set the correct transfer parameters. Dry blotting was developed with the end user in mind. Set up is limited to placing a polyacrylamide gel in a pre-assembled transfer stack, placing the transfer stack inside the transfer device, and choosing a pre-programmed transfer method before hitting “Go.”

Setting up to use the iBlot 3

These improvements make protein transfer set-up much more convenient but pale in comparison to the clean-up improvements. To ensure wet tank electrodes do not corrode, the user must clean the tank with soap and water. Usually this is completed while the user is washing incubation trays, graduated cylinders, beakers and spatulas – all dirtied from the protein transfer method. Dry blotting utilizes disposable, self-contained transfer stacks which limit the clean-up procedure to two steps; discarding the transfer stack and tray and wiping down the top lid of the device with a damp cloth.

Bottom line: dry transfer offers high transfer efficiency in a fraction of the time.

Switching from wet to dry blotting for western blot transfers offers significant improvements in performance, productivity, reliability, and ease-of-use. By simplifying the setup and cleanup processes, dry blotting offers an attractive option for both novice and experienced researchers, enhancing the overall efficiency and quality of protein research.

The dry transfer technology of the iBlot ™ 3 transfer system provides exceptional transfer efficiency across a wide range of molecular weight proteins. It combines ease of use with a simple, fast setup and cleanup process. It offers a suite of customizable settings, providing the flexibility required to meet various experimental needs. All these features make the iBlot ™ 3 dry transfer system an excellent alternative to wet transfer.

Learn more about dry blotting and the iBlot 3 »

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For Research Use Only. Not for use in diagnostic procedures.