This widely used immunoassay to probe samples for specific proteins and posttranslational modifications has potential time and material savings associated with variations of the transfer step. Whether it’s a simple buffer switch, or a more involved equipment swap - it’s always worthwhile to explore lab workflow efficiencies. Just take care not to risk a successful blot!

Your samples and validated antibodies represent greater lab resources than any blot transfer method can conserve

It might sound obvious, but it’s worth saying. If an eco-friendlier variation of a Western blot workflow saves time and reduces hazardous waste, but results in failed Westerns, is it really more sustainable? Of course not.

The basic Western Blot protocol steps entail sample prep, polypeptide separation by molecular weight using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transfer from the gel to a membrane, membrane blocking, antibody probing, and detection.

Nowadays Western blot protocols have diversified - but the fundamentals stay the same. Impressive new instruments and next-generation techniques have been introduced to support high throughput research and develop clinically relevant applications. The three key interdependent drivers of Western blot success are the same. These are antigen preservation, transfer blotting efficiency, and antibody-based detection. (1)

Your primary antibody is the finely honed tool you use to reveal your results. A primary antibody is validated for use in defined protocol conditions to recognize an epitope presentation specifically and reliably. For example, low expression targets often require higher specificity which can be achieved with lower affinity and higher avidity antibody profiles. Primary antibodies are often paired with secondary antibodies that facilitate chemiluminescence or fluorescence signal detection.  (Most labs have moved away from radioactivity-based detection.) The key is that antibody development for use in Western blots does not take place in a vacuum.

Your samples constitute your experiment’s objective. Naturally, proteins and their post-translational modified forms will vary in size and abundance in your samples based on your experiments. Your sample lysis, sample buffer, and the percentage of acrylamide in your SDS-PAGE gel will facilitate the separation of your denatured proteins. After generating and processing your samples, the transfer step should be a stepping stone to success, not a stumbling block.

Respectively, the transfer from the gel to the membrane will impede or promote how an epitope ultimately displays on your blot. Any change to the transfer method requires antibody validation in that new context. Not every transfer method will work well for every sample target. (2)

The point is that generating useful samples and validating antibodies are the most resource-intensive processes involved in Western blot experiments. Since using commercial antibodies represent a massive saving in development time, materials, and validation work - it’s imperative to carefully examine the advantages and limitations that hinge around the new transfer methods.

The wrong transfer conditions can ruin your results. Download the Western blot Application Guide from my former lab mates at Cell Signaling Technology (CST) and check out the troubleshooting section Transfer... How Much Time Are You Really Saving? Transfer conditions represent lower scientific resources overall. Transfer variations with advantages to save time, material, and energy, must be examined holistically.

Conserve wisely with a transfer that preserves your signal

Let’s see how the transfer step can impact your time, costs, waste streams, and space and energy usage. Then let’s compare how the two main membrane types differ. Finally, we will touch upon blot transfer controls.

How wet tank transfer works:

This is an electroblotting process using a tank apparatus and external power supply. Tank cassettes are assembled with a layered sequence of sponge, filter paper, the all-important gel, membrane, second filter paper and second sponge. The closed cassettes are fully immersed in transfer buffer vertically with the membrane side closer to the positively charged red anode of the tank apparatus. (Run to red!) Electric current is conducted by transfer buffer salt (i.e. Tris, CAPS) powering an electrical field and generating heat as a side effect.

• Pros:

Tank transfer is the optimal technique to migrate net negatively charged polypeptides from an SDS-PAGE gel to a membrane blot. We can reasonably assume that a self-contained cooling apparatus setup decreases lab energy consumption over the use of refrigerated space. Wet transfer equipment is relatively inexpensive.

• Cons:

Green chemistry-wise, the higher volumes of transfer buffer used in tank transfer can add to your lab’s hazardous lab waste stream due to the toxicity of methanol used in most buffers.

Energy-wise, the transfer tank apparatus must be kept cold while powered or it can overheat samples. You either need space at 4C or an apparatus that incorporates a frozen ice pack, or a cooling coil with a refrigerated water recirculatory system. Helpfully, these are available from Biorad®.

Tick tock. Wet tank transfer takes at least one hour.* That can vary slightly given the dimensions and percent acrylamide of your gel, the molecular weight of your target protein, transfer buffer conditions, and membrane characteristics. Tank transfer can also be run overnight at lower power.

How semi-dry blot transfer works:

Horizontal cassette assemblies containing layered filter paper, an equilibrated gel, a pre-wetted membrane, and filters all soaked in transfer buffer are laid directly between two electrode plates in a semi-dry transfer instrument. (3, 4)

• Pros:

This instrument-based method transfers proteins from gel to membranes at room temp. It’s fast. The transfer takes place in 7 minutes for mixed molecular weights or 10 minutes for high molecular weights. Transfer buffer hazardous waste is minimized by the small volumes required. No energy is needed for cold temp.

• Cons:

Can require more optimization work for sample targets and re-validation of antibodies compared to wet transfer. Does not allow for universal quantitative transfer proteins. The risk increases the closer your target protein is to the extremes ends of molecular weight ranges. Some state-of-the-art stain-free detection protocols require gels formulated for use with specific instruments. Certain instruments can have limited transfer optimization capabilities.

How dry blot transfer works:

These instruments with built-in power supplies pair with transfer stacks that contain top and bottom gel matrixes with transfer buffer incorporated. Your protein gel is put on the bottom of the stack without needing to be equilibrated in transfer buffer first. In fact, no transfer buffer prep is needed on your part. Filter paper does need to be pre-soaked with DI water. The assembled stack must have electrical contacts aligned with blotting contacts.  

• Pros:

  User-friendly setup. Very fast transfer. The default setting is seven minutes but optimization is available in newer models. Not having to equilibrate gels in buffer saves even more time. No worries about oxygen bubbles since there is no oxygen gas released from the copper anode. A copper recycling program is offered for transfer stacks.

• Cons:

  System cost. The transfer stacks are consumables that can’t be replicated in your lab. Optimization work can be challenging for sample targets and antibodies.

Can ethanol replace methanol in Western blot transfer buffers?

Most of the time, the answer is yes. But just like everything in science, the rationale is nuanced. 

Methanol in the transfer buffer reliably promotes the dissociation of SDS from gel proteins to improve their adsorption onto membranes. That said many protein targets won’t be strongly impacted by substituting methanol with ethanol. This can reduce your hazardous waste stream. Methanol-free transfer buffers are another option that can be useful in certain contexts. (5)

Since I formerly worked in the labs of Cell Signaling Technology making polyclonal and monoclonal antibodies for research use, I reached out to hear CST’s current thinking on transfer methods.

It’s putting it mildly to say that Elisabeth Russell, Senior Group Leader of the CST Tissue Culture & Western Core, and her team conduct high numbers of Western blots. When I inquired about the technical outcomes of using ethanol in place of methanol, Elisabeth shared “My team has switched to a transfer buffer containing ethanol instead of methanol, and in our experience, it seems to work well across a broad range of protein targets. When we use semi-dry over a wet transfer system, the major advantage is time savings, and therefore the ability to complete more experiments per day.” Other research similarly supports that “methanol free” based transfer buffers are useful in specific contexts. (5) In case it is relevant to your target, it’s worth noting that transfer with a buffer containing no methanol or ethanol is most risky for protein targets that do not have high molecular weights. (6,7)

“My team has switched to a transfer buffer containing ethanol instead of methanol, and in our experience, it seems to work well across a broad range of protein targets. When we use semi-dry instead of a wet transfer system, the major advantage is time savings, and therefore the ability to complete more experiments per day.”

̶  Elisabeth Russell, Senior Group Leader of the Cell Signaling Technology Tissue Culture & Western Core

Should I reuse my buffer solutions to reduce lab waste?

Another, somewhat contentious issue around hazardous waste reduction is whether methanol-based transfer buffer can be re-used, and how many times. The safest answer is no. Reusing will affect its pH and diminish transfer. On a related note, investigators have shown that gel electrophoresis buffer CAN be safely reused. (8)

stop and smell the roses - Both Nitrocellulose and Polyvinylidene difluoride (PVDF) membranes offer solid support

Let’s stop here and smell the roses. Those who know their life science history appreciate that it originally took many hours of handling to stain, de-stain, and dry high-resolution polyacrylamide protein gels - so protein blotting itself offered terrific time savings and important assay versatility!

These two main membrane types are used in Westerns to immobilize proteins. They differ in binding ability, cost, and biodegradability. Both types are typically manufactured with 0.2 µm pore–size or .45 um pore sizes that reflect the varied sizes and absorption requirements of target polypeptides.

• Nitrocellulose is the paper of the gods. (I kid!)  It has the same binding ability as PVDF for medium molecular weight proteins and greater binding for low molecular weight proteins.  It is non-toxic. However, dry nitrocellulose membranes are highly flammable, and burning nitrocellulose membranes immersed in a solvent can release toxic fumes - and that’s no joke. Follow storage guidance.

• PVDF membranes are essentially non-toxic, non-hazardous, chemically stable sheets of plastic, otherwise known as a thermoplastic fluoropolymers.  PVDF has a higher binding capacity than nitrocellulose. The higher gray background is often the downside. It is better for glycoproteins and higher molecular weight proteins and lowly expressed targets. These membranes are more expensive than nitrocellulose. Disposal by incineration can release carbon dioxide, carbon monoxide, and halogenated compounds. Overall, the chemical compatibility, protein retention, and physical strength are superior to nitrocellulose.

This is science - know your Blot transfer controls

Investigators have called for more informative western blot figures in published studies. (8) Whichever transfer method you use, here are some controls worth considering.

• Loading pre-stained molecular weight protein ladders

• Loading Control Antibodies

• Staining membranes for total protein (Amido black, MemCode, Ponceau S)

• Checking gels after transfer with a total protein stain. A reversible Ponceau red stain is highly recommended.

• Adding a second membrane behind the first can be useful to detect gel overloading or too long a transfer time when the sample appears in the second membrane.

Antibody development does not take place in a vacuum

The transfer step should be considered holistically since it is integral to both the reproducibility and sustainability of Western blotting protocols. High-tech instruments and automation are absolutely awesome when they can transform translational research discoveries into clinical tools. Saving time and materials are the virtues of semi-dry and dry blotting. Intuitively their simplicity ignites confidence in experimental reproducibility. Wet transfer systems are evolving. Semi-dry transfer works really well but is not desirable for high MW proteins Let’s also acknowledge that dry transfer methods add to the challenge of experiment reproducibility by adding complexity to establishing a consensus on antibody standards.

Your samples and antibodies are high-value resources. As the old saying goes, don’t put the cart before the horse.

Citations:

1. Kurien BT, Scofield RH. Western blotting: an introduction. Methods Mol Biol. 2015;1312:17-30. doi: 10.1007/978-1-4939-2694-7_5. PMID: 26043986; PMCID: PMC7304528.

2. Fredrik Edfors et al. Enhanced validation of antibodies for research applications. Nature Comm. 9, Article number: 4130 (2018)

3. Singh, K.K., Gupta, A., Bharti, C. et al. Emerging techniques of western blotting for purification and analysis of protein. Futur J Pharm Sci 7, 239 (2021). https://doi.org/10.1186/s43094-021-00386-1

4. Drábek, Jiří et al. Laboratory Techniques in Cellular and Molecular Medicine. (2022) ISBN 978-80-244-6049-9

5. Villanueva MA. Electrotransfer of proteins in an environmentally friendly methanol-free transfer buffer. Anal Biochem. 2008 Feb 15;373(2):377-9. doi: 10.1016/j.ab.2007.08.007. Epub 2007 Aug 10. PMID: 17850757.

6. Kurien, B.T., Scofield, R.H. (2015). Western Blotting of High and Low Molecular Weight Proteins Using Heat. In: Kurien, B., Scofield, R. (eds) Western Blotting. Methods in Molecular Biology, vol 1312. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2694-7_26

7. Ghanshyam D Heda et al. (2020) Optimization of western blotting for the detection of proteins of different molecular weight. Biotechniques, vol. 68, No. 6. https://doi.org/10.2144/btn-2019-0124

8. Heda GD, Omotola OB, Heda RP, Avery J. Effects of Reusing Gel Electrophoresis and Electrotransfer Buffers on Western Blotting. Journal of Biomolecular Techniques: JBT. 2016 Sep;27(3):113-118. DOI: 10.7171/jbt.16-2703-004. PMID: 27582639; PMCID: PMC4972471

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