The Ultimate Quick Tips & Tricks Guide to Western Blots

The western blot is a research linchpin across all biomedical fields. While the overall workflow is the same, labs are swapping homemade reagents for commercial, clunky running systems for streamlined, and film for digital. As data incurs more scrutiny than ever before, validated antibodies, appropriate controls, and clear, noise-free, quantifiable blots are critical to the advancement of experiments. Although short-cuts now abound, attention to detail is still paramount to generating and analyzing the western blot.

“Speed, workflow simplification [and] robustness are becoming more critical in the market,” says Sayuri Otaki, Global Product Manager & Segment Lead, Protein Separations, at Merck. “Researchers are adopting techniques to speed up their workflow such as fast gel casting kits, fast transfer systems, fast or automated immunodetection systems, multiplexing and multimode imaging.” Still, she notes, “While the western blotting workflow appears simple, to achieve quantifiable results, every step of the workflow requires careful optimization.”

Otaki sums up honing the western blot experiment: “This starts with sample preparation, SDS-PAGE gel chemistry, gel percentage, and amount of sample loaded,” she says. “Choice of blotting membrane, transfer method and buffer composition, as well as transfer time have a significant impact on the final outcome.”

Here, we’ll explore how scientists can streamline and improve their western blotting experiments while generating reliable and informative data.

Casting gels and running blots

When making your own gels, the misalignment of plates in the casting device can cause leaks. The Millipore^® mPAGE^® Caster has a unique alignment guide and clamping mechanism to prevent this problem.

Otaki also references old reagents as common problem makers and says to always use fresh APS and TEMED solutions, since they could be degraded. Some may choose a different approach. The Millipore^® mPAGE^® Lux Casting system doesn’t require hazardous reagents, like those mentioned. It also speeds up the process. “The system makes ready-to-use gels for SDS-PAGE in less than three minutes by using photoinitiator via UV light. It replaces the traditional gel casting process with faster, simpler, and safer methods, with more reproducible results.”

Take the time to understand the proteins you are trying to detect, their molecular weights, and any modified isoforms. “There is an ideal gel for your sample type; evaluate the properties of the different gel chemistries and percentages to get the perfect version for your application,” advises Katie Schaefer, Ph.D., Product Manager, Imaging, at Bio-Rad Laboratories.

“It is important to ensure that the western blot being run is a true reflection of the results, reducing the risk of misinterpretation. As well as following standard protocol steps, the operator should include a loading control, protein transfer check [with] Ponceau staining, use a range of relevant primary antibody concentrations, and include a negative lane, omitting the primary antibody, and positive control lysate,” explains Jane Shatford, Product Development Manager at Everest, an Absolute Biotech company.

“Western blotting is a tried-and-true technique for protein quantitation,” adds Schaefer. “Over the past few years, more accessible options for expediting and optimizing the workflow have emerged, such as [Bio-Rad’s] Stain-Free Western Blotting workflow.” With this system, a compound in the gel makes proteins fluoresce through photoactivation allowing the user to visualize proteins at any point during the process, including following electrophoresis and transfer. Later, it serves as the basis for total protein normalization (instead of relying on housekeeping proteins). “Given the increased scrutiny of western blot data submissions, total protein normalization is objectively the most transparent normalization method for western blotting data analysis.”

There is a lot of discussion around whether to use housekeeping proteins, like beta-actin, or total protein, comments Otaki, and reports suggest that normalization using total protein is more reproducible than using housekeeping proteins. “But as with all things western blotting, one has to carefully examine that the protein applied is within the linear binding capacity of the membrane to provide quantifiable results independent of the normalization method used.”

Transfer, blocking & immunoreagents

When selecting transfer reagents such as buffers and membranes, scientists must continue to appraise their needs and the characteristics of the target proteins. Schaefer explains, nitrocellulose works for many applications and doesn’t dry out quickly, but may have less overall binding capacity. PVDF and LF-PVDF have high mechanical strength and higher binding capacity. Still, the user must assemble the transfer stack expeditiously, as they are highly hydrophobic and susceptible to drying out.

“Our Millipore^® Immobilon^® Transfer Membranes include a wide range of products in terms of functionality and format,” says Otaki. They range in composition and size. There is even an Immobilon^® Narrow Roll format and dispenser designed to eliminate membrane waste.

Otaki also reminds researchers that protein molecular weight can influence buffer choices. “Transfer buffer additives such as concentration of alcohol and the concentration of SDS must be carefully balanced,” she explains. “High alcohol concentration may impede high molecular weight proteins from transferring efficiently out of the gel, while SDS in the buffer may prevent smaller proteins from binding to the membrane.”

Another Millipore device, the SNAP i.d.^® 2.0 Protein Detection System for Western Blotting uses a vacuum to drive reagents through the membrane. “While traditional western blotting requires 4 to 24 hours for the blocking, antibody incubation, and washing steps, the SNAP i.d.^® 2.0 protocol takes only 30 minutes with no loss of signal intensity or reduction in blot quality,” points out Otaki. All immunodetection steps after protein transfer to a membrane can be performed with the SNAP i.d.^® 2.0 System.”

Blocking

Blocking reagents appear to be shifting from homemade to commercially available, an easy way to ensure researchers are using the correct formulation. “The blocking buffer needs to be carefully evaluated to assure compatibility with the analysis to be performed,” advises Otaki. “Scientists have many choices from the traditional protein-based blockers such as non-fat dry milk to non-protein blockers and signal enhancers.”

Blocking reagents can also help optimize blots for new targets while minimizing background, according to Schaefer. “Using a high-quality blocking reagent can also ensure reproducible results over the course of all necessary experiments. Bio-Rad’s EveryBlot Blocking Buffer provides a 5-minute block, resulting in optimal background blocking to maximize sensitivity regardless of the detection method chosen.” Schaefer adds, since this product doesn’t contain phosphate-based buffers, it is compatible with phosphate-specific antibodies.

Dr. Tobias Polifke, Managing Director at CANDOR Bioscience, believes that milk has no place in the western blot workflow and recommends CANDOR’s The Blocking Solution, instead. “Choose high-quality blocking buffers without batch-to-batch inconsistencies for membrane blocking. Use modern assay diluents such as LowCross-Buffer^® for dilution of antibodies to avoid false bands. Don’t use any foodstuff in scientific work only to save [on the] costs of reagents,” he advises. “Milk powder doesn’t have a place in the lab, only in the coffee mug.”

Antibodies

Over the last few years, antibody companies are meeting the need for better, validated antibodies, as well as corresponding controls. “The increased selection and availability of tissue and cell lysates, and a greater knowledge of which of these are the most suitable to select, for the particular target protein of interest makes the western blot experiment more focused and relevant,” according to Shatford.

Antibodies, to a great extent, continue to dictate the relative ease and amount of troubleshooting required to generate a successful blot. That’s why scientists should adequately research and understand the qualities and quirks of their antibodies, such as species, affinity, and phosphorylation state. “As the antibody field becomes more and more aware of reproducibility issues with antibody experiments, the importance of antibody validation is only increasing,” explains Shatford. “Researchers are placing more stock in having their antibodies validated for the specific applications they are planning to use, including western blotting.” Everest offers batch-specific western blot validation data for their entire range of goat polyclonal antibodies. To ensure reproducibility, Everest is also developing a line of batch-specific positive control lysates proven to work with the corresponding antibodies. “This level of specificity will be unique to the industry, as far as I’m aware, and will help researchers to secure successful western blots,” adds Shatford.

Otaki again emphasizes optimization at every step of the workflow and the need to carefully assure target specificity of antibodies. Affinity must also be evaluated and refined so that appropriate dilutions and selection of reagents of varying sensitivity can be chosen for subsequent steps such as visualization. “Scientists can further optimize and preserve their precious primary antibody by utilizing a more sensitive detection reagent. This strategy can also be used when the amount of sample is limited,” points out Otaki.

The proper dilution will alleviate issues down the line, as well. “One of the most common questions we get is how to reduce high nonspecific background,” says Shatford. “Researchers should make sure that their primary and secondary antibodies are diluted far enough. By diluting the antibody, you are asking for higher affinity interactions, and the lower affinity interactions (to remotely similar epitopes) will not last at lower antibody concentrations.” In other words, only true antibody-epitope interactions will appear on the blot and your background will be minimalized. She warns, however, that background can depend on more than antibodies—“Poor experimental conditions will incur random noise not usually related to the primary antibody. Omitting blocking components, or contaminated buffers, usually are the culprit. There is danger for antibodies being dismissed prematurely because of not dealing with background and noise separately.”

Imaging

Labs are moving away from radiographic film and embracing digital imaging systems, cutting down on hazardous waste and expensive film. These systems also provide flexibility. “Most imaging systems incorporate a gel doc system and chemiluminescence as well as fluorescent imaging capabilities. Onboard software automates many tasks and dedicated analysis packages simplify analysis,” says Otaki.

In the realm of digital photography, there is some debate over complementary metal-oxide-semiconductor (CMOS) sensors versus charge-coupled devices (CCD) regarding image quality, processing speed, and power consumption. Both versions are available in digital imaging systems through a variety of vendors. CMOS technology has improved over the years, however. Schaefer says CMOS is replacing CCD for a variety of reasons, all of which “yield performance enhancements in sensitivity, acquisition times, and resolution for gel and blot images.”

“Bio-Rad Laboratories will launch our most sensitive chemiluminescent imaging system utilizing the CMOS technology this year,” she reveals. “The forthcoming ChemiDoc Go Imaging System offers high-resolution and high-sensitivity imaging for chemiluminescence and all standard gel applications in an attractive compact benchtop design.”

In the lab, Schaefer often sees scientists select the highest resolution settings for chemiluminescence, and this isn’t necessary. Instead, she advises selecting a higher binning setting for higher sensitivity when working with a faint bot. With the Bio-Rad Imagers, the user has the ultimate flexibility in selecting the optimal balance of resolution and speed for detecting their particular band of interest. “I also recommend zooming in as close as possible if your camera has a physical or optical zooming mechanism, which will ensure you are using all pixels of your detector for data collection.”

Moving forward while remembering the basics

Researchers may be considering automating some of the steps in the western blot process, but the experts say automation can mean less flexibility. Otaki says there are automated immunodetection systems available, but the process isn’t any faster. Instead, you can speed up the process with advancements like those described here, such as the Millipore^® SNAP i.d.^® 2.0 Immunodetection System, which reduces time-consuming steps to half an hour while maintaining versatility.

Schaefer remarks, “There are significant advancements in making your blot work harder for you through multiplexing.” Bio-Rad’s ChemiDoc MP imaging, in conjunction with StarBright Secondary Antibodies, is one such streamlined solution for simultaneously detecting up to three protein targets of interest on one blot, normalized against the total protein.

Clearly researchers have a lot of options when it comes to devising the best set-up for western blotting experiments. In the meantime, Schaefer reminds everyone to remember the basics: “Be sure to thoroughly clean all blotting equipment, including incubation trays, tetra cells, and TransBlot Turbo cassettes after each use. And refrain from skimping on reagents, wash steps, and incubation times. Consistency is key!”