The Next Generation of Western Blot Normalization

The western blot is a lab linchpin. With the right combination of antibodies, optimized loading, and newer technology, including automation and digitization, immunoassays are a more formidable tool than ever before. “Readers should know just how much information they can get from their western blots!” says Tram Tran, Associate Marketing Manager at Azure Biosystems. As an example, she cites more sensitive chemiluminescent detection with greater linear dynamic ranges. “Combined with digital imaging, chemiluminescent western blots can provide much more than yes/no answers.” Then there is multi-fluorescent detection, which allows scientists to “assess multiple target proteins in a single experiment and save so much time compared to the old days of stripping and reprobing.”

But sometimes the data that westerns produce can be misleading. As we’ll discuss here, normalization, a critical component of the quantitative blot workflow, may lead to deceptive conclusions when control proteins are not properly vetted and validated. Total protein normalization (TPN) is a newer method researchers should strongly consider.

Housekeeping proteins—controls with caveats

Housekeeping proteins (HKP), like actin and GAPDH, were loading control mainstays for three decades. But there have long been issues related to their use for normalization, such as saturation due to their high expression. “Normalizing western blots using HKPs really only works if the HKP and protein of interest are expressed at a similar level,” says Tran. Both signals must be within the linear range of detection, which can be difficult with huge amounts of a particular HKP. A saturated HKP signal equals a faulty control, and the blot will need to be repeated with reduced protein loading concentrations.

Large amounts of protein can be more problematic when using chemiluminescent detection. Again, a saturated signal will not be linear with the amount of loaded protein. Tran points out HKP saturation is less likely when using multicolor fluorescence detection. Azure offers the Sapphire FL Biomolecular Imager, which lets the user “adjust the exposure time in the appropriate fluorescent channel when detecting the HKP and signal saturation.”

Although possible, choosing an appropriate HKP control can be challenging, says Kelly Gardner, Senior Director and Business Unit Leader at Bio-Techne. “Many HKP controls fluctuate in response to cell stimuli or cell state producing misleading normalized immunoassay results that undermine research quality,” she explains. Tran notes if HKP expression changes at the same time as the protein of interest, normalization will be invalid.

There are still some good HKP options out there, says Chelsea B. Pratt, Ph.D., Biopharma Market Development Manager at Bio-Rad Laboratories, if it can be ensured they are within the linear range, are not producing saturated signals, and do not change over time or with the experimental condition.

Total protein normalization is the new normal

“Scientists are moving away from HKPs because total protein normalization (TPN) is easier to validate and more reliable,” says Pratt. Based on the amount of protein transferred to the blot, “the entire protein signal is used as a normalization factor instead of just one HKP.” This method does not use additional antibodies or require further blotting. Tran adds that using TPN “automatically corrects for variations that might occur during the western blotting protocol such as variations in electrophoresis, protein transfer, or sample loading.”

Versatile stains sidestep additional HKP blots

There are several ways to carry out TPN. According to Pratt, staining with Ponceau S is one traditional method. “However, this requires additional steps to both stain and de-stain the membrane.” Tran agrees the extra steps required may be a deterrent, however, the staining process can be a quick one. Azure’s TotalStainQ fluorescent stain, available for both PVDF and nitrocellulose membranes, requires only 20 minutes and is carried out prior to blocking. “The dye has an excellent linear range up to 50 micrograms of lysate so low-abundance proteins can be detected and normalized with accuracy even at high protein loads.” Tran reminds potential users, “This is much less time than is required to strip and reprobe a chemiluminescent blot with a second set of antibodies to detect a HKP.”

Another fluorescent stain option is AzureRed. This dye is for use with very low protein concentrations of up to one microgram per lane, can be used on 1-D or 2-D gels, and is compatible with western blotting, mass spectrometry, and Edman sequencing.

Stain-free protein visualization

If you want to avoid extra stain washing steps, Pratt recommends Bio-Rad's Stain-Free Western Blotting system. After SDS-PAGE, the gel is exposed to UV light, causing proteins to form covalent labels with a trihalo compound in the gel, leading to protein fluorescence. Once activated, the total protein can be visualized in the gel and on the membrane after transfer, without any additional staining. “This enables the entire proteome to be used as a reference giving a more accurate representation of the loaded and transferred lane. … Bio-Rad currently [offers] the only commercially available UV-activated in-gel total protein solution. We have taken steps to provide scientists with an easy-to-follow process along with imaging software analysis to harness the power of TPN for their experiments.” It’s worth noting that Bio-Rad offers the option of commercially available stain-free gels or using the trihalo compound in hand-cast gels.

Capillary westerns eliminate the transfer step

“Simple Western™ capillary westerns are the future of western blotting and many users adopt the platforms to enable effortless total protein normalization,” according to Gardner.

In this platform, proteins are not transferred to a membrane. Instead, tiny amounts of protein sample, less than one microgram of lysate per well, are separated within capillaries and then crosslinked to the capillary walls. Samples are blocked, washed, and incubated with conventional primary and conjugated secondary antibodies and are then detected through chemiluminescence or fluorescence.

Bio-Techne offers the only fully automated capillary western platform on the market allowing for channel-based or sequential TPN on the same small amount of sample, around 3 microliters. Channel-based uses a separate fluorescence channel dedicated to TPN and is used in the same capillary alongside chemiluminescence, near-infrared, and/or infrared immunoassay detection. The sequential approach relies on RePlex stripping buffer to remove antibodies, and permits a second cycle dedicated to chemiluminescence-based TPN. “Both strategies are automated, generating reproducible and fully quantitative results in just a few hours.”

Advantages of Simple Western™ include less variability since there's no membrane transfer step, use of very small sample amounts, multiplex capability, and built-in TPN.

Digitization enhances quantification

As more labs switch from film to digital imaging, western blotting is becoming a more efficient and powerful tool. “The scientific community has embraced the transition from traditional film to digital image acquisition,” says Pratt. She underscores how this eliminates the problems that come with film use, such as saturated signals, the need for expensive equipment and maintenance, as well as the use of hazardous chemicals. Software can also help to sidestep potential mistakes and simplify the TPN process. Bio-Rad’s software, for example, uses red spots to flag when a signal is saturated. Another benefit, according to Pratt, “Digitalization has allowed for lower cost re-imaging to optimize exposure time, and with our ImageLab analysis software, normalization to total protein signal is a breeze.”

Bio-Techne’s Our Compass™ software for Simple Western™ Platforms automates quantification of immunoassay peak areas and total protein signal for “reproducible normalized western blotting data that is secure and immune to user-to-user variations or improper manipulation.” Azure Imaging Systems and Azure’s Sapphire FL Biomolecular Imager also provide software to facilitate normalization.

Although these systems can also help optimize and validate HKPs, the road can be longer when using this approach over TPN. Scientists should also consider the requirements of specific scientific journals. “Journals are moving toward requesting blot validation data, which can upend your time to publication,” says Pratt. “Best practice is to either transition to TPN or start the process of validating your HKP before your initial experiments.”