Multiplexing Western Blots: More Data, Less Sample

September 15, 2018 (Vol. 38, No. 16)

DigiWest Protocol Uses Luminex’ Bead-Based Technology for High-Throughput Protein Profiling

Western blots have been a tried-and-true method for separating and quantifying proteins based on molecular weight, but they have not kept up with the continually increasing output demands of modern experimentation. The technique has some severe limitations. For example, it is not automated, making it time consuming and laborious for practitioners. Moreover, it requires additional material to run a new blot for each additional question asked. For an application like cellular signaling analysis, in which scientists must characterize many proteins, the volume of protein required for repeated examination exceeds what is available to most scientists.

To overcome these challenges, our team at the Natural and Medical Sciences Institute (NMI) at the University of Tübingen has developed a multiplexed version of the Western blot in a protocol we call DigiWest. We have established dedicated labs for DigiWest, but in principle, any scientist with access to the necessary equipment and antibodies can use the method.

Unlike other high-output options for protein characterization, such as mass spectrometry and reverse-phase protein microarrays, this method was designed to keep the best elements of the Western blot—in particular, data concerning the molecular weight of each protein. By pairing Western blot results with those from a robust bead-based multiplexing technology, DigiWest allows for the analysis of hundreds of proteins at a time despite requiring only as much protein as would be needed for a single Western blot.

This highly parallel technique can be used for protein expression profiling and phospho-status determination. It has applications as diverse as biomarker discovery, compound modes-of-action analyses, antibody screening and characterization, and cell signaling studies.

Protocol

To keep the analysis as close as possible to the classical Western blot, the DigiWest multiplexing approach begins with the same gel preparation steps that every molecular biology student learns performing his or her first Western. Proteins are blotted onto the membrane as usual.

After that, the method shifts to a different type of technology to enable the highly parallel analysis. Our team chose a multiplex bead-based assay from Luminex since we have extensive experience with that platform. We theorized that if we could transfer the proteins from a particular part of the membrane onto the surface of one bead population, the experiment should yield comparable sensitivity to the conventional blot. By loading all the proteins from a 0.5 mm membrane fraction of one sample lane onto one distinct bead color, and doing this for 96 size fractions onto 96 bead colors in a single experiment, it becomes possible to run a complex assay, essentially performing hundreds of different Western blots at the same time, all with only the amount of protein required for a single gel.

The detailed protocol and subsequent validation were described in a Nature Communications paper.¹ Briefly, here’s how it works:

1. Standard sample extraction and protein lysis
2. Gel electrophoresis (SDS-PAGE) and Western blotting
3. Bead coating with neutravidin
4. Biotinylation of the membrane, slicing of each sample lane into 96 strips
5. Protein elution and immobilization on 96 bead populations, pooling of beads
6. Aliquoting of beads for hundreds of assays, primary and secondary antibody incubations
7. Detection using Luminex’ FLEXMAP 3D instrument (Figure)
8. Data analysis

Since the original publication, NMI scientists have continued to use the DigiWest approach, validating over 1000 antibodies for use in experimental materials from human, mouse, and rat, as well as other organisms and with utility for several types of experiments. Users can quantify as many as 800 analytes from just 60 µg of protein, and the analysis process can identify more than 260 post-translational modifications, including phosphorylations.

Applications

Multiplexing the Western blot makes for a valuable discovery tool with many possible uses. In basic research, the approach was inspired by a need for more information about cellular signaling—an application for which DigiWest is quite well suited. In drug discovery workflows, it is deployed for activities ranging from lead compound characterization and predictive toxicology to biomarker screening, drug mechanism-of-action studies, and pathway profiling.

For cellular signaling, the method allows scientists to compare diseased tissue to healthy tissue, generating signaling information for many more molecules and from far less sample than would be possible with traditional approaches. Because of these advantages, cancer researchers have been among the earliest adopters of this multiplexed technique.

The approach is also an excellent choice for characterizing antibodies at scale. The DigiWest protocol makes it possible for scientists to screen hundreds of antibodies in a reasonably short period and to produce a lot of very detailed information about each candidate.

Pharma and biotech companies have also adopted this method, often for characterizing new compounds to identify their molecular targets, their function in cells, potential side effects, and more. For these applications, the DigiWest method nicely complements the many genomic techniques available for drug characterization but allowing researchers to interrogate these compounds at the level of proteins and—even more interesting—activated/phosphorylated proteins, which adds an essential layer of information to the high-stakes drug discovery pipeline.

DigiWest at Work

Scientists in several organizations have already implemented this method and generated valuable results. A few examples nicely illustrate the utility of a multiplexed Western blot.

In a publication from researchers at Bayer,² the method was used to learn more about the antitumor function of two different drugs, regorafenib and sorafenib, in hepatocellular carcinoma models.

The team used mice with patient-derived xenografts and monitored survival times as well as other metrics. Incorporating the bead-based Western blot technique led to the discovery that protein expression signatures were quite different for each drug. The study recapitulated and may explain what has been seen in clinical use—that while both drugs can be effective in treating hepatocellular carcinoma, regorafenib has been successful in patients who do not respond to or have become resistant to sorafenib.

Another example comes from scientists at the Karolinska Institute, who reported in a paper³ that the DigiWest method supported an effort to identify candidate biomarkers to aid in the diagnosis of ovarian cancer. The investigators focused on platelet proteins, which are known to support cancer growth, and aimed to stratify patients with ovarian cancer (stage 3 or stage 4) from those with benign adnexal lesions. The Western multiplex method proved valuable for high-throughput validation of candidate biomarkers on an independent set of samples.

In one last case, scientists at the German Cancer Research Center turned to multiplexing for a study of autocrine Wnt signaling.⁴ They hoped to learn more about the secretion of Wnt ligands and its relationship to genomic instability as well as the formation of teratoma tumors, a common side effect of using embryonic stem cells to cure disease.

The team produced mouse embryonic stem cells—some deficient in Evi, the protein that transports Wnt ligands, and some overexpressing Evi—and analyzed them with the DigiWest method. Profiling results for 167 total and phosphorylated proteins helped the team understand mechanisms of Wnt signaling and the role of Evi; importantly, the data highlighted vital proteins that were missed by genomics-based methods.

Conclusion

Multiplexing has been a significant improvement for many conventional methods in molecular biology, making even decades-old techniques useful in an era of high-throughput genomics and proteomics. By multiplexing the Western blot, scientists can now produce high-quality, scalable protein profiling results from small amounts of sample in a quick, cost-effective workflow.

Christoph Sachse, Ph.D. ([email protected]), is site head, NMI TT Pharmaservices. Markus Templin, Ph.D ([email protected]), is head, Assay Development Department, Natural and Medical Sciences Institute, University of Tübingen.