Electrophoresis 2.0: Faster, Stronger, and More Reproducible

In 2010, researchers around the world were competing to finish the race to purify BRCA2, a protein whose mutant form is associated with more than 50% of hereditary breast cancer. But only one of these scientists—Ryan Jensen, then a postdoc at University of California, Davis— was able to cross the finish line first with a powerful research paper published in Nature.1

At the center of Jensen’s discovery story is a simple and unlikely star: a protein electrophoresis gel. It was a new type of gel that Jensen was beta testing in his lab that played a pivotal role in his findings.

Revolutionary changes in gel chemistry, as well as in protein electrophoresis equipment, have drastically expanded what is possible with electrophoresis—empowering discovery in all areas of life science. Read on to learn more about Jensen’s story and find out about other ways that gels and advances in protein electrophoresis technology can transform your workflow.

Gel innovations propel cancer research

Jensen, who made waves with his BRCA2 findings several years ago, is now an assistant professor of therapeutic radiology at the Yale School of Medicine, where he focuses on DNA repair and homologous recombination as they relate to breast cancer. His lab frequently uses precast gels for 1-DE (1-dimensional electrophoresis) to purify the proteins involved in DNA repair.

Figure 1 – Western blot of 2XMBP-BRCA2 from total cell lysate (lanes 1–5) and an amylose pulldown of 2XMBP-BRCA2 (lanes 6–10). Bands near the gel’s bottom are RAD51 paralog proteins (proposed to assist BRCA2 in DNA repair). Lanes 6–10 show BRCA2 interacting with the RAD51 paralog proteins. 2XMBP refers to the two maltose binding affinity tags affixed to the N-terminus of BRCA2. Prior to Western blotting, the samples were run on Mini-PROTEAN TGX precast gels from Bio-Rad Laboratories. (Image courtesy of Ryan Jensen.)

Jensen said he landed his Yale position in part because of his breakthrough discovery at UC Davis, when he was studying under Stephen Kowalczykowski. His mission was to purify BRCA2, a very large and fragile protein. The protein is integral in DNA repair, especially in homologous recombination, which is the cell’s response to the most catastrophic type of DNA damage: double strand breaks.

Purifying BRCA2 had eluded research groups for years. The protein is large   (3418 amino acids, or nearly 400 kDa), has low expression levels in human cell lines, and has a tendency to degrade very easily. Like many researchers, Jensen previously used his own hand-cast polyacrylamide gels to trace the purification of BRCA2. During many of these attempts, the higher molecular weight proteins, including BRCA2, simply disappeared.

This changed, though, when Jensen incorporated Mini-PROTEAN TGX (Tris-Glycine eXtended) precast gels (Bio-Rad Laboratories, Hercules, CA) into his process. He immediately started noticing tight, crisp bands corresponding to BRCA2, and his research took a turn for the best (Figure 1).

The precast TGX gels proved tremendously effective in the study, due in no small part to their sensitivity and the higher resolution than Jensen’s hand-poured gels. This effectiveness was especially important because of the small amount of protein Jensen had at his disposal. He decided to use the gels for all the figures in his Nature paper.

Reproducible 2-DE gels arrive due to enhancements in gel chemistry and IEF

Another benefit of modern protein electrophoresis technology—improved reproducibility—can be seen with the method known as two-dimensional electrophoresis (2-DE). 2-DE includes a number of manual steps in which errors can be introduced, making analysis between gels, especially between different labs, tricky and frustrating.

To improve the reproducibility of 2-DE for quantitative analysis, Dr. Stefan Lehr, head of proteomics at the German Diabetes Center in Düsseldorf, and his colleagues have developed a robust workflow called 2D-ToGo (Figure 2). Through this workflow, sources of variability are squeezed out through a more efficiently designed workflow incorporating recent developments in the field of protein electrophoresis. When evaluating the reproducibility of the 2D-ToGo workflow, the correlation coefficients of 18 gels analyzed between three different labs using a standard E. coli sample were between 0.91 and 0.95.2 The workflow clearly works.

Figure 2 – Entire 2D-ToGo workflow, a complete solution for robust and reproducible 2-DE that utilizes precast reagents. (Image courtesy of Stefan Lehr.)

The authors attribute many of the gains in reproducibility of 2D-ToGo to the PROTEAN i12 IEF system in the first dimension and precast SDS gels with stain-free technology (Bio-Rad) in the second dimension.

The first dimension, isoelectric focusing (IEF), is often one of the most critical due to its impact on the quality and resolution of 2-DE separation. Dr. Lehr’s team used the i12 IEF system to avoid some of the variability sometimes seen with conventional IEF instruments. By incorporating the i12 into the 2D-ToGo workflow, they were able to independently control and monitor both the current and voltage profile of each immobilized pH gradient (IPG) strip during separation. This allows researchers to evaluate whether all samples/strips profiles achieve a threshold separation performance, which enhances reproducibility. Samples that don’t meet the ideal profile during the run can be identified and frequently “rescued” by adjusting the strip specific separation parameters.

The second dimension, which generally involves sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), is another major source for gel-to-gel variability. During gel casting, uneven polymerization often causes distortions within the gel matrix, which in turn causes distortions within the protein spot pattern.3 Yet another source of variability, manual protein staining, is often used to visualize protein spots. To eliminate the variability that can occur either during gel casting or downstream steps of protein visualization, Dr. Lehr relies on standardized stain-free precast gels (TGX® Any kD Stain-Free® Precast Gels® from Bio-Rad).

Composed of proprietary gel and buffer chemistries, the TGX Stain-Free Precast Gels help improve positional spot reproducibility by eliminating the need for manual protein staining. Gel staining requires careful manual handling and can artificially alter spot quantities. The TGX Stain-Free Precast Gels contain chemicals in the gel that bind to tryptophan molecules in the proteins as they migrate. Using a stain-free enabled imager from Bio-Rad such as the ChemiDoc MP or Gel Doc EZ imaging systems, researchers can visualize the gels without staining them. The gel system is also compatible with commonly used fluorescence staining methods.

New developments in gel chemistry and transfer systems improve Western blotting

Following 1-DE or 2-DE separation of protein mixtures, researchers often turn to Western blotting analysis for protein identification and validation. Like 2-DE, Western blots involve a series of complex, hands-on steps that introduce variability. Confidence in the final results depends to a large degree on the effective protein transfer to the solid support membranes used for Western blotting.

Figure 3 – Comparison of transfer efficiency of the Trans-Blot Turbo and System B protein transfer systems using Criterion TGX Any kD gels (stain-free SDS-PAGE). Replicate 2-DE analyses of rat liver lysates were transferred to nitrocellulose membranes using the Trans-Blot Turbo (left panel) or System B (right panel). Protein transfer efficiency was determined by visualizing residual protein remaining on gel by Coomassie staining of post-transfer gels or SYPRO Ruby staining of posttransfer blots. Protein spots on the blots were counted using PDQuest 2-D analysis software.

Protein transfer efficiency hinges not only on the physicochemical properties of the proteins, but also on external factors such as the gel, the transfer system, e.g., tank   or semi-dry, and the transfer buffer composition.4 To improve the efficiency, researchers from Bio-Rad Laboratories sought to evaluate and identify the optimal protein transfer system and gel chemistry for transfer across pI and molecular weight of a 2-DE gel.

The researchers evaluated the performance of two leading rapid semi-dry transfer systems: the Trans-Blot Turbo (Bio-Rad Laboratories) and a protein transfer system we’ll call System B (see Figure 3). They chose these protein transfer systems because they offer the fastest protein transfers with reported transfer times of 7–10 min using medium-sized gels.

Following 2-DE of a rat liver lysate   sample, they transferred replicate gels to PVDF membranes using either Trans-Blot Turbo or System B. They then stained the blots using SYPRO® Ruby (Life Technologies, Carlsbad, CA) to visualize proteins transferred to the PVDF membranes. To visualize the proteins that remained on each gel—that is, proteins not transferred to PVDF—they stained the gels with Bio-Safe™ Coomassie (Bio-Rad). Thus, they could determine the efficiency of protein transfer by looking at the absence of detectable protein spots on the original gels post-transfer as well as the presence of protein spots on PVDF membranes.

They found that the post-transfer gel processed with System B had considerably more Coomassie-visualized spots than that processed with Trans-Blot Turbo, pointing to a lower protein transfer efficiency with System B. Further analysis on SYPRO Ruby-stained blots detected roughly twice as many proteins transferred with Trans-Blot Turbo than with System B (1066 with Trans-Blot Turbo versus 555 with System B). Thus, the Trans-Blot Turbo system demonstrated higher transfer efficiencies across protein pI and molecular weight ranges.

New possibilities in electrophoresis

Gel electrophoresis technology has come a long way in recent years. It has played a vital role in enhancing protein purification, quantitative proteomic analysis, and Western blotting. In fact, any workflow that involves electrophoresis can benefit from this new technology. What can it do for you?

References

  1. Jensen, R.; Carreira A. et al. Purified human BRCA2 stimulates RAD51-mediated recombination. Nature2010, 467, 678–83.
  2. Posch, A.; Lehr, S. et al. 2D-ToGo workflow: increasing feasibility and reproducibility of 2-dimensional gel electrophoresis. Arch. Physiol. Biochem.2013, 119(3), 108-13.
  3. Sun, C.; Berkelman, T. et al. http:// www.biocompare.com/Application- Notes/136942-Optimized-combination-ofprotein- transfer-system-and-SDS-PAGEchemistry- for-high-efficiency-proteintransfer- following-2-D-electrophoresis/

Ryan Jensen is Assistant Professor of Therapeutic Radiology and of Pathology, Yale School of Medicine, New Haven, CT, U.S.A. Stefan Lehr is Head of the Proteomics Unit of the German Diabetes Center in Düsseldorf, Germany. Anton Posch is a Senior Scientist at Bio-Rad Laboratories, 4000 Alfred Nobel Dr., Hercules, CA 94547, U.S.A.; tel.: 510-741-1000; e-mail: Anton_Posch@bio-rad. com; www.bio-rad.com

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