Posts tagged ‘V3 Western Workflow’
Bio-Rad Laboratories recently announced the launch of a new simplified and robust workflow to more effectively evaluate antibodies used for measuring residual host cell protein (HCP) impurities in biotherapeutics. The two-dimensional electrophoresis (2-DE)- and western blotting (WB)-based workflow was unveiled during the Well Characterized Biotechnology Pharmaceutical (WCBP) Symposium in Washington, D.C. January 29–31.
Regulatory agencies worldwide including the U.S. Food and Drug Administration (FDA) require that residual HCP impurities in biotherapeutics remain below levels detectable using a highly sensitive analytical method (PTC 1997). Failure to meet this requirement puts patients at risk, an issue recently documented by a biopharmaceutical company forced to put a clinical trial on hold when patients developed a positive response to its product’s HCP.
“It can take a company several months and tens of thousands of dollars to develop an adequately immunospecific host cell protein reagent, so there is a lot time and money riding on the validity of that reagent,” said Nadine Ritter, senior CMC consultant at the Biologics Consulting Group.
The most widely used quality control method for determining the presence of HCPs is the ELISA, a test that uses a polyclonal anti-HCP antibody mixture raised against HCPs. To ensure the accuracy of HCP ELISA results, ICH Q6B guidelines state that polyclonal antibodies must be “capable of detecting a wide range of protein impurities.” The standard workflow for evaluating this range — defined as the percentage of HCPs that are detectable by polyclonal antibodies — involves 2-DE separation of host cell protein preparations, then transfer of the proteins from the gel to a solid support membrane, followed by western blotting with the polyclonal anti-HCP antibodies.
Although this workflow is widely used, several key issues remain, including time-intensive optimization of the components of 2-DE separation (IEF and SDS-PAGE) and variable protein transfer efficiencies. These issues reduce the overall reliability of 2-DE and western blotting results and thus lower confidence in the evaluation of anti-HCP antibodies. By integrating unique instruments and consumables into a workflow solution for addressing these issues, Bio-Rad offers highly confident evaluation of the range of protein impurities detectable by anti-HCP antibodies.
Results Presented at WCBP Demonstrate Workflow’s Effectiveness
Bio-Rad researchers presented results at WCBP that demonstrate the effectiveness of their 2-DE/WB-based workflow for evaluating polyclonal anti-HCP antisera. Bio-Rad senior staff scientist Tom Berkelman and his colleagues generated optimized host cell protein separations via 2-DE and integrated them with rapid semi-dry blotting, fluorescent protein staining, and western blotting to confidently evaluate the antisera within two days.
“Bio-Rad’s 2-DE/WB-based technique allows the detection of HCP impurities often unseen in one-dimensional electrophoresis,” said Karin Abarca Heidemann, PhD, R&D director at antibody development firm, Rockland Immunochemicals, Inc. “This is a significant benefit in the development of our HCP antibodies, as two-dimensional electrophoresis delivers improved insights into the quality and immunocoverage of antibodies against host cell contamination. Rockland is collaborating successfully with Bio-Rad on developing an improved standardized immunoassay for HCP detection using its 2-DE/WB workflow, the result of which will be presented during the WCBP Symposium.”
Bio-Rad’s New Workflow
Bio-Rad’s HCP antibody evaluation workflow consists of the following steps and takes two days to complete:
- Separate proteins based on isoelectric point (pI) in IPG strips using the PROTEAN® i12™ IEF system (the first dimension of 2-DE). The individual lane control feature — unique to the PROTEAN i12 system — enables fast optimization of isoelectric focusing (IEF) conditions and easy application of different optimized conditions for highly reproducible, simultaneous IEF of multiple samples.
- Separate proteins based on molecular weight (MW) by SDS-PAGE on TGX™ precast gels (the second dimension of 2-DE). The unique chemistry of Bio-Rad’s TGX gels enables fast electrophoresis times(less than 30 minutes) and highly efficient protein transfer.
- Process the gel with the Trans-Blot® Turbo™ system to transfer the 2-DE separated proteins to a nitrocellulose or PVDF membrane in less than 10 minutes. The unique combination of TGX precast gels and the Trans-Blot Turbo system enables highly efficient and reproducible transfer of proteins across a broad pI/MW range, which is essential for confidence in 2-DE/WB results.
- Perform fluorescent protein staining with SYPRO Ruby (to confirm the efficiency of protein transfer) and western blotting using polyclonal anti-HCP antibodies on a nitrocellulose or PVDF membrane.
- Image using the ChemiDoc™ MP imager and analyze with PDQuest™ software to determine the percentage of matching spots between those detected by membrane-bound protein staining and those visualized by immunodetection.
“Bio-Rad’s solution provides a more sensitive means to confirm the effective transfer of all proteins from the gel to the membrane, which is a crucial step in this study,” Ritter said. “If you see a low degree of immunoreaction on the blot, you won’t know if it is due to limited coverage of the antibodies or the absence of immobilized protein. Bio-Rad’s system assures that the proteins will be present on the blot.”
Independent Study Reports V3 Western Workflow™ with Stain-Free Technology Yields Superior Western Blotting Results
Scientists at Flinders University in Bedford Park, Australia recently demonstrated the superiority of a method employing Bio-Rad’s stain-free technology to use total protein as a loading control in semiquantitative western blotting.
In their report in Analytical Biochemistry, published online on September 15, Dr. Alex Colella and his colleagues describe how the stain-free approach allowed them to assess the quality of both electrophoresis and western transfer before committing to subsequent steps of the western blotting procedure, providing more accurate blot data in fewer steps and less time in comparison with traditional loading control methods.
“Our research shows that Bio-Rad stain-free gels can improve the quality of data from semiquantitative western blotting experiments,” said Dr. Tim Chataway, head of the Flinders University School of Medicine Proteomics Laboratory.
To ensure accuracy in semiquantitative western blotting, one of the most important steps is the determination of the protein load in each gel. Loading controls are used to normalize for loading errors that can result from imprecise protein estimation, pipetting inaccuracy, or uneven protein transfer. Traditionally, researchers have relied on two loading control methods: reprobing of membranes with an antibody against a housekeeping protein such as β-actin, or the use of total protein stains such as SYPRO Ruby, Ponceau S, or Coomassie Blue.
To use a housekeeping protein as a loading control, it must be established that the level of this protein remains constant when the experimental variable of interest is changed. In some cases, the expression level of a housekeeping protein may be too high or low in comparison with the protein(s) being studied, or such controls are unavailable or inappropriate, for example, when comparing the abundance of a specific protein in different tissue extracts in which the protein concentrations differ. While membrane protein stains do not suffer the same issues as housekeeping proteins, the method still adds significant cost and time to western blotting experiments.
Dr. Chataway’s results indicated that the stain-free approach provides a superior alternative to existing loading control methods because the quality of electrophoresis and western transfer results can be confirmed before applying antibodies for detection. Identifying any problems at each step allows researchers to target troubleshooting efforts. Scientists also have more time to optimize their workflows, as the stain-free method can cut the time required for this protocol in half, from two days to one.
Chataway and his team, led by postdoctoral researcher Colella, conducted a series of western blots using purified protein from rat retinas in amounts ranging from 10–40 µg. The proteins were separated by electrophoresis, transferred by blotting, and visualized using Bio-Rad’s V3 Western Workflow, a portfolio of stain-free enabled western blotting products that allows researchers to visually monitor proteins throughout all steps of an experiment. Researchers can simultaneously image their protein(s) of interest and total protein on each blot for easy normalization. Because the V3 Western Workflow requires fewer handling steps, the probability of procedural errors is lower than in the SYPRO Ruby and antibody methods.
Dr. Chataway stated that stain-free gels and SYPRO Ruby staining yielded equivalent performance, but the use of stain-free gels reduced the time required to perform semiquantitative western blotting by 1.5 hours compared with SYPRO Ruby, and by one day compared with antibody detection of the housekeeping protein β-actin. The stain-free method also reduced overall costs by eliminating the use of expensive reagents.
“We found that stain-free technology provides a faster and more cost-effective alternative to traditional methodologies, producing an overall superior performance as a loading control compared with SYRPO Ruby or antibody,” said Dr. Chataway.
For more information on how the V3 Western Workflow can increase your rate of success in western blotting experiments, visit www.bio-rad.com/ad/V3pr.
Western blotting, the backbone of protein research, is a universal lab procedure. While the premise of a western blot is simple — target proteins are identified and quantitated via antibody-antigen interactions — the traditional workflow is labor intensive and time consuming. Researchers have long sought a faster solution — an archetype that would streamline the entire process of separation, transfer, and visualization of results without compromising data quality.
One of my earliest experiences in the lab was as a summer student performing tons and tons of western blots. The technique seemed so repetitive and boring. Pour gel, prepare samples, load gel, run gel, transfer proteins, block, incubate, wash, wash, wash, wash, incubate, wash, wash, wash, develop, expose to film and…….BAD RESULTS! REPEAT, REPEAT, REPEAT!!!!! All my friends were finished their jobs working in a local day camp and here I was washing a PVDF membrane and praying to the gods of x-ray development. I was bored out of my brain and I felt like I was wasting my time and my talents. Does this resonate with anybody? There had to be a better way.
Thankfully, Bio-Rad Laboratories has now released a novel approach to Western Blotting. The V3 Western Workflow cuts Western blotting time in half and significantly improves results. Don’t believe me? Checkout the gels below. See the difference?
Care to read about a real life example? Checkout the article Novel V3 Western Workflow Revolutionizes COPD Protein Research.