Quantitative Western Blotting Using Stain-Free Technology
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Dr. Aldrin Gomes PhD, an associate professor at the University of California at Davis, presented a talk entitled, “Can Western Blots be Trusted?” Dr. Robyn Murphy, PhD, an associate professor at La Trobe University in Victoria, Australia, discussed the use of stain-free total protein loading controls for conducting single cell western blotting.
Bringing Back the Trust
Dr. Gomes’ talk addressed common western blot pain points that were also presented in a survey conducted by Biocompare, which found that 41 percent of scientists say their blots fail 25 percent of the time. “We have multiple problems at the western blot level that are holding back the field and giving way to poor quality blots with inaccurate results,” said Gomes during his presentation.
Dr. Gomes said the most common pitfalls preventing quantitative western blots are failure to validate antibodies, the use of housekeeping proteins for normalization, and reliance on film. For this reason, Dr. Gomes has adopted Bio-Rad’s V3 Western Workflow™, which allows him to perform blots within three hours once his antibodies have been validated. The V3 Workflow uses total protein normalization and the stain-free enabled ChemiDoc™ MP Digital Imager to improve blot quantitation. Dr. Gomes says this approach has several advantages over using film, which he discussed in detail during his presentation.
Dr. Murphy discussed the challenges of performing quantitative western blots on samples from individual muscle cells.
By using a four-point calibration curve, Dr. Murphy says western blotting can accurately measure subtle changes in protein amounts even from single cells. Using Bio-Rad’s Criterion™ TGX Stain-Free™ Precast Gels, she can normalize her western blots against the total protein in each lane, which she finds to be more accurate than normalizing against the housekeeping protein GADPH. Stain-free gels also give her the ability to verify each step in the experiment. For example, using the stain-free gels she is able to quickly determine, after the electrophoresis step, whether or not the sample is present. She says this step is a necessity due to the challenging nature of collecting single cells.
“My take-home is that if it’s appropriately undertaken, western blotting can be quantitative, very reliable, and extremely valuable in our world of science,” Dr. Murphy said.
Recordings of these presentations can be viewed above or on Bio-Rad’s Life Science Research YouTube page.