Detecting Low Abundant Proteins in Precious Pediatric Cancer Samples
"Some of the proteins we wanted to detect are low in abundance and it’s really hard to get a good amount of protein for traditional Western blot. The low protein concentrations required by [Simple Western] makes it easier to save precious samples ensuring protein detection."
- Debbie Hicks, Ph.D., Faculty Fellow, Wolfson Childhood Cancer Research Centre, Northern Institute for Cancer Research, Newcastle University
Advancing the Treatment of Childhood Cancers
The work of the Paediatric Brain Tumour group (PBTG) at the Wolfson Childhood Cancer Research Centre focuses on developing highly effective treatments for childhood cancers, reducing both toxicity and side effects of treatments. Debbie Hicks, Faculty Fellow, studies the molecular basis of pediatric brain tumor development and the application of molecular diagnostics for improved individualized treatment regimes. Together with graduate students Azira Ramli and Gemma Llargues-Sistac, they aim to develop novel therapies for treating pediatric tumors.
High-Quality Low Abundant Protein Detection From Precious Samples
The PBTG used traditional Western blots to analyze their proteins of interest but struggled with low abundant protein detection in their limited samples. They implemented Simple Western™ to detect their low abundant proteins of interest from nuclear, cytoplasmic, or total protein extractions faster and more efficiently than they could with traditional Western blotting. The low protein concentrations required by Simple Western made it easier to detect proteins of interest in their precious samples. They plan to expand the use of Simple Western to validate the expression levels of key proteins in their pediatric brain tumor cell line models.
One of the low abundant proteins under investigation is very small, 16 kDa, and hardly detectable by conventional Western blotting. Simple Western offers a higher resolution that enables the detection of this low abundant protein using low protein concentrations of as little as 0.8 μg/mL of cell lysate.
Saving Sample, Time, and Money
When working with traditional Western blots, it took the team three days to generate results, and sometimes the experiment needed to be repeated as they could not detect any protein on the membrane. With Simple Western, they can run up to 25 samples at the same time, using very low protein concentrations. Moreover, with multiplexing, they can measure multiple low abundant proteins at the same time, saving time and enabling them to get the most out of their samples. The team gets quantitative results in three hours, without all the hands-on steps associated with traditional Westerns. The in-built quantification system enables them to accurately detect, quantify low abundant proteins and analyze results without the need for additional software. In addition to the low protein concentration and time savings, the researchers noticed they were saving money on antibody costs due to the reduced amount of detection antibody required to detect their low abundant proteins of interest compared to the amounts needed in traditional Westerns.
Simple Western is keeping busy at Wolfson Childhood Cancer Research Centre. Based on the impressive results from the PBTG, the technology is being implemented by many different research groups at the Centre.