Desirable outcomes from novel therapeutic approaches are often trackable at the protein level, whether through modulation of signalling pathways, changes in protein expression or abundance, or alterations in biophysical properties like complexation. At o2h Discovery, our expertise in cell signalling, microscopy, and biophysical assay techniques allows us to apply the precise methods needed to detect protein-level changes central to your novel pipeline therapeutic strategy.

Western Blotting Services at o2h

Western blotting is a fundamental technique in protein analysis, enabling the detection and quantification of specific proteins within complex biological samples. At o2h, we offer both traditional Western blotting and the advanced automated Western blotting using the JESS Simple Western™ system. Our expertise ensures accurate, reliable, and reproducible data tailored to meet your research needs.

While traditional Western blotting remains a gold standard for low-throughput, customized workflows that require high sensitivity and flexibility, it is particularly useful for exploratory studies where optimizing parameters like antibody selection and transfer conditions is essential. On the other hand, the JESS system automates the process, delivering faster results with lower sample and reagent consumption. It provides consistent, quantitative data, making it an excellent choice for high-throughput studies and biomarker validation.

Below are some representative case studies that highlight our Western blotting expertise. For example, we conduct biomarker validation using Western blotting, analyse different phospho-epitope binding antibodies to assess the phosphorylation status of target proteins, and examine tissue lysates to study biomarker changes in pharmacodynamic mouse models. Additionally, we investigate mitochondrial protein expression and optimize extraction methods for both whole-cell and nuclear fractions.

o2h also has extensive experience in target validation through CRISPR-Cas9 knockout (KO) or siRNA/shRNA knockdown (KD) approaches. We study the expression of target proteins and their downstream effects in various cell lines. Furthermore, we specialize in immunoprecipitation for target protein enrichment to study protein-protein interactions, complementing these studies with expression analyses based on drug compound activity.

Case Studies

1. Assessing Biomarkers for Drug Compound Activity

Using traditional western blotting, we worked with a client to evaluate biomarkers associated with their drug compound’s activity. By optimising protein extraction and transfer conditions, we detected target protein responses in a dose-dependent manner, providing critical insights into the compound’s mechanism of action. This approach demonstrated the adaptability of traditional western blotting for nuanced exploratory research.

Figure 1. Biomarker compound effects using traditional western blotting. (A) Biomarker western blotting panel using compound X in increasing concentration, as compared to controls. (B) Timecourse biomarker panel for compound X at 24 and 48 hours.

2. In Vivo Compound Activity in Mice

Combining JESS with traditional western blotting, we analysed protein post-translational modifications in mouse tissue samples to assess the dose and timing of a client’s small molecule. Traditional blotting was used to confirm initial findings, while JESS provided quantitative, high-throughput analysis of key biomarkers. This dual approach ensured robust, reproducible data, informing dosing strategies for the client’s preclinical studies.

Figure 2. Drug activity in vivo. (A) Traditional western blotting used to assess compound activity in mice on phosphorylated target over 4 timepoints. (B) JESS-based probing using alternative antibody epitope to phosphorylated target in (A). Compound tested at low and high dose at 60 and 240 min.

3. Mitochondrial Protein Analysis with JESS

A client required detailed insights into mitochondrial protein expression. Using JESS, we achieved precise, quantitative detection of mitochondrial markers, even with limited sample quantities. The automated system’s high sensitivity allowed for clear differentiation between nuclear and mitochondrially-encoded proteins, facilitating the client’s research into cellular bioenergetics.

Figure 3. Detection mitochondrial proteins. (A) Lane images from automated western blotting using JESS for mitochondrial proteins MFN2 and SDHA in the presence of decoupling agent CCCP which stimulates mitophagy. Beta actin used as a loading control. (B) Quantified relative protein intensities from (A).

Figure 3. (C) o2h scientist working on newly installed JESS equipment at Hauxton House

4. Optimising Extraction Methods in Cancer Cell Lines

We employed JESS to assess protein extraction methods in two cancer cell lines, comparing whole cell and nuclear fractions. By detecting two nuclear proteins across the fractions, we demonstrated how extraction techniques impact protein localisation and quantification. These findings helped the client optimise their experimental workflows for cancer biology research.

Figure 4. JESS-based assay optimisation. Using JESS to optimise extraction conditions of 2 nuclear proteins in 2 cancer cell lines.

For more information or to explore collaboration opportunities, reach out to us at discovery@o2h.com.