Advanced Western Blotting Solutions for Cancer & Immuno-Oncology

Tumor heterogeneity poses a significant challenge to the development of effective cancer therapies. Further, the tumor microenvironment creates a complex ecosystem of diverse tumor and immune cell types. Methodologies like flow cytometry have limitations such as off-target binding of variants/ isoforms and difficulties in finding validated antibodies. In this application note, Simple Western and Single-Cell Western systems partner to get you critical answers on the immune cell populations found in the tumor microenvironment offering both population level views and single-cell level perspectives on cell subtypes. Request pricing or more information on Simple Westerns and Single-Cell Westerns.

The new and rapidly expanding field of immuno-oncology requires high-resolution, quantitative analytical tools at both the population and single-cell level to parse the complex interplay between cancer, immune, and stromal cells. As a result of the increased linear range, small sample size requirements and quantification, Simple Western systems have been involved in breakthrough immunotherapies, including those involving immune checkpoint inhibitors, CAR T-cell therapy, bispecific antibodies, and macrophage switching.

Meanwhile, Single-Cell Western with Milo provides insight into population heterogeneity at the protein level and has been used to study the molecular underpinnings of malignant cell transformation with unprecedented detail. This Scientific Review highlights recent studies that demonstrate the use of Simple Western and Single-Cell Western assays to make breakthroughs in immuno-oncology.

Laser capture microdissection (LCM) is a powerful technique that has become instrumental to cancer researchers interested in comparing the molecular profiles of tumor tissue to surrounding non-tumor cells. As such, LCM analysis is leading the way for the development of tailored treatment and personalized medicine. Simple Western requires only a few microliters of LCM sample to generate multiple data points. Simple Western also has the sensitivity to analyze low abundance proteins that are difficult to detect by traditional Western blot. And it’s all wrapped up in a simple workflow that minimizes your hands-on time. In this application note, we show two examples of how Simple Western was ideally suited for the analysis of LCM samples. Request pricing or more information on Simple Westerns.

Quantitative measurements of protein expression are extremely difficult with traditional western blotting due to variability during membrane transfer. Further, simultaneous phospho-protein and total protein measurements on the same sample are challenging on conventional western blots due to loss of protein and non-uniformity during stripping & reprobing. With Simple Westerns, proteins are immobilized after separation, reducing variability and enabling quantitative measurements of protein abundance. Further, multiplexing is easy using multiple detection channels or RePlexing cycles to enable phospho/total measurements in the same capillary. Total protein measurements can also be made on the same sample for more accurate and robust normalization. Request pricing or more information on Simple Westerns.

The Milo Single-Cell Western system helps detect discrete cell signaling states by measuring multiple phospho-proteins in individual cells within a population.  Because cells are lysed in Single-Cell Westerns, membrane permeabilization is not required and both intracellular and intranuclear proteins are accessible for antibody probing, making it possible to use phospho- antibodies validated for western blotting and simplifying some challenging intracellular flow assays. This application note demonstrates how Milo adds single cell resolution and heterogeneity information to cell signaling studies traditionally done with conventional western blotting and simplifies challenging phospho-flow assays done with flow cytometry. Request pricing or more information on Single-Cell Westerns.