Western blotting or protein immunoblotting remains the gold standard methodology of choice for protein detection and characterization, remaining relatively unchanged since 1979.
A traditional western blot is a multi-step procedure that typically involves the following:
- Sample preparation (extraction of protein from the desired sample) from cells or tissue
- Separation of proteins by size using a polyacrylamide gel by electrophoresis and immobilization on a PVDF or nitrocellulose membrane
- Detection of proteins using a primary antibody specific to the target and a conjugated secondary antibody suitable for the method of detection.
- Detection of the signal from the secondary antibody, often chemiluminescence and fluorescence.
- Analysis of the bands to give qualitative and semi-quantitative results.
The interaction of a specific antibody (primary) to its epitope enables detection within complex protein mixtures, commonly cell or tissue lysates. Qualitative and semi-quantitative data about a protein of interest is obtainable using this technique and is applicable to all disease areas.
Automated and Quantitative Approach
Immunoblotting has been used routinely used since the 1970’s. The specificity of the interaction allows the protein expression of the target to be determined within complex mixtures, however this technique is not truly quantitative. The next step in the evolution of western blot, the Simple-Western system uses capillary-based separation, requires only a small sample size and can implement multiplexed/multichannel detection. The Simple-Western system allows for a faster, automated, and importantly truly quantitative approach to protein expression analysis.
Figure 1) Cells treated with increasing concentrations of SNS-THAL-032 prior to lysis and subsequent analysis by Simple Western. Quantification of CDK9 (normalised to β-Actin) showed that SNS-THAL-032 is a potent and reproducible degrader of CDK9 with an average DC50 of 16.1 nM ± 7.6 nM and Dmax of 99% across three biological replicates.
The desire to increase throughput while maintaining physiological relevance can be met with the use of flow cytometry, allowing protein expression within individual cells to be determined. At Charnwood Discovery, our high-throughput flow cytometry capability enables the measurement of protein expression of multiple targets within mixed cell populations and to also analyze other key cell health markers simultaneously, such as apoptosis. Flow cytometry is therefore a valuable tool for PROTAC screening.
Figure 2) PROTAC treatment results in a dose dependent shift in the median fluorescence intensity (MFI) of the target protein as measured by flow cytometry. This shift can be quantified to determine DC50 constants for degrader molecules.
The enzyme-linked immunosorbent assay (ELISA) is a commonly used technique to determine the concentration of an analyte, whether that be a protein or antigen. Using specialized plates, the analyte is immobilized to the well surface, enabling quantification.
The bead based AlphaLISA® immunoassay works in a similar manner as the ELISA but has been specifically designed with drug discovery in mind. The AlphaLISA® is a chemiluminescent, no wash assay, with greater sensitivity, wider dynamic range, and smaller sample size than an ELISA. At Charnwood Discovery, this technology has been used to screen against targets of interest, producing highly reproducible data and allowing effective small molecule development.
Figure 4) AlphaLISA schematic example illustrating transfer of energy from donor to acceptor when in close proximity. Quantification of the protein of interest in response to therapeutic treatment demonstrates a dose dependent response.
In early drug discovery bioassays not only reveal whether a compound acts on a biological target but also how effective it is and if it has any potentially off target effects. In turn, this can influence decisions about whether the candidate warrants further study.
The importance of bioassays in early drug discovery may be clear, yet what is less clear is how they should be developed to ensure they are optimised for their specific purpose.
Assay design and development is a complex process, involving many considerations that can each determine the specificity, sensitivity, and reliability of a compound’s action on a target. As such, if you develop your own assays, what you produce can mean the difference between valid and invalid results that help or hinder your drug discovery research.