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Flexibility is our middle name when it comes to antibody selection!

At Theralink, we pride ourselves on our extensive selection of over 700 antibodies, allowing customers to study a wide range of protein pathways. So far, we have tested antibodies involved pathways such as JAK-STAT, NF-kB, DNA damage response, B-cell receptor signaling, and more. When a customer needs to analyze a biomarker not already covered by our library, we have the capability to validate new antibodies in-house. Today, we’ll walk you through what it takes to ensure all new antibodies meet Theralink’s standard of quality and customer service.

Choosing an antibody: the “client first” approach

To begin, we need to decide which vendor and antibody to use. In the best-case scenario, we use an antibody the client has already validated with their own work. This minimizes experimental variation between results we provide and any prior data the client already has, adding confidence to a study’s outcome.

However, there are several reasons a client’s antibody may not be suitable. Antibodies used for Western blots may exhibit off-target binding, which will appear as multiple bands in a blot. While this isn’t always an issue with Western blots as the desired protein can be identified by its expected molecular weight, array-based techniques such as reverse-phase protein arrays (RPPA) will be unable to distinguish off-target binding from the target biomarker. In addition, antibodies validated for immunohistochemistry (IHC) may not be optimal for RPPA, as IHC antibodies recognize native protein conformation while RPPA uses denatured lysate. Based on this, we look for antibodies that have been validated on Western blots with minimal off-target signals.

Phase I: screening by Western blot

Once we have selected the best candidate antibody, we start our in-house validation process, beginning with Western blots. We use a positive control provided by the antibody vendor if available; if not, we search the literature to identify tissues or cell lines where the protein is expected to be present and obtain those samples for testing. We’ll do the same process for our negative controls, looking for a single clean band on the positive controls (or the predicted sizes of a cleavage product if relevant) and no bands on the negative controls. If the client has their own control samples to use, we can also include these in our validation process as well.

Figure 1: Flowchart of our antibody validation process.

Phase II: Optimization for RPPA analysis

Once the antibody passes our Western blot inspection, it moves on to array testing. We print the same negative and positive control lysates used for Western blot validation, as well as a few additional calibration lysates to ensure we stay within the quantitative range of the RPPA scanner. If we are testing a phosphorylation-specific antibody, we also add a sample treated with phosphatase or stimulant to look for a dose response, ensuring the antibody detects the phosphorylated state and not simply total protein. We then test a range of antibody concentrations on our arrays. We look for a concentration that is high enough to reliably detect our protein but is low enough to avoid antibody binding to off-target proteins. This optimization ensures our results are quantitative with minimal input requirements, allowing us to accurately measure biomarkers in patients even when tissue quantity is limited, such as when a core needle biopsy is used.

Figure 2: Sample Western blot and RPPA array
A) An example Western blot testing a HER2/ErbB2 antibody. The left, middle, and right cell lines are expected to have high, medium, or no HER2 expression, respectively. (Source: https://www.thermofisher.com/antibody/product/ErbB2-HER-2-Antibody-Polyclonal/PA5-14635).
B) An example RPPA slide printed, scanned, and ready for analysis.

We’ll work with you and your experiments!

If you are thinking about planning your next experiment and want comprehensive and accurate pathway profiling, reach out to our team here to learn more. We’ll work with your antibodies when possible or help you to select an excellent alternative to enable high-throughput protein biomarker quantification.