Why Immunoassay Controls Matter and How to Use Them Effectively

There’s nothing quite like the thrill of genuine scientific discovery, regardless of the scale – it’s that moment when you glimpse something truly novel. But as wonderful as the moment are, we have to temper them with an important consideration: validation. Because behind every great result is a backbone of rigorous controls. Without these, even the most exciting findings might crumble under scrutiny.

The Role of K_V Channels in Cellular Excitability

In a 2024 study, Jansen and colleagues looked at the regulation of K_V4.2 and K_V4.3 channels, which are needed for cellular excitability and contribute to shaping action potentials (1). These channels are finely regulated by a balance between post-translational SUMOylation and phosphorylation. By examining how different post-translational modifications impact K_V channel function, the team made significant strides in understanding the balance between SUMOylation and phosphorylation in regulating ion channel function. They managed to identify the protein inhibitor of activated stat proteins 3 (PIAS3) as a SUMO ligase, one of the principal downstream mediators controlling surface expression of K_V4 channels in HEK cells and cardiomyocytes. Their results show the precision of ion channel regulation and the importance of controls, such as IgG controls, in validating these results.

Why Controls Are Crucial for Immunoassays

This work stands on its own as good research into cell signaling, but it’s also a great example of the use of great controls in immunoassay work. Biological systems are inherently complex, with interconnected pathways that can easily skew experimental results if we don’t have controls in place. When we modify something – like an ion channel or a specific signaling pathway – the ripples of that affect downstream molecules in unexpected ways.

This is where immunoassay controls become indispensable. In their study, Jansen et al. used our Anti-K_V4.2 (#APC-023) and Anti-K_V4.3 (#APC-017) antibodies for immunoprecipitation (IP) and western blot (WB), but they also made good use of isotype control to ensure the validity of their data. Specifically, they included IgG controls, which were critical for ruling out non-specific binding and ensuring the antibodies were targeting K_V4.2 correctly (Figure 1A and C). Without these controls, their findings on the SUMOylation and phosphorylation interplay could have been compromised​.

_{Figure 1. Increasing PIAS3 expression results in enhanced SUMOylation at KV4.2-K579. A) Representative results for IP followed by western blot experiments using protein lysates from HEK cells expressing Kv4.2 TC with or without PIAS3. IP and western blot (WB) antibodies were as indicated. WBs were first probed with anti-SUMO, imaged, stripped, and re-probed with anti-KV4.2. B) Bar graphs show the mean normalized fraction of SUMOylated KV4.2 ± SEM. The O.D. for each signal was measured. The SUMO O.D. was divided by the KV4.2 O.D. to obtain the fraction of SUMOylated channels for a given lysate. All data points were then divided by the mean for the control treatment group. Each symbol in the graph represents an independent transfection and IP/western blot experiment. *, significantly different, ns, not significant (SUMO1: 1.00 ± 0.2 vs. 2.7 ± 0.4; t-test, p = 0.021; SUMO2: 1.00 ± 0.29 vs. 1.2 ± 0.18; t-test, p = 0.5032). C) Representative results for IP followed by western blot experiments using protein lysates from HEK cells expressing KV4.2-K579R TC with or without PIAS3. IP and WB antibodies were as indicated. The protocol was as described in A. D) Bar graphs show the mean normalized fraction of SUMOylated KV4.2-K579R ± SEM. Measurements and analyses were as in B. ns, not significant (SUMO1: 1.00 ± 0.3 vs. 1.05 ± 0.34, t-test, p = 0.9146; SUMO2: 1.00 ± 0.32 vs. 0.89 ± 0.38, t-test, p = 0.8373). Figure and legend from Jansen et al. (2024).}

The IgG Control in Focus

An IgG control is a powerful negative control known as an isotype control. Isotype controls are antibodies of the same isotype as your detection antibody – like IgG – but they lack specific target binding. This control helps you to account for unwanted Fc receptor interactions, so you can distinguish non-specific background noise from target signal. Isotype controls are essential for immunohistochemistry (IHC), flow cytometry (FC), and immunoprecipitation (IP) – techniques where there is a high risk of background staining – but they are valuable immunoassay controls for all immunoassay techniques.

In Jansen’s work, Rabbit IgG Isotype Control was used to confirm that the signal they observed in their experiments was specific to the anti-K_V4.2 and anti-K_V4.3 antibodies and not the result of random protein binding to the antibody. Using this sort of control, the team could confirm that the SUMOylation of K_V4.2 channels was valid and specific by comparing signals from their anti-K_V4.2 antibody with an IgG control. Without this negative control, the authors could not definitively rule out non-specific interactions – undermining their conclusion that SUMOylation specifically targets K_V4.2 channels​.

Immunoassay Controls Build Credibility

Controls aren’t glamorous, but they are necessary. The excitement of discovering novel ion channel regulatory mechanisms – like the crosstalk between phosphorylation and SUMOylation seen in this study – only holds up if you have rigorous controls to back it up. By using IgG and other controls, you can build a foundation of credibility for your findings. Without them, even a breakthrough study could lose any impact when scrutinized.

At Alomone, we recognize the importance of these controls, which is why our K_V4.2 and K_V4.3 antibodies come validated for use in key assays like immunoprecipitation. Properly selected controls ensure that the findings you present are built on a foundation of scientific rigor. Whether you’re studying ion channel modulation or any other target, always remember that thoughtful controls will prepare your research for the unknown.

Invest in the Right Tools for Immunoassay Controls

Using properly validated antibodies and adding in rigorous controls might feel like an extra step, but it’s time well spent. We have a range of antibodies and controls – all developed in house – to support your work. We’ve even created a page dedicated to helping your pick the right controls for your research. Yes, controls are upfront investment, but they’ll save you valuable time and resources later on by ruling out false positives and strengthening the reliability of your data.

When the time come to plan your next immunoassays, take a moment to make sure you’ve included the proper controls, so your groundbreaking findings hold up in the long run.

Visit our immunoassay control page!

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Reference

1. L.-A. R. Jansen, M. A. Welch, L. D. Plant, D. J. Baro, Crosstalk between PKA and PIAS3 regulates cardiac Kv4 channel SUMOylation. Cell Commun Signal22, 422 (2024). DOI: https://doi.org/10.1186/s12964-024-01795-4.