Choosing Immunoassay Controls

Your guide to picking the right control for the right assay so you get better results

We’ve said this before, but your results are only as good as the controls you have in place. Great results are meaningless if you haven’t controlled for a bunch of variables that could mean your “eureka” moment was actually just a random anomaly, or worse, an error. 

Thankfully, there are plenty of great positive and negative controls to choose from when it comes to running immunoassays like immunohistochemistry (IHC), immunocytochemistry (ICC), and western blot (WB). And to help you navigate the pros and cons of the various controls, we’ve put together this little guide to choose the right control for the right experiment. 

Blocking Peptides 

Right at the top of the list are blocking peptides and isotype antibody controls. Blocking peptides are the immunizing antigens we use during antibody generation and are effective negative reagent controls. Blocking peptides help you confirm antibody specificity as they compete with the detection antibody. 

Blocking Peptide Pros and Cons

Pros

• Targeting: Blocking peptides allow epitope validation, ensuring that the antibody recognizes the specific region of interest on the target protein.

• Detailed information: They provide in-depth information about antibody binding at the epitope level, helping researchers better understand the antibody’s specificity.

• Epitope validation: Useful when there’s a need to help confirm binding specificity to a known epitope.

• Customization: You can design blocking peptides based on the antibody’s known epitope.

Cons

• Labor-intensive: Preparing and validating blocking peptides yourself can be time-consuming, especially for custom sequences. (Thankfully, Alomone already has these for every primary antibody in our catalog.)

• Epitope knowledge: Requires prior knowledge of the antibody’s epitope, something we have plenty of!

Choose a blocking peptide when you need high precision and detailed epitope validation, particularly for custom or specialized antibodies. They are suitable for situations where you want to assess antibody binding to specific regions of a protein.

Figure 1. IHC with a TRPV4 control. Mouse cortical collecting duct cell line stained with Anti-TRPV4 Antibody (#ACC-034) (top row), and with the antibody plus the TRPV4 Blocking Peptide (#BLP-CC034) (bottom row). Adapted from Y. Li et al. PLoS ONE 11, e0155006 (2016).

Isotype Controls

An isotype control is an antibody of the same isotype as your detection antibody – such as IgG – but it doesn’t have any specific target binding. Isotype controls help you account for unwanted Fc receptor interactions, which can generate non-specific background signal. Blocking the Fc receptor should be considered if the cells of interest highly express Fc receptor on their surface, especially for flow cytometry.

Isotype Antibody Pros and Cons

Pros

• Easy access: Isotype control antibodies are readily available and convenient for general specificity checks.

• Standardized controls: They offer standardized negative controls, ensuring consistency across experiments.

• Simple: Ideal for situations where a quick assessment of general specificity is sufficient.

• Flexible: Can be conjugated to the same reporters as the primary antibodies you use.

Cons

• Lack of epitope specificity: Isotype controls do not provide information about the specific region of target protein recognition.

Choose an isotype control antibody when you need a quick and easy way to assess overall antibody specificity. Isotype controls are especially useful in flow cytometry for distinguishing between specific antibody signal and nonspecific background signal.

Figure 2. IHC of perfusion-fixed frozen rat spinal cord sections with Anti-P2Y1 receptor (extracellular)-ATTO Fluor-488 Antibody (#APR-021-AG), (1:80). P2RY1 immunoreactivity (green), appears along the superficial layer of dorsal horn (arrows). B. Staining of sequential sections with Rabbit IgG Isotype Control-ATTO Fluor-488 (#RIC-001-AG), (1:80), shows background signal. Cell nuclei are stained with DAPI (blue).

Western Blot Positive Controls

Positive controls for WB, which are often recombinant proteins, give you a nice and clear expectation of what to expect from your primary antibody (showing you expected molecular weight of the target protein and ensuring the assay’s integrity) and also to confirm that your secondary antibodies are performing as expected. 

WB Positive Controls Pros and Cons

Pros

• Benchmarking: These set a clear expectation for your primary antibody’s target, including the expected molecular weight.
  
• Secondary antibody validation: They can play a crucial role in verifying the performance of secondary antibodies. By ensuring that secondary antibodies can detect the primary antibody-bound protein, positive controls help you confirm the effectiveness of the entire detection system.
  
• Consistency and reliability: This applies to all controls really, but using positive controls in your WB promotes consistency across different experiments and offers a standard against which to compare results.

Cons

• Cost and availability: It can be hard getting high-quality recombinant proteins – they can also be relatively expensive, especially for less common proteins. This cost factor can be a significant consideration for labs with limited budgets.
  
• Misleading interpretations: While positive controls help you confirm a system’s functionality, they might not always accurately reflect the native protein’s behavior in complex biological samples. Differences in post-translational modifications or protein folding between the control and the native protein can lead to misleading interpretations – always have this in mind!
  
• Over-reliance: You can sometimes put too much stock in your positive control if used in isolation. Unless you think more broadly, it can be easy to neglect other crucial aspects of validation, like assessing antibody specificity using additional methods like peptide blocking or knockout models.

Choose a WB positive control when using a new antibody, reagent, or setup to make sure things are working as expected, or when there’s a possibility of cross-reactivity due to closely related proteins or nonspecific binding.

Western blot using GDNF Positive Control

Figure 3. WB using GDNF positive control. Lanes 1 and 2: 10 µL (corresponding to 50 ng) per lane of GDNF Positive Control for Western Blot (#PCP-G240), was run in SDS-PAGE and detected in a Western blot using Anti-GDNF Antibody (#ANT-014), (1:200 dilution).

Our Immunoassay Controls in the Literature

Figure 4. TRPA1 expression in human cardiac fibroblasts (hCFs) A. Immunofluorescence of hCFs using anti-TRPA1 (extracellular) antibody (ACC-037). The right picture is a magnified version of the left picture. B. hCFs were co-incubated with TRPA1 (extracellular) Blocking Peptide (#BLP-CC037). Note that co-incubation with a blocking peptide decreased the staining. C. Negative control in the absence of the primary antibody. G. Oguri et al. Heliyon 7, e05816 (2020)

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Figure 5. Control for non-specific binding by rabbit anti-Kv4.3 antibody. A. Fields of optic nerve that were longitudinally sectioned. The Anti-KV4.3 Antibody (#APC-017) in the upper panel was preincubated with Kv4.3 Blocking Peptide (#BLP-PC017). B. Western-blotted lanes of optic nerve lysate. The anti-Kv4.3 antibody was preincubated with the blocking peptide (“+immunogen”) or without this preincubation (“–immunogen”). G. Ogata et al. Front Neuroanat 16, 958986 (2022)

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Figure 6. Expression of SHANK3 in mouse retina. A. Western blot confirmed SHANK3 expression in retinal homogenates via Anti-Shank3 Antibody (#APZ-013). No band when the anti-Shank3 antibody was pre-absorbed with Shank3 Blocking Peptide (#BLP-PZ013). B. Confocal fluorescence microphotographs of a vertical section labeled with anti-Shank3 antibody. C. No signal when anti-Shank3 antibody was pre-absorbed with the corresponding blocking peptide. D. No signal was detectable when the primary antibody was omitted. E. No signal was detectable in Shank3-mutant control. Y. Xu et al. Front Cell Neurosci 16, 795668 (2022)

Making Your Research Robust

When we talk about “robust research” we’re talking about research that’s accurate, research that reflects the true nature of what we’re investigating, and research that others can reproduce. Understanding and implementing the right controls in your experiments is more than absolutely central to that. By rigorously validating antibody specificity and function, we can push the boundaries of what’s known, one accurate result at a time.

Take a look at our comprehensive range of immunoassay controls and see how you can make your research that much more robust.

Looking for more references? Here’s a list of papers where you can see our immunoassay controls in action:

J. Abdinghoff, D. Servello, T. Jacobs, A. Beckmann, T. Tschernig, Evaluation of the presence of TRPC6 channels in human vessels: A pilot study using immunohistochemistry. Biomed Rep 16, 42 (2022). DOI: https://doi.org/10.3892/br.2022.1525.

T. K. Acharya, S. Pal, A. Ghosh, S. Kumar, S. Kumar, N. Chattopadhyay, C. Goswami, TRPV4 regulates osteoblast differentiation and mitochondrial function that are relevant for channelopathy. Front Cell Dev Biol 11, 1066788 (2023). DOI: https://doi.org/10.3389/fcell.2023.1066788.

I. Andriulė, D. Pangonytė, A. Gwanyanya, D. Karčiauskas, K. Mubagwa, R. Mačianskienė, Detection of TRPM6 and TRPM7 Proteins in Normal and Diseased Cardiac Atrial Tissue and Isolated Cardiomyocytes. Int J Mol Sci 23, 14860 (2022). DOI: https://doi.org/10.3390/ijms232314860.

Y. S. Cho, H. M. Han, S. Y. Jeong, T. H. Kim, S. Y. Choi, Y. S. Kim, Y. C. Bae, Expression of Piezo1 in the Trigeminal Neurons and in the Axons That Innervate the Dental Pulp. Front Cell Neurosci 16, 945948 (2022). DOI: https://doi.org/10.3389/fncel.2022.945948.

N. D’Onofrio, C. Sardu, M. C. Trotta, L. Scisciola, F. Turriziani, F. Ferraraccio, I. Panarese, L. Petrella, M. Fanelli, P. Modugno, M. Massetti, L. V. Marfella, F. C. Sasso, M. R. Rizzo, M. Barbieri, F. Furbatto, F. Minicucci, C. Mauro, M. Federici, M. L. Balestrieri, G. Paolisso, R. Marfella, Sodium-glucose co-transporter2 expression and inflammatory activity in diabetic atherosclerotic plaques: Effects of sodium-glucose co-transporter2 inhibitor treatment. Mol Metab 54, 101337 (2021). DOI: https://doi.org/10.1016/j.molmet.2021.101337.

R. M. De Guzman, Z. J. Rosinger, K. E. Parra, J. S. Jacobskind, N. J. Justice, D. G. Zuloaga, Alterations in corticotropin-releasing factor receptor type 1 in the preoptic area and hypothalamus in mice during the postpartum period. Horm Behav135, 105044 (2021). DOI: https://doi.org/10.1016/j.yhbeh.2021.105044.

L. Liu, Y. Zhao, W. An, M. Zhao, N. Ding, H. Liu, N. Ge, J. Wen, X. Zhang, S. Zu, W. Sun, Piezo2 Channel Upregulation is Involved in Mechanical Allodynia in CYP-Induced Cystitis Rats. Mol Neurobiol 60, 5000–5012 (2023). DOI: https://doi.org/10.1007/s12035-023-03386-9.

G. Ogata, G. J. Partida, A. Fasoli, A. T. Ishida, Calcium/calmodulin-dependent protein kinase II associates with the K+ channel isoform Kv4.3 in adult rat optic nerve. Front Neuroanat 16, 958986 (2022). DOI: https://doi.org/10.3389/fnana.2022.958986.

G. Oguri, T. Nakajima, H. Kikuchi, S. Obi, F. Nakamura, I. Komuro, Allyl isothiocyanate (AITC) activates nonselective cation currents in human cardiac fibroblasts: possible involvement of TRPA1. Heliyon 7, e05816 (2020). DOI: https://doi.org/10.1016/j.heliyon.2020.e05816.

A. Pegoraro, E. Orioli, E. De Marchi, V. Salvestrini, A. Milani, F. Di Virgilio, A. Curti, E. Adinolfi, Differential sensitivity of acute myeloid leukemia cells to daunorubicin depends on P2X7A versus P2X7B receptor expression. Cell Death Dis 11, 876 (2020). DOI: https://doi.org/10.1038/s41419-020-03058-9.

D. Peixoto-Neves, P. Kanthakumar, J. M. Afolabi, H. Soni, R. K. Buddington, A. Adebiyi, KV7.1 channel blockade inhibits neonatal renal autoregulation triggered by a step decrease in arterial pressure. Am J Physiol Renal Physiol 322, F197–F207 (2022). DOI: https://doi.org/10.1152/ajprenal.00568.2020.

K. Tate, B. Kirk, A. Tseng, A. Ulffers, K. Litwa, Effects of the Selective Serotonin Reuptake Inhibitor Fluoxetine on Developing Neural Circuits in a Model of the Human Fetal Cortex. Int J Mol Sci 22, 10457 (2021). DOI: https://doi.org/10.3390/ijms221910457.

P. Wang, N. Kljavin, T. T. T. Nguyen, E. E. Storm, B. Marsh, J. Jiang, W. Lin, H. Menon, R. Piskol, F. J. de Sauvage, Adrenergic nerves regulate intestinal regeneration through IL-22 signaling from type 3 innate lymphoid cells. Cell Stem Cell30, 1166-1178.e8 (2023). DOI: https://doi.org/10.1016/j.stem.2023.07.013.

Y. Xu, Y. Wang, G. Tong, L. Li, J. Cheng, L. Zhang, Q. Xu, L. Wang, P. Zhang, Expression of SH3 and Multiple Ankyrin Repeat Domains Protein 3 in Mouse Retina. Front Cell Neurosci 16, 795668 (2022). DOI: https://doi.org/10.3389/fncel.2022.795668.M. Zhao, N. Ding, H. Wang, S. Zu, H. Liu, J. Wen, J. Liu, N. Ge, W. Wang, X. Zhang, Activation of TRPA1 in Bladder Suburothelial Myofibroblasts Counteracts TGF-β1-Induced Fibrotic Changes. Int J Mol Sci 24, 9501 (2023). DOI: https://doi.org/10.3390/ijms24119501