Updated on November 12, 2024.
Your immunoassay results are only as accurate as the controls you use. High-quality immunoassay controls are essential for distinguishing between valid data and misleading results. These controls help you to eliminate errors and increase confidence in your findings. To ensure reliable and reproducible data, it’s crucial to select the right immunoassay controls for every experiment.
Explore our comprehensive range of immunoassay controls designed to give you confidence in your results, whether you’re performing immunohistochemistry, flow cytometry, or any other immunoassay technique.
Positive immunoassay controls serve as reference points, allowing you to compare your results against known standards, confirm the target protein’s presence, and verify that your antibodies are working as expected. Currently, we offer several recombinant proteins for use as positive controls in western blot. When your antibodies are functioning properly, these controls produce a clear band at the expected molecular weight of the target protein, confirming antibody performance.
Negative immunoassay controls are crucial for demonstrating epitope-antibody interaction, especially for commercial antibodies. Below we cover two critical negative immunoassay controls: isotype controls and blocking peptides.
Isotype controls are primary antibodies that match the isotype of your detection antibody – such as IgG – but lack specific target binding. These controls help you distinguish non-specific background noise from the target signal by accounting for unwanted Fc receptor interactions.
Isotype controls are particularly valuable in techniques prone to high background staining, like flow cytometry (FC) and immunohistochemistry (IHC). When selecting an isotype control, make sure it matches the reporter conjugate of your detection antibody, such as FITC to FITC or APC to APC, to maintain consistency across your experiments.
An isotype control is an antibody of the same isotype as your detection antibody – such as IgG – but which lacks specific target binding.
This control helps you account for unwanted Fc receptor interactions, helping you to 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.
Make sure your isotype control matches the reporter conjugate of your detection antibody, such as FITC to FITC or APC to APC.
An isotype control is an antibody of the same isotype as your detection antibody – such as IgG – but which lacks specific target binding.
This control helps you account for unwanted Fc receptor interactions, helping you to 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.
Make sure your isotype control matches the reporter conjugate of your detection antibody, such as FITC to FITC or APC to APC.
Blocking peptides – also known as immunizing peptides or negative control antigens – are essential immunoassay controls that validate antibody specificity. These peptides are the immunizing antigens used during antibody generation, and they act as effective negative reagent immunoassay controls by competing with your detection antibody.
Using blocking peptides allows you to confirm that your antibody is binding specifically to its target. Persistent staining indicates specific binding. Note that preincubating the antibody with an excess of blocking peptide may inhibit off-target binding and the recognition of related epitopes. It confirms specificity – but not selectivity – and should be used cautiously in certain cases.
You can find blocking peptides for all the antibodies in our catalog: simply search for your antibody to locate the corresponding blocking peptide.
Blocking peptides – also known as immunizing peptides or negative control antigens – are the immunizing antigens we use during antibody generation. They are effective negative reagent immunoassay controls that help you confirm antibody specificity, because they compete with the detection antibody.
You can find blocking peptides for all the antibodies in our catalog: simply search for your antibody to locate the corresponding blocking peptide.
Selecting the appropriate immunoassay control depends on the specific needs of your experiment. Consider the type of assay, potential sources of background noise, and the importance of confirming antibody specificity and functionality. Our experts are available to help you choose the right controls to achieve the most accurate and reproducible results. Please contact us.
Cat # | APR-095-F |
Host | Rabbit |
Clonality | Polyclonal |
Cat # | APR-095-F |
Host | Rabbit |
Clonality | Polyclonal |
Cat # | APR-095-F |
Host | Rabbit |
Clonality | Polyclonal |
Microglia, the resident macrophages of the central nervous system (CNS), make up approximately 10% of the CNS cell population and play a critical role in maintaining homeostasis, responding to injury and inflammation, and clearing cellular debris.
Dysregulation of microglia function has been implicated in a range of neurological disorders, including Alzheimer’s disease and multiple sclerosis. Given their vital function in brain health and disease, there is a growing interest in developing tools and techniques for identifying and studying microglia.
Microglia markers are specific proteins or molecules that are expressed by microglia and that are commonly used to identify and study these cells, to distinguish microglia from other cells, and to provide insights into dysfunctional states. The use of microglia markers can also help identify changes in microglial function that occur during injury or disease.
Microglia-specific markers include surface markers, intracellular markers, and functional markers. Surface markers are proteins found on the outer membrane of microglial cells and play a crucial role in identifying and characterizing cell types, activation and functional states, and disease-related implications. Intracellular markers are located inside microglial cells and are used to investigate specific cellular processes, signaling pathways, and activation states. Functional markers are associated with specific microglial functions, including inflammatory response, neuroprotection, tissue repair, and disease-associated changes. Microglia-specific markers serve as valuable tools for research, diagnostics, and potential therapeutic interventions targeting microglial activity in neurological conditions.
A widely used technique to analyze microglia markers is immunofluorescence (IF), which allows for the visualization and localization of specific molecules within cells or tissues. IF uses fluorescently labeled antibodies to detect and target specific proteins or antigens of interest. The obtained fluorescence images can be analyzed and quantified using image analysis software. Colocalization analysis can determine the degree of overlap between different microglia-specific markers, indicating potential interactions or colocalization within cells or tissues. Fluorescence intensity measurements can provide insights into relative protein expression levels and their subcellular localization patterns.
Microglia markers play a useful role in investigating various neurological diseases. Microglia play a crucial role in the pathogenesis of Alzheimer’s disease, which is characterized by the accumulation of amyloid beta plaques and neurofibrillary tangles in the brain. Under normal conditions, microglia can phagocytose and clear amyloid beta plaques. In Alzheimer’s disease, microglia often exhibit an impaired ability to efficiently remove these toxic protein aggregates. The dysfunction of microglia in Alzheimer’s disease is believed to be a result of the chronic inflammatory environment and dysregulated immune response, which can exacerbate neurodegeneration.
In Parkinson’s disease, a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra of the brain, microglia impairment decreases the clearance capacity of Lewy bodies, misfolded alpha-synuclein aggregates which are the hallmark of Parkinson’s disease. Chronic neuroinflammation mediated by microglia is thought to contribute to the progression of neurodegeneration in Parkinson’s disease.
Microglia respond to CNS injuries, such as traumatic brain injury (TBI) and spinal cord injury. Upon injury, microglia become activated and undergo morphological changes, migrating to the site of injury and releasing various factors, such as cytokines, chemokines, and reactive oxygen species, which can contribute to secondary damage and neuroinflammation after CNS injuries. Microglia also have beneficial functions in CNS injuries, such as promoting tissue repair, clearance of cellular debris, and releasing neurotrophic factors that support neuronal survival and regeneration. Modulating microglial activation and inflammatory responses is an area of active research for potential therapeutic interventions for CNS injuries.