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Controls for Immunoassays

Giving you confidence in your results

Your results are only as good as the controls you use. To be confident your results are accurate and reproducible, you need the right controls for every experiment.

Take a look at some of the immunoassay controls we offer and how you should use them.

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.

RIC-001_IHb
Immunohistochemistry with anti-P2RY1 vs an IgG isotype control Cell nuclei are stained with DAPI (blue) in both panels. A. Perfusion-fixed frozen rat spinal cord sections with Anti-P2Y1 receptor (extracellular)-ATTO Fluor-488 Antibody (#APR-021-AG), (1:80 dilution). 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.
RIC-001-AG_THP1
Direct flow cytometry to establish the effectiveness of an IgG isotype control Rabbit IgG Isotype Control-ATTO Fluor-488 (#RIC-001-AG), alongside cell surface detection of P2RX7 in a live intact human THP-1 monocytic leukemia cell line. A. In the absence of an Fc block. B. In the presence of an Fc block. Black = Cells. Blue = Cells + Rabbit IgG Isotype Control-ATTO Fluor-488 (#RIC-001-AG), (2.5 µg). Green = Cells + Anti-P2X7 Receptor (extracellular)-ATTO Fluor-488 Antibody (#APR-008-AG), (2.5 µg).
RIC-001_IP
Immunoprecipitation of rat brain lysates using SV2A and isotype control antibodies
  1. Brain lysate
  2. Brain lysate + Protein A beads + Rabbit IgG Isotype Control (#RIC-001), (4 µg).
  3. Brain lysate + Protein A beads + Anti-SV2A (#ANR-095), (4 µg).
Black arrow indicates the SV2A protein, Red arrow indicates the IgG heavy chain. Western blot was conducted using Anti-SV2A (#ANR-095), (1:500 dilution).

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.

Immunohistochemistry with anti-P2RY1 vs an IgG isotype control
Cell nuclei are stained with DAPI (blue) in both panels. A. Perfusion-fixed frozen rat spinal cord sections with Anti-P2Y1 receptor (extracellular)-ATTO Fluor-488 Antibody (#APR-021-AG), (1:80 dilution). 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.
Direct flow cytometry to establish the effectiveness of an IgG isotype control
Rabbit IgG Isotype Control-ATTO Fluor-488 (#RIC-001-AG), Rabbit IgG Isotype Control-ATTO Fluor-488 (#RIC-001-AG) alongside cell surface detection of P2RX7 in a live intact human THP-1 monocytic leukemia cell line. A. In the absence of an Fc block. B. In the presence of an Fc block.
Black = Cells. Blue = Cells + Rabbit IgG Isotype Control-ATTO Fluor-488 (#RIC-001-AG), (2.5 µg). Green = Cells + Anti-P2X7 Receptor (extracellular)-ATTO Fluor-488 Antibody (#APR-008-AG), (2.5 µg).
Immunoprecipitation of rat brain lysates using SV2A and isotype control antibodies
  1. Brain lysate
  2. Brain lysate + Protein A beads + Rabbit IgG Isotype Control (#RIC-001), (4 µg).
  3. Brain lysate + Protein A beads + Anti-SV2A (#ANR-095), (4 µg).
  4. Black arrow indicates the SV2A protein, Red arrow indicates the IgG heavy chain. Western blot was conducted using Anti-SV2A (#ANR-095), (1:500 dilution).
Direct flow cytometry to establish the effectiveness of an IgG isotype control
Rabbit IgG Isotype Control-ATTO Fluor-488 (#RIC-001-AG), Rabbit IgG Isotype Control-ATTO Fluor-488 (#RIC-001-AG) alongside cell surface detection of P2RX7 in a live intact human THP-1 monocytic leukemia cell line. A. In the absence of an Fc block. B. In the presence of an Fc block.
Black = Cells. Blue = Cells + Rabbit IgG Isotype Control-ATTO Fluor-488 (#RIC-001-AG), (2.5 µg). Green = Cells + Anti-P2X7 Receptor (extracellular)-ATTO Fluor-488 Antibody (#APR-008-AG), (2.5 µg).

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.

Immunohistochemistry with anti-P2RY1 vs an IgG isotype control
Cell nuclei are stained with DAPI (blue) in both panels. A. Perfusion-fixed frozen rat spinal cord sections with Anti-P2Y1 receptor (extracellular)-ATTO Fluor-488 Antibody (#APR-021-AG), (1:80 dilution). 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.
Direct flow cytometry to establish the effectiveness of an IgG isotype control
Rabbit IgG Isotype Control-ATTO Fluor-488 (#RIC-001-AG), Rabbit IgG Isotype Control-ATTO Fluor-488 (#RIC-001-AG) alongside cell surface detection of P2RX7 in a live intact human THP-1 monocytic leukemia cell line. A. In the absence of an Fc block. B. In the presence of an Fc block.
Black = Cells. Blue = Cells + Rabbit IgG Isotype Control-ATTO Fluor-488 (#RIC-001-AG), (2.5 µg). Green = Cells + Anti-P2X7 Receptor (extracellular)-ATTO Fluor-488 Antibody (#APR-008-AG), (2.5 µg).
Immunoprecipitation of rat brain lysates using SV2A and isotype control antibodies
  1. Brain lysate
  2. Brain lysate + Protein A beads + Rabbit IgG Isotype Control (#RIC-001), (4 µg).
  3. Brain lysate + Protein A beads + Anti-SV2A (#ANR-095), (4 µg).
  4. Black arrow indicates the SV2A protein, Red arrow indicates the IgG heavy chain. Western blot was conducted using Anti-SV2A (#ANR-095), (1:500 dilution).

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.

ACC-037_WB
Western blot analysis of rat brain lysate (lanes 1 and 3) and mouse brain lysate (lanes 2 and 4):
1-2. Guinea Pig Anti-TRPA1 (extracellular) Antibody (#ACC-037-GP), (1:200).
3-4. Guinea Pig Anti-TRPA1 (extracellular) Antibody (#ACC-037-GP), preincubated with TRPA1 (extracellular) Blocking Peptide (#BLP-CC037).
ACC-037_IH
Immunohistochemical staining of perfusion-fixed frozen mouse brain sections with Guinea Pig Anti-TRPA1 (extracellular) Antibody (#ACC-037-GP), (1:1000), followed by goat anti-guinea pig-Alexa-594. A. TRPA1 immunoreactivity (red) appears in the wall of the fourth cerebral ventricle (arrows). B. Pre-incubation of the antibody with TRPA1 (extracellular) Blocking Peptide (#BLP-CC037), suppressed staining. Cell nuclei are stained with DAPI (blue).

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.

AQP4 control
Western blot of pre-absorbed AQP4 control
Mouse (lanes 1 and 3) and rat (lanes 2 and 4) brain lysates: 1,2. Anti-Aquaporin 4 (AQP4) (300-314) Antibody (#AQP-014), (1:200). 3,4. Anti-Aquaporin 4 (AQP4) (300-314) Antibody preincubated with Aquaporin 4/AQP4 (300-314) Blocking Peptide (#BLP-QP014).
TRPV4 control
Immunocytochemistry with a TRPV4 control
mCCDcl1 cells stained with Anti-TRPV4 Antibody (#ACC-034) (top row), and with the antibody plus the TRPV4 Blocking Peptide (#BLP-CC034) (bottom row). Adapted from Li, Y. et al. (2016) PLoS ONE 11, e0155006
Positive controls – these are recombinant proteins that allow you to check that your primary and secondary antibodies are working as expected. Positive immunoassay controls like these also give you a clear band at the expected molecular weight of the target protein.
PCP-G240
Western blot 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).
PCP-B257
Western blot using proBDNF Positive Control Lanes 1 and 2: 10 µL (corresponding to 50 ng) per lane of proBDNF Positive Control for Western Blot (#PCP-B257), was run in SDS-PAGE and detected in a Western blot using Anti-proBDNF Antibody (#ANT-006), (1:200 dilution).
Positive Controls– these are recombinant proteins that allow you to check that your primary and secondary antibodies are working as expected. Positive immunoassay controls like these also give you a clear band at the expected molecular weight of the target protein.
GDNF Positive Control
Western blot 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).
proBDNF Positive Control
Western blot using proBDNF Positive Control
Lanes 1 and 2: 10 µL (corresponding to 50 ng) per lane of proBDNF Positive Control for Western Blot (#PCP-B257), was run in SDS-PAGE and detected in a Western blot using Anti-GDNF Antibody (#ANT-014), (1:200 dilution).

Tips

Positive Controls

Confirm the target protein’s presence and that your reagents work as intended.

  • Reference points: positive controls often aid visualization across various applications like Western blot, overexpression studies, and knock-down experiments, giving you an idea of what to expect from experiments.  
  • Anatomical positive control: The gold standard: it uses a tissue known to express the protein of interest. It confirms antigen presence in a known location, either within the specimen (internal) or in a separate specimen (external), thereby ensuring staining specificity.

Negative Controls

Crucial for demonstrating epitope-antibody interaction, especially for commercial antibodies.

  • “No primary antibody” pitfall: Omitting the primary antibody alongside a specimen actually controls for secondary antibody binding, not primary antibody specificity.  
  • Preabsorption control for nonspecific binding: This test involves preincubating the antibody with an excess of the blocking peptide. Reduced staining indicates specific binding, but it may inhibit off-target binding and recognize related epitopes. It confirms specificity – but not selectivity – and should be used cautiously in certain cases.

Immunoassay Controls Coming Soon

Currently we are working on developing positive controls for Western blot in Xenopus oocytes. We’ll give you more details as soon as we have some data to share. Make sure to keep an eye on this section.
Microglia-Markers-graph.png
Live intact mouse BV-2 cells were simultaneously stained by adding 5μg of the mentioned antibodies for 1 hour at 4°C. The cells were rinsed and analyzed by flow cytometry.

Anti-P2Y12 Receptor (extracellular)-PE Antibody

Cat # APR-095-F
Host Rabbit
Clonality Polyclonal
Cat # APR-095-F
Host Rabbit
Clonality Polyclonal
Cat # APR-095-F
Host Rabbit
Clonality Polyclonal

Subheading Lorem Ipsum

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.

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