ADCY8 Antibody

What is the molecular function of ADCY8 and why is it a significant research target?

ADCY8 belongs to the adenylyl cyclase class-4/guanylyl cyclase family and functions as a calcium-stimulated adenylyl cyclase. It catalyzes the formation of cAMP in response to calcium entry, leading to cAMP signaling activation that affects processes such as synaptic plasticity and insulin secretion. ADCY8 plays crucial roles in numerous brain functions, including learning, memory, drug addiction, and anxiety modulation through regulation of synaptic plasticity . It modulates long-term memory and long-term potentiation (LTP) through CREB transcription factor activity regulation .

In peripheral tissues, ADCY8 plays a central role in insulin secretion by controlling glucose homeostasis through glucagon-like peptide 1 and glucose signaling pathways. It maintains insulin secretion through calcium-dependent PKA activation leading to vesicle pool replenishment . Recent research also indicates that cardiac-specific overexpression of ADCY8 can enhance cardiac performance while activating protective mechanisms against stress .

What are the validated applications and species reactivity of ADCY8 antibodies?

Commercial ADCY8 antibodies have been validated for multiple applications with varying species reactivity:

Most commercially available antibodies show reactivity to human, mouse, and rat ADCY8 . Some antibodies may potentially cross-react with monkey tissues due to sequence homology, though this requires experimental validation . The calculated molecular weight of ADCY8 is approximately 140 kDa, and observed molecular weight in Western blots typically ranges from 140-150 kDa, though some antibodies may detect it at lower molecular weights .

What are the optimal storage conditions for maintaining ADCY8 antibody stability?

For optimal maintenance of ADCY8 antibody performance, the following storage conditions are recommended:

• Long-term storage: -20°C for up to one year

• Short-term/frequent use: 4°C for up to one month

• Avoid repeated freeze-thaw cycles as they significantly degrade antibody performance

• Most antibodies are provided in buffers containing:

  • PBS with 0.02% sodium azide as preservative

  • 50% glycerol to prevent freezing damage

  • Some formulations may contain 0.1-0.5% BSA for additional stability

According to manufacturer recommendations, aliquoting may be unnecessary for -20°C storage for some formulations, but it's generally advisable for antibodies that will be used multiple times to minimize freeze-thaw cycles .

How should I optimize immunohistochemistry protocols for ADCY8 detection?

For optimal ADCY8 detection in immunohistochemistry applications, consider the following protocol optimization steps:

• Sample preparation and fixation:

  • Formalin-fixed, paraffin-embedded (FFPE) tissues are commonly used

  • Fixation times should be standardized across samples for consistent results

• Antigen retrieval:

  • Heat-induced epitope retrieval (HIER) using Tris/EDTA buffer at pH 9.0 for 45 minutes at 95°C

  • Allow 20 minutes cooling at room temperature after heating

  • This step is critical for unmasking epitopes in formalin-fixed tissues

• Blocking and antibody incubation:

  • Use 5-10% normal serum (from the species of the secondary antibody) for blocking

  • Primary antibody dilutions typically range from 1:50-1:300 for IHC applications

  • Incubation can range from 30 minutes at room temperature to overnight at 4°C

• Detection system:

  • HRP-polymer systems work well for visualization

  • DAB is typically used as the chromogen with 5-minute development time

  • Counterstain nuclei with hematoxylin for contrast

• Controls:

  • Include positive controls (brain tissue, particularly hippocampus)

  • Use negative controls (primary antibody omitted, replaced with diluent)

  • Consider peptide competition controls to verify specificity

Validation images from manufacturers show successful staining of human brain tissue and salivary gland with ADCY8 antibodies using these optimized protocols .

What approaches can validate the specificity of ADCY8 antibodies?

Validating antibody specificity is crucial for ensuring reliable experimental results. For ADCY8 antibodies, consider these validation approaches:

• Peptide competition assays:

  • Pre-incubate your antibody with excess immunogenic peptide

  • Compare staining patterns with and without peptide blocking

  • Specific staining should be abolished or significantly reduced

• Genetic approaches:

  • Test the antibody on tissues/cells from ADCY8 knockout models

  • Use ADCY8 knockdown cells (siRNA or morpholino-mediated)

  • Compare wild-type vs. knockout/knockdown samples

• Orthogonal methods:

  • Correlate protein detection with mRNA levels (RT-PCR, RNA-seq)

  • Compare staining patterns using multiple antibodies against different epitopes of ADCY8

  • Use mass spectrometry to confirm the identity of immunoprecipitated proteins

• Expected molecular weight and localization verification:

  • Confirm that Western blots show bands at the expected molecular weight (140-150 kDa)

  • Verify that subcellular localization matches known ADCY8 distribution patterns

As demonstrated in validation studies, ADCY8 antibodies have been tested with blocking peptides to confirm specificity in both immunofluorescence and immunohistochemistry applications .

How do different antibody formats (polyclonal vs. monoclonal) affect ADCY8 detection?

The choice between polyclonal and monoclonal ADCY8 antibodies significantly impacts detection outcomes:

For optimal experimental design:

• Western blotting: Both formats work well, but polyclonals may detect multiple forms of the protein simultaneously

• IHC/IF: Monoclonals often provide cleaner background but might be more sensitive to fixation methods

• Immunoprecipitation: Monoclonals typically yield more specific pull-downs

• Critical experiments: Validate findings with both antibody types if possible

Epitope accessibility is particularly important for monoclonal antibodies, which require their specific epitope to be accessible, while polyclonals can often detect the protein even if some epitopes are masked.

How can ADCY8 antibodies be employed to study neurological functions and disorders?

ADCY8 plays critical roles in neurological processes and has been implicated in various disorders. Here are methodological approaches using ADCY8 antibodies for these studies:

• Synaptic plasticity and long-term potentiation:

  • Use dual immunofluorescence to examine ADCY8 colocalization with synaptic markers

  • Analyze presynaptic vs. postsynaptic distribution using confocal microscopy

  • The endogenous hippocampal ADCY8 is preferentially enriched in the presynaptic active zone and also detected in extrasynaptic fractions

  • Correlate ADCY8 levels with electrophysiological measures of synaptic plasticity

• Axon guidance and pathfinding:

  • ADCY8 is required for axonal pathfinding before axons reach their targets

  • Knockdown of zADCY8 induces abnormal ipsilateral retinal projections that would normally cross the ventral midline

  • ADCY8 is part of a pathway that antagonizes repellent signals expressed at the ventral midline

  • Use ADCY8 antibodies to track expression during developmental stages

• Neurodegenerative disease models:

  • Examine ADCY8 expression in Alzheimer's disease models

  • Decreased methylation of ADCY8 may contribute to upregulation of hippocampal ADCY8 in AD patients

  • Despite upregulation, CREB (which regulates genes involved in learning and memory) is impaired in AD patients

  • Investigate correlations between ADCY8 levels and cognitive performance

• Drug addiction mechanisms:

  • Administer drugs of abuse and track ADCY8 expression changes

  • ADCY8 knock-out mice exhibit abnormal anxiety-related behavioral responses

  • Correlate molecular changes with addiction-like behaviors

• Calcium signaling integration:

  • Investigate how ADCY8 couples calcium entry to cAMP production

  • Store-operated calcium entry (CCE) activates ADCY8 in neurons, likely through STIM and Orai proteins

  • Use ADCY8 antibodies alongside calcium imaging techniques

These approaches enable detailed investigation of ADCY8's roles in normal neurological function and pathological conditions, providing insights into potential therapeutic targets.

What methods can be used to study ADCY8's role in pancreatic β-cell function and insulin secretion?

ADCY8 is involved in pancreatic β-cell function and insulin secretion. The following methodological approaches can be used to investigate this role:

• Expression analysis in healthy vs. diabetic models:

  • ADCY8 expression is decreased in Goto-Kakizaki (GK) rat islets compared with healthy Wistar controls

  • Use ADCY8 antibodies for comparative expression analysis in pancreatic sections or isolated islets

  • Correlate expression levels with insulin secretion capacity

• Transcriptional regulation studies:

  • Farnesoid X Receptor (FXR) directly binds to ADCY8 promoter and recruits histone acetyltransferase SRC1

  • This results in increased acetylation of histone H3 in ADCY8 locus, promoting gene transcription

  • ADCY8 expression is suppressed by knockdown of FXR in INS-1 832/13 cells and islets from FXR knockout mice

  • Conversely, ADCY8 expression increases with FXR overexpression or activation

  • Use chromatin immunoprecipitation with ADCY8 antibodies to study regulatory mechanisms

• Signaling pathway analysis:

  • ADCY8 mediates the effects of nutrients and insulinotropic peptides like GLP-1 on insulin secretion

  • ADCY8 is activated by calcium increase induced by glucose

  • Knockdown of ADCY8 abrogates insulin secretion stimulated by glucose

  • Use ADCY8 antibodies alongside antibodies for downstream effectors (PKA, CREB)

• Co-localization with insulin secretory machinery:

  • Examine ADCY8 distribution relative to insulin granules and calcium channels

  • Investigate potential interactions with proteins involved in exocytosis

  • Track dynamics during glucose stimulation

These approaches can provide insights into how ADCY8 contributes to normal β-cell function and how its dysregulation may contribute to diabetes pathogenesis.

How can ADCY8 antibodies be used to investigate cardiac adaptations and protection mechanisms?

ADCY8 plays important roles in cardiac function and adaptation. Here are approaches to investigate these processes:

• Cardiac-specific overexpression models:

  • Cardiac-specific overexpression of ADCY8 in mice (TG AC8) shows remarkable adaptive responses

  • Despite chronically increased AC activity, hearts maintain enhanced performance (30% increases in heart rate, ejection fraction, and cardiac output) for up to a year without heart failure

  • Use ADCY8 antibodies to confirm expression levels and localization

• Protein expression and modification analysis:

  • ADCY8 overexpression triggers complex adaptive circuitry

  • Western blot analysis using ADCY8 antibodies shows that AC8 expression was markedly increased (by 8–9-fold in TG AC8 vs. WT)

  • AC activity in TG AC8 was 50% higher than in WT

  • Examine co-expression with other proteins in the cAMP pathway

• Cellular adaptation mechanisms:

  • TG AC8 hearts show:

    • Increased protein synthesis (40% higher than WT)

    • Enhanced proteasome activity and autophagy

    • Increased Nrf-2, Hsp90α, and ACC2 protein levels

    • Metabolic shift from fatty acid oxidation to aerobic glycolysis

  • Use immunofluorescence with ADCY8 antibodies to examine cellular distribution

• Cellular proliferation studies:

  • TG AC8 hearts have reduced LV cavity volume with thicker LV walls

  • They harbor increased numbers of small cardiac myocytes and a network of small interstitial proliferative non-cardiac myocytes

  • Use dual staining with ADCY8 and proliferation markers

These approaches can help elucidate how ADCY8 contributes to cardiac adaptation and protection against stress, with potential implications for treating heart disease.

How do I address weak or non-specific staining with ADCY8 antibodies?

When encountering issues with ADCY8 antibody staining, consider these troubleshooting approaches:

• For weak or no signal:

  • Optimize antigen retrieval - HIER using Tris/EDTA buffer at pH 9.0 for 45 minutes at 95°C is recommended for ADCY8

  • Increase antibody concentration - try higher concentrations within the recommended range (1:50-1:300 for IHC)

  • Extend incubation time - overnight at 4°C may improve signal compared to shorter incubations

  • Check antibody storage conditions - improper storage can lead to degradation

  • Verify sample expression - use tissues known to express ADCY8 (brain, pancreas) as positive controls

• For high background or non-specific staining:

  • Optimize blocking conditions - increase blocking time or concentration

  • Try different blocking agents (BSA, normal serum, casein)

  • Increase washing steps or duration

  • Dilute primary antibody further within recommended ranges

  • Consider pre-absorbing antibody with non-specific proteins

  • Use monoclonal antibodies for potentially higher specificity

• For unexpected staining patterns:

  • Verify antibody specificity with peptide competition assays as demonstrated in validation studies

  • Compare with literature reports of ADCY8 localization

  • Use additional antibodies recognizing different epitopes

  • Check cross-reactivity with other adenylyl cyclase isoforms

Proper storage of ADCY8 antibodies is crucial - store at -20°C for long-term storage, 4°C for up to one month for frequent use, and avoid repeated freeze-thaw cycles .

What considerations are important when using ADCY8 antibodies in knockout/knockdown studies?

When using ADCY8 antibodies in genetic manipulation studies, consider these important factors:

• Validation of knockout/knockdown efficiency:

  • Use ADCY8 antibodies to confirm the absence or reduction of protein

  • Compare with wild-type controls using multiple methods (WB, IHC)

  • Be aware that protein may persist longer than mRNA after knockdown initiation

  • Design sampling times accordingly based on protein half-life

• Knockdown design and verification:

  • Antisense morpholinos can be designed to block pre-mRNA splicing

  • Target exons within the first guanylate cyclase domain of ADCY8

  • This causes shifts in reading frame and truncation of the translated protein

  • Use antibodies that recognize regions upstream of the targeted exon for validation

• Compensatory mechanisms:

  • Check if other adenylyl cyclase isoforms are upregulated in response to ADCY8 knockout

  • Use isoform-specific antibodies to assess expression changes

  • Consider functional assays to measure total adenylyl cyclase activity

• Phenotypic analysis correlation:

  • Knockdown of ADCY8 makes retinal axons insensitive to SDF1

  • ADCY8 knockdown induces abnormal ipsilateral projections of retinal axons

  • ADCY8 knockout mice show abnormal anxiety-related behaviors

  • Correlate molecular findings with functional/behavioral outcomes

• Rescue experiments:

  • Design rescue constructs resistant to knockdown strategy

  • Use ADCY8 antibodies to verify expression of rescue construct

  • Compare phenotypic recovery with molecular restoration

These considerations help ensure robust interpretation of results from knockout/knockdown studies involving ADCY8.