ICA1 Antibody

What is ICA1 and why is it important in research?

ICA1 (Islet Cell Autoantigen 1), also known as ICA69 or ICAp69, is a cytosolic protein associated with the Golgi complex and immature secretory granules. The protein contains an N-terminal BAR domain (amino acids 1-256) with lipid-binding ability and a C-terminal ICAC domain (amino acids 257-480) with highly conserved evolutionary sequences . ICA1 was first identified as a cross-reacting protein in cloned rat β-islet tumor cell extracts using rat anti-bovine serum albumin antiserum .

ICA1 serves as an important research target for multiple reasons: it functions as an autoantigen in insulin-dependent diabetes mellitus (Type 1 diabetes), primary Sjögren's syndrome, and rheumatoid arthritis . Additionally, recent research has implicated ICA1 in neurodegenerative diseases, particularly Alzheimer's disease, where decreased ICA1 expression has been observed in patient brains and AD mouse models .

What types of ICA1 antibodies are available for research applications?

Researchers have access to various types of ICA1 antibodies optimized for different applications:

• Monoclonal antibodies:

  • ICA1 Monoclonal Antibody (C7) (MA5-41622) - validated for Western blot applications

• Polyclonal antibodies:

  • Human ICA1 Antigen Affinity-purified Polyclonal Antibody (AF4084) - validated for flow cytometry, IHC, and Western blot

  • ICA1 Polyclonal Antibody (163525) - validated for Western blot with human samples

  • ICA1 Polyclonal Antibody (PA5-79426) - validated for Western blot with human, mouse, and rat samples

  • Anti-ICA1 IgG Polyclonal Antibody - rabbit IgG for human, mouse, and rat samples

  • Rabbit Polyclonal Anti-ICA1 Antibody (HPA017646) - validated for IHC and WB with enhanced validation

  • ICA1 Polyclonal Antibody (E-AB-19237) - validated for IHC in human brain samples

These antibodies are generated using different immunogens, including E. coli-derived recombinant human ICA1 (Ser2-Ala482) and fusion proteins containing specific ICA1 sequences .

Where is ICA1 expressed and localized in cells?

ICA1 is widely expressed throughout the body, with particularly high expression in the pancreas (especially islets of Langerhans), muscles, digestive tract, and brain . At the subcellular level, immunoelectron microscopy has revealed ICA1 localization in multiple compartments:

• Cytosol (predominant form)

• Golgi apparatus membrane

• Secretory vesicle membrane

• Synaptic vesicle membrane

This subcellular distribution suggests ICA1's involvement in cellular protein transport and processing within the secretory pathway . In immunohistochemistry applications, ICA1 shows specific staining in the islets of Langerhans in pancreatic tissue, consistent with its role in diabetes pathophysiology .

What are the optimal conditions for Western blot detection of ICA1?

For optimal Western blot detection of ICA1, researchers should consider the following protocol parameters:

• Antibody dilutions:

  • Primary antibodies: 1:500 to 1:2000 range, with 1:1000 being commonly used for polyclonal antibodies

  • Secondary antibodies: 1:10000 dilution for HRP-conjugated anti-species antibodies

• Sample preparation:

  • Protein loading: 25μg per lane is recommended

  • Validated sample types include human cell lines (SH-SY5Y, RT4, MCF-7, K562), rat tissues (pancreas, brain), and mouse tissues (pancreas, brain)

• Detection parameters:

  • Blocking buffer: 3% nonfat dry milk in TBST

  • Detection system: ECL Basic Kit

  • Exposure time: Starting with approximately 1 second

The expected molecular weight of ICA1 is approximately 69 kDa, although variations may occur due to post-translational modifications or alternative splicing . For reproducible results, researchers should validate antibody specificity using positive control lysates from SH-SY5Y, RT4, MCF-7, or K562 cells .

How should ICA1 antibodies be stored and handled for maximum stability?

To maintain optimal reactivity and stability of ICA1 antibodies, follow these storage and handling recommendations:

• Long-term storage:

  • Store at -20°C to -70°C

  • Use a manual defrost freezer to avoid temperature fluctuations

  • Avoid repeated freeze-thaw cycles

  • Valid for 12 months from date of receipt under these conditions

• Working storage:

  • After reconstitution, store at 2-8°C for up to 1 month under sterile conditions

  • For longer periods (up to 6 months), store at -20°C to -70°C under sterile conditions

• Reconstitution parameters:

  • For lyophilized antibodies: Reconstitute with 0.2 mL of distilled water to yield 500 μg/mL

  • Many ICA1 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Handling precautions:

  • Aliquot antibodies into single-use volumes to minimize freeze-thaw cycles

  • When working with the antibody, keep on ice

  • Allow antibodies to equilibrate to room temperature before opening vials

Following these guidelines will help maintain antibody functionality and extend shelf life for experimental use.

What controls should be used when working with ICA1 antibodies?

Implementing appropriate controls is essential for validating experimental results with ICA1 antibodies:

• Positive controls:

  • Cell lysates: Human SH-SY5Y, RT4, MCF-7, K562 whole cell lysates

  • Tissue extracts: Rat pancreas, rat brain, mouse pancreas, mouse brain

  • Recombinant ICA1 protein when available

• Negative controls:

  • Isotype control antibody: For example, AB-108-C is mentioned as an appropriate isotype control for flow cytometry applications

  • Tissue sections known to have minimal ICA1 expression

  • Samples from ICA1 knockdown studies using siRNA

• Application-specific controls:

  • For flow cytometry: Compare staining pattern between ICA1 antibody (filled histogram) and isotype control (open histogram)

  • For IHC: Include counterstaining (like hematoxylin) to provide context for ICA1-positive structures

  • For Western blot: Include molecular weight markers to confirm the expected size of ICA1 (approximately 69 kDa)

• Experimental validation controls:

  • When studying ICA1 overexpression, compare with empty vector transfected cells

  • When using PKC inhibitors like Go 6983 or aprinocarsen sodium that affect ICA1 function, include vehicle-treated controls

These controls ensure experimental rigor and help distinguish specific ICA1 signals from background or non-specific interactions.

How can ICA1 antibodies be used to study neurodegenerative diseases?

ICA1 antibodies have emerged as valuable tools for investigating neurodegenerative conditions, particularly Alzheimer's disease, through multiple approaches:

• Expression analysis in disease models:

  • Western blot analysis has revealed decreased ICA1 expression in AD patient brains compared to normal individuals

  • ICA1 antibodies can detect reduced expression in the cortex and hippocampus of APP23/PS45 mice compared to wild-type C57 mice

  • These findings suggest ICA1 reduction may contribute to AD pathology

• Mechanistic studies of APP processing:

  • ICA1 antibodies can detect changes in APP processing when ICA1 is overexpressed or knocked down

  • Research has demonstrated that ICA1 affects APP processing through the PICK1-PKCα signaling pathway, shifting APP processing to non-amyloidogenic pathways

  • Detection of APP, APP-CTFs, ADAM10, ADAM17, BACE1, and PS1 levels in relation to ICA1 expression changes provides insights into disease mechanisms

• Signaling pathway investigation:

  • ICA1 antibodies can help study the G protein-coupled receptor signaling pathway regulated by ICA1

  • Western blotting with antibodies against ICA1, PKCα, and phosphorylated PKCα can reveal how ICA1 increases PKCα protein levels and phosphorylation

  • These studies support ICA1 as a potential therapeutic target for AD by modulating APP processing

• Therapeutic intervention assessment:

  • ICA1 antibodies can evaluate the effects of PKCα inhibitors (such as Go 6983 or aprinocarsen sodium) on ICA1-mediated APP processing

  • This application is particularly valuable for developing and validating novel therapeutic approaches targeting the ICA1-PICK1-PKCα pathway

What is the role of ICA1 antibodies in diabetes and autoimmune disease research?

ICA1 antibodies play a critical role in research on diabetes and other autoimmune conditions:

• Autoantibody detection and characterization:

  • ICA1 was originally identified as an autoantigen in type 1 diabetes

  • Antibodies against ICA1 can detect and quantify autoantibodies in patient samples

  • Research has shown ICA1 is also an autoimmune target in primary Sjögren's syndrome and rheumatoid arthritis

• Risk assessment and disease prediction:

  • Islet autoantibodies, including those against ICA1, help identify people at increased risk for developing type 1 diabetes or requiring insulin treatment

  • Studies show that combinations of multiple islet autoantibodies provide stronger predictive value than single autoantibodies

  • ICA1 antibodies contribute to these risk stratification approaches

• Standardization and methodology development:

  • Efforts to standardize ICA1 detection have been important for reliable research and clinical applications

  • Earlier claims about ICA69 (ICA1) being a target of islet cell antibodies (ICA) were not confirmed by all research groups or in standardization workshops

  • This highlights the importance of validated antibodies and methodologies

• Longitudinal studies of disease progression:

  • ICA1 antibodies help track changes in autoimmune responses over time

  • Research has shown that losing certain autoantibody reactivity can be associated with delayed progression to type 1 diabetes

  • These studies provide insights into the natural history of autoimmune diseases

• Pancreatic islet research:

  • ICA1 antibodies can localize ICA1 in pancreatic tissues, particularly in islets of Langerhans

  • This helps understand the relationship between ICA1 expression and islet function in both normal and disease states

How can ICA1 antibodies be utilized to study membrane trafficking pathways?

ICA1 antibodies provide valuable tools for investigating membrane trafficking due to ICA1's association with various secretory compartments:

• Subcellular localization studies:

  • ICA1 exists in both cytosolic and membrane-bound forms, associated with the Golgi complex, secretory granules, and synaptic vesicles

  • Immunofluorescence or IHC with ICA1 antibodies can reveal its distribution across these compartments

  • The N-terminal BAR domain of ICA1 (amino acids 1-256) has lipid-binding ability, suggesting direct interaction with membranes

• Vesicle trafficking investigation:

  • ICA1 shares similarity with arfaptins (ADP-ribosylation factor-interacting-proteins), indicating its involvement in membrane trafficking

  • Antibodies can help track ICA1's association with different vesicle populations during transport processes

  • This is particularly relevant in neurotransmitter secretion, where ICA1 may play a functional role

• Protein transport and processing studies:

  • ICA1's localization suggests its involvement in cellular protein transport and processing

  • Antibodies can help identify cargo proteins that co-traffic with ICA1

  • This application is valuable for understanding both normal secretory processes and disease-related dysfunctions

• Rab GTPase interactions:

  • Rab GTPases control membrane trafficking by recruiting effector proteins like sorting adaptors, tethering factors, and motors

  • ICA1 antibodies can help investigate potential interactions between ICA1 and Rab GTPases

  • This connection provides insights into how ICA1 might influence vesicle budding, uncoating, motility, and fusion

• Secretory pathway dynamics:

  • In specialized secretory cells like pancreatic islets or neurons, ICA1 antibodies can track secretory process regulation

  • The relationship between ICA1 expression and secretory function can be assessed through quantitative imaging approaches

  • This application bridges cellular trafficking mechanisms with tissue-specific functions

How can non-specific binding be minimized when using ICA1 antibodies?

Non-specific binding is a common challenge when working with antibodies. For ICA1 antibodies specifically, consider these optimization strategies:

• Antibody dilution optimization:

  • Start with manufacturer-recommended dilutions

  • For Western blot: 1:500-1:2000 range for primary antibodies

  • For IHC: 1:50-1:100 for certain polyclonal antibodies

  • Titrate to find the optimal balance between specific signal and background

• Blocking protocol refinement:

  • For Western blot: 3% nonfat dry milk in TBST has been validated

  • For IHC/ICC: Consider 5-10% normal serum from the same species as the secondary antibody

  • Extend blocking time if background remains high

• Washing optimization:

  • Increase number and duration of washes

  • Ensure appropriate detergent concentration in wash buffers

  • For intracellular staining, paraformaldehyde fixation followed by saponin permeabilization has been validated

• Secondary antibody selection:

  • Use highly cross-adsorbed secondary antibodies to minimize cross-reactivity

  • Validated examples include HRP Goat Anti-Rabbit IgG (H+L) at 1:10000 dilution for Western blot

  • For detection, ECL Basic Kit with short exposure times (approximately 1 second) has proven effective

• Include appropriate controls:

  • Isotype controls (such as AB-108-C for flow cytometry)

  • Negative controls omitting primary antibody

  • When possible, include ICA1 knockdown samples as biological negative controls

• Sample preparation considerations:

  • For pancreatic tissues, proper fixation is crucial to preserve islet architecture while maintaining epitope accessibility

  • For brain tissues, perfusion fixation may provide improved results over immersion fixation

  • Cell permeabilization with saponin rather than harsher detergents may better preserve membrane-associated ICA1

What are best practices for detecting ICA1 in different cell and tissue types?

Detection of ICA1 requires optimization based on the specific cell or tissue type being studied:

• Pancreatic tissue:

  • ICA1 shows specific staining in islets of Langerhans

  • For IHC: 0.5 µg/mL antibody concentration overnight at 4°C has been validated

  • Anti-Goat IgG VisUCyte™ HRP Polymer Antibody with hematoxylin counterstaining works effectively

• Brain tissue:

  • ICA1 is expressed in various brain regions

  • Detection in AD patient brains shows decreased expression compared to controls

  • For comparing cortical and hippocampal regions, matched tissue processing is essential

• Cell lines:

  • A172 human glioblastoma cell line: Validated for flow cytometry

  • Requires paraformaldehyde fixation and saponin permeabilization for intracellular staining

  • For stably transfected cell lines (2EB2, 20E2, SAS), antibody concentration may need adjustment based on expression levels

• Multiple application approaches:

  • Western blot: Effective for quantification across different samples

  • IHC/ICC: Provides spatial information about ICA1 distribution

  • Flow cytometry: Allows quantitative assessment in cell populations

• Cross-species considerations:

  • Human ICA1 shows 89% and 94% amino acid identity to mouse and canine ICA1, respectively

  • Most antibodies are validated for human, mouse, and rat samples

  • Verify species cross-reactivity before applying to comparative studies

• Special processing considerations:

  • For membrane-bound ICA1, gentle detergent extraction is recommended

  • For subcellular fractionation studies, validate fraction purity with compartment-specific markers

  • For dual-labeling studies, select compatible antibody pairs from different host species

How can ICA1 antibodies be validated for experimental applications?

Rigorous validation of ICA1 antibodies ensures reliable and reproducible research results:

• Specificity validation:

  • Western blot analysis showing a single band at the expected molecular weight (approximately 69 kDa)

  • Verification using multiple antibodies targeting different epitopes

  • Confirmation using genetic approaches (siRNA knockdown, CRISPR knockout)

• Application-specific validation:

  • For IHC: Demonstrate specific staining in known positive tissues (islets of Langerhans, brain regions)

  • For flow cytometry: Compare with isotype control antibody (such as AB-108-C)

  • For Western blot: Include positive control lysates (SH-SY5Y, RT4, MCF-7, K562 cells)

• Cross-validation with recombinant proteins:

  • Several recombinant ICA1 proteins are available for validation:

    • E. coli-derived recombinant human ICA1 (Ser2-Ala482)

    • His-tagged partial ICA1 fragments

  • These can serve as positive controls and for antibody characterization

• Functional validation:

  • Overexpression studies: Verify that antibody signal increases with ICA1 overexpression

  • Inhibitor studies: Confirm antibody can detect changes induced by PKCα inhibitors like Go 6983

  • Co-localization studies: Validate antibody detects ICA1 in expected subcellular compartments

• Enhanced validation approaches:

  • Some commercial antibodies undergo "Enhanced Validation"

  • This typically includes orthogonal validation (verification by an independent method)

  • Genetic validation (using knockout/knockdown approaches)

  • Independent antibody validation (using antibodies recognizing different epitopes)

What are promising new research areas involving ICA1 antibodies?

Emerging research areas utilizing ICA1 antibodies include:

• Alzheimer's disease therapeutic development:

  • Recent findings suggest ICA1 as "a novel target for the treatment of AD"

  • ICA1 antibodies can help assess experimental therapeutics targeting the PICK1-PKCα pathway

  • Studies show ICA1 shifts APP processing to non-amyloidogenic pathways, making it a promising intervention point

• Diabetes-neurodegeneration connection:

  • ICA1 provides a molecular link between autoimmune diseases and neurodegenerative conditions

  • Antibodies can help investigate common pathways across these historically separate disease areas

  • The decreased expression of ICA1 in AD brains suggests potential shared mechanisms with autoimmune conditions

• G protein-coupled receptor signaling pathways:

  • Transcriptome sequencing shows ICA1 regulates G protein-coupled receptor signaling

  • This pathway has broad implications across multiple physiological systems

  • ICA1 antibodies can help map this regulatory network in different tissues and disease states

• Secretory pathway dynamics:

  • ICA1's localization across secretory compartments suggests roles in vesicle biogenesis and transport

  • Antibodies can help track temporal changes in ICA1 distribution during secretory processes

  • This research area has implications for understanding both normal physiology and disease states

• Multiplex biomarker development:

  • Combined analysis of multiple islet autoantibodies provides stronger predictive value than single antibodies

  • ICA1 antibodies can contribute to multiplex panels for early disease detection

  • This approach has particular relevance for diabetes risk stratification and personalized medicine

How might technical advances enhance ICA1 antibody applications?

Technical innovations are expanding the utility of ICA1 antibodies in research:

• High-resolution imaging techniques:

  • Super-resolution microscopy can provide nanoscale localization of ICA1 in cellular compartments

  • This is particularly valuable for studying ICA1's association with vesicular structures

  • These approaches can reveal previously undetectable details of ICA1 distribution and trafficking

• Multiparameter analysis:

  • Multiplexed antibody panels can situate ICA1 within broader regulatory networks

  • Mass cytometry or multiplexed immunofluorescence allows simultaneous detection of ICA1 with multiple markers

  • This provides contextual information about ICA1's function in different cellular states

• Live-cell imaging approaches:

  • Combining antibody-based detection with live-cell compatible systems

  • Can provide dynamic information about ICA1 trafficking in real-time

  • Particularly valuable for studying neurotransmitter secretion and vesicle dynamics

• Enhanced antibody engineering:

  • Development of recombinant antibodies with improved specificity

  • Creation of domain-specific antibodies targeting BAR versus ICAC domains

  • Generation of antibodies that distinguish between cytosolic and membrane-bound ICA1 forms

• Integration with multi-omics approaches:

  • Combining antibody-based detection with proteomic or transcriptomic analyses

  • This can connect ICA1 protein levels with broader molecular signatures

  • Particularly valuable for understanding ICA1's role in complex disease processes