AP180 Antibody

What is AP180 and why is it important in cellular research?

AP180, also known as SNAP91 or CALM, is a 907 amino acid cell membrane protein that plays a crucial role in receptor-mediated endocytosis. It facilitates the assembly of clathrin-coated pits and vesicles by binding to clathrin triskelia through its N-terminal clathrin binding site, promoting the formation of 60-70 nm coats necessary for vesicle transport. This function is essential for maintaining cellular homeostasis and facilitating neurotransmitter release in neurons, making AP180 a significant target for research in cellular biology and neuroscience. The protein exists in three alternatively spliced isoforms, highlighting its functional diversity across different cellular contexts and developmental stages .

What species reactivity do AP180 antibodies typically demonstrate?

AP180 antibodies like the LP2D11 and AP180-I clones demonstrate consistent reactivity across mouse, rat, and human samples. This cross-species reactivity reflects the evolutionary conservation of AP180 protein structure and function, with the gene located on human chromosome 6q14.2 and mouse chromosome 9 E3.1. Some AP180 antibodies, such as the AP180-I clone, are also recommended for detection in additional species like bovine samples, although this may require further validation. The conservation across mammalian species makes these antibodies versatile tools for comparative studies in different model organisms .

What are the primary applications for AP180 antibodies in research?

AP180 antibodies are validated for multiple research applications including:

• Western blotting (WB) - typically used at starting dilutions of 1:200 (range 1:100-1:1000)

• Immunoprecipitation (IP) - using 1-2 μg per 100-500 μg of total protein

• Immunofluorescence (IF) - starting at 1:50 dilution (range 1:50-1:500)

• Immunohistochemistry (IHC) - including paraffin-embedded sections (same dilution range as IF)

• Electron microscopy - used at dilutions of 1:25 to 1:50

These applications enable researchers to study the expression, localization, and interactions of AP180 in various cellular contexts and experimental systems .

What is the molecular weight of AP180 and how does this impact detection methods?

The molecular weight of AP180 is approximately 180 kDa, which is important to consider when designing detection protocols. When performing Western blotting, researchers should use appropriate molecular weight markers to properly identify AP180 bands (typically between 170-190 kDa). The high molecular weight of this protein may require special considerations for gel separation, such as using lower percentage polyacrylamide gels (6-8%) to achieve adequate resolution. Additionally, longer transfer times may be necessary when moving these large proteins from gel to membrane during Western blotting procedures .

How can researchers effectively differentiate between AP180 and its homolog CALM in experimental systems?

Differentiating between AP180 and CALM (Clathrin Assembly Lymphoid Myeloid leukemia protein) requires careful antibody selection and experimental design. While both proteins contain ENTH domains and function in clathrin-mediated endocytosis, they have distinct subcellular distributions and functions. Researchers should use antibodies specifically validated against each target, such as clone LP2D11 or AP180-I for AP180 and specific CALM antibodies (e.g., #sc5395 or #sc6433). Co-immunolabeling experiments can reveal differential localization patterns, with AP180 more predominantly expressed in neuronal tissues. For definitive differentiation, researchers can employ siRNA knockdown approaches targeting unique nucleotide sequences, such as nucleotides 2157-2175 of rat AP180, followed by antibody detection to confirm specificity of signals. Western blotting with careful attention to molecular weight differences and distinct band patterns can also help distinguish between these structurally related proteins .

What strategies can be employed for quantitative analysis of AP180 subcellular distribution in electron microscopy?

For quantitative analysis of AP180 subcellular distribution using electron microscopy, researchers should implement a systematic approach as follows:

• Sample Preparation: Process hippocampal sections using postembedding techniques following established protocols as described in previous studies.

• Immunogold Labeling: Incubate sections with AP180 antibody at 1:25 dilution at 4°C overnight, followed by thorough washing and incubation with gold (10 nm)-conjugated secondary antibodies.

• Visualization: Contrast sections with uranyl acetate and lead citrate and examine on the electron microscope at magnifications of 25,000× or 50,000×.

• Quantification: Systematically count gold particles in defined subcellular compartments, such as presynaptic terminals, postsynaptic densities, and endocytic zones.

• Statistical Analysis: Compare the distribution patterns across different developmental stages (e.g., P10 vs. P37 rat samples) or experimental conditions.

This approach allows researchers to precisely quantify the spatial distribution of AP180 protein at the ultrastructural level, providing insights into its functional role in clathrin-mediated endocytosis at synapses and other cellular compartments .

How can researchers design experiments to investigate the differential roles of AP180 ENTH domain in clathrin assembly?

To investigate the differential roles of AP180's ENTH (epsin N-terminal homology) domain in clathrin assembly, researchers can employ a multi-faceted experimental approach:

• Domain Deletion Constructs: Generate AP180-ANTHΔ-HA constructs by PCR amplification and ligation into expression vectors (e.g., pcDNA3.1 using appropriate restriction sites like EcoI and NotI).

• Domain Swapping: Create chimeric proteins by replacing the ENTH domain with homologous domains from related proteins to assess functional specificity.

• Site-Directed Mutagenesis: Introduce specific mutations in conserved residues within the ENTH domain to identify critical amino acids for clathrin binding.

• Transfection Studies: Express these constructs in neuronal cultures or cell lines to assess effects on clathrin coat formation using immunofluorescence and electron microscopy.

• Vesicle Formation Assays: Quantify the size and number of clathrin-coated vesicles formed in the presence of wild-type versus mutant AP180.

• Co-immunoprecipitation: Determine how ENTH domain modifications affect AP180's interaction with clathrin and other endocytic proteins.

This comprehensive approach enables researchers to dissect the specific contribution of the ENTH domain to AP180's function in organizing clathrin assembly and vesicle formation, distinguishing it from other functional domains of the protein .

What are the optimal conditions for Western blotting using AP180 antibodies?

For optimal Western blotting results with AP180 antibodies, researchers should follow these methodological guidelines:

• Sample Preparation:

  • Use fresh tissue lysates from brain samples (human brain, rat cerebellum, or mouse brain extracts serve as positive controls)

  • Include protease inhibitors in lysis buffers to prevent degradation of this large protein

• Gel Electrophoresis:

  • Use 6-8% polyacrylamide gels to properly resolve the 180 kDa protein

  • Load 20-50 μg of total protein per lane for adequate detection

• Transfer and Blocking:

  • Perform wet transfer at lower voltage (30V) for extended periods (overnight) to ensure complete transfer of large proteins

  • Use UltraCruz® Blocking Reagent (sc-516214) for optimal blocking

• Antibody Incubation:

  • Use AP180 antibody at a starting dilution of 1:200 (range 1:100-1:1000)

  • For detection, use appropriate secondary antibodies such as m-IgGκ BP-HRP (sc-516102) at dilutions of 1:1000-1:10000

• Visualization:

  • Develop with Western Blotting Luminol Reagent (sc-2048)

  • For near-infrared detection, use appropriate BP-CFL 680 secondary antibodies

Following these guidelines will help ensure specific detection of AP180 protein with minimal background interference .

What controls and validation approaches should be included when using AP180 antibodies in immunofluorescence studies?

When performing immunofluorescence studies with AP180 antibodies, researchers should implement the following controls and validation approaches:

• Positive Controls:

  • Include known AP180-expressing tissues such as brain sections (cerebellum is particularly recommended)

  • Use cell lines with confirmed AP180 expression

• Negative Controls:

  • Omit primary antibody incubation to assess non-specific binding of secondary antibodies

  • Use tissue samples from AP180 knockout models if available

  • Include AP180 siRNA-treated samples as specificity controls

• Validation Through siRNA Knockdown:

  • Transfect cells with AP180-specific siRNA (targeting nucleotides 2157-2175 of rat AP180)

  • Compare immunofluorescence signals between knockdown and control cells

• Co-localization Studies:

  • Perform dual labeling with markers of clathrin-coated vesicles or endocytic compartments

  • Include markers like clathrin (clone TD.1), EEA1, or TGN38

• Signal Specificity Verification:

  • Use competing peptides to confirm antibody specificity

  • Compare staining patterns with multiple AP180 antibodies targeting different epitopes

These validation approaches ensure that the observed immunofluorescence signals accurately represent AP180 localization and are not artifacts of non-specific binding or cross-reactivity .

How should researchers optimize immunoprecipitation protocols for AP180 protein interactions?

For effective immunoprecipitation of AP180 and its interaction partners, researchers should optimize their protocols as follows:

• Lysis Conditions:

  • Use mild lysis buffers containing 1% NP-40 or 0.5% Triton X-100 to preserve protein-protein interactions

  • Include phosphatase inhibitors if studying phosphorylation-dependent interactions

• Antibody Selection and Binding:

  • Use 1-2 μg of AP180 antibody per 100-500 μg of total protein (1 ml of cell lysate)

  • Pre-clear lysates with Protein A/G PLUS-Agarose (sc-2003) to reduce non-specific binding

  • Allow antibody-antigen binding to occur overnight at 4°C for maximum capture efficiency

• Washing Stringency:

  • Optimize wash buffer composition based on interaction strength:

    • For strong interactions: higher stringency with 300-500 mM NaCl

    • For weak interactions: lower stringency with 150 mM NaCl

• Interaction Verification:

  • Confirm precipitated proteins by Western blotting with antibodies against:

    • AP180 itself (to confirm successful IP)

    • Clathrin (clone TD.1) to verify functional interactions

    • Other suspected interaction partners (e.g., adaptor proteins)

• Negative Controls:

  • Perform parallel IPs with isotype-matched irrelevant antibodies

  • Use lysates from cells expressing AP180 mutants lacking specific interaction domains

This optimized approach allows researchers to reliably isolate AP180 protein complexes and identify physiologically relevant interaction partners in different experimental contexts .

What are common challenges in detecting AP180 in Western blotting and how can they be overcome?

Researchers often encounter specific challenges when detecting AP180 in Western blotting due to its large molecular weight and expression characteristics:

Additionally, researchers should consider that the three alternatively spliced isoforms of AP180 may appear as closely spaced bands. When optimizing protocols, start with known positive control tissues such as brain extracts where AP180 is highly expressed before moving to samples with potentially lower expression levels .

How can researchers address non-specific binding in immunohistochemistry applications with AP180 antibodies?

Non-specific binding in immunohistochemistry with AP180 antibodies can be addressed through the following systematic approach:

• Antigen Retrieval Optimization:

  • Compare different antigen retrieval methods (heat-induced with citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

  • Adjust retrieval time and temperature based on tissue fixation conditions

• Blocking Enhancements:

  • Extend blocking time to 1-2 hours at room temperature

  • Add 0.1-0.3% Triton X-100 to blocking buffer to reduce hydrophobic interactions

  • Include 5% serum from the same species as the secondary antibody

  • Consider adding 0.1% BSA to reduce non-specific protein interactions

• Antibody Dilution and Incubation:

  • Test serial dilutions (starting at 1:50, ranging to 1:500)

  • Extend primary antibody incubation to overnight at 4°C

  • Perform incubations in humid chambers to prevent section drying

• Washing Modifications:

  • Increase number of washes (5-6 times, 5 minutes each)

  • Add 0.05% Tween-20 to wash buffers to reduce non-specific binding

• Detection System Adjustments:

  • Use m-IgGκ BP-HRP (sc-516102) with DAB, 50X (sc-24982)

  • Consider Immunohistomount (sc-45086) or Organo/Limonene Mount (sc-45087) for optimal results

  • Reduce substrate development time if background is high

These systematic modifications can significantly improve signal-to-noise ratio in immunohistochemical detection of AP180, particularly in tissues with complex architecture like neuronal tissues .

What strategies can address inconsistent results in AP180 subcellular localization studies?

Inconsistent results in AP180 subcellular localization studies can arise from various methodological factors. To address these challenges, researchers should implement the following strategies:

• Fixation Protocol Standardization:

  • Compare paraformaldehyde (4%) alone versus combined glutaraldehyde/paraformaldehyde fixation

  • Optimize fixation time based on tissue type and thickness (typically 10-30 minutes)

  • Test permeabilization conditions (0.1-0.3% Triton X-100 for 5-15 minutes)

• Antibody Validation Using Multiple Approaches:

  • Confirm specificity using siRNA knockdown (targeting nucleotides 2157-2175 of rat AP180)

  • Compare localization patterns using multiple antibodies against different AP180 epitopes

  • Validate with tagged AP180 constructs (HA-tagged or VSVG-tagged) to confirm antibody staining patterns

• Co-localization Controls:

  • Include established markers for relevant subcellular compartments:

    • Clathrin (clone TD.1) for coated vesicles

    • EEA1 for early endosomes

    • GM130 for Golgi apparatus

    • TGN38 for trans-Golgi network

    • AP1γ for clathrin-associated adaptor complexes

• Technical Optimization:

  • Use confocal microscopy with appropriate z-stack sampling to avoid misinterpretation of overlapping signals

  • Implement quantitative co-localization analysis with appropriate statistical tests

  • Consider super-resolution microscopy techniques for precise localization

• Biological Variables Control:

  • Account for cell type-specific expression patterns

  • Consider developmental stage differences (compare P10 versus P37 patterns)

  • Control for cell cycle stage in dividing cells

By implementing these strategies, researchers can establish reliable and reproducible protocols for AP180 subcellular localization studies, enabling accurate interpretation of its distribution and trafficking in different cellular contexts .

How can researchers distinguish between the roles of AP180 and other clathrin assembly proteins in endocytosis?

To differentiate between the specific functions of AP180 and other clathrin assembly proteins in endocytosis, researchers can employ these sophisticated approaches:

• Selective Gene Silencing:

  • Design highly specific siRNAs targeting AP180 (e.g., targeting nucleotides 2157-2175 of rat AP180)

  • Implement sequential knockdown of AP180 and other clathrin assembly proteins

  • Create rescue experiments with RNAi-resistant AP180 constructs to confirm specificity

• Domain-Specific Functional Analysis:

  • Express AP180-ANTHΔ-HA constructs that lack specific functional domains

  • Compare these effects with similar manipulations of other clathrin assembly proteins

  • Create chimeric proteins with domain swaps between AP180 and related proteins

• Quantitative Endocytosis Assays:

  • Measure transferrin uptake rates in cells with modified AP180 expression

  • Analyze receptor internalization kinetics with fluorescently-labeled ligands

  • Employ live-cell imaging to track vesicle formation in real-time

• Ultrastructural Analysis:

  • Perform electron microscopy to compare clathrin-coated pit morphology

  • Measure pit size and distribution using immunogold labeling with AP180 antibody (1:25 dilution)

  • Compare these parameters with those affected by other assembly proteins

• Interaction Network Mapping:

  • Conduct comparative immunoprecipitation followed by mass spectrometry

  • Identify unique versus shared interaction partners between AP180 and related proteins

  • Use proximity ligation assays to visualize protein interactions in situ

These approaches allow researchers to delineate the unique contributions of AP180 to clathrin-mediated endocytosis distinct from those of other assembly proteins, providing a comprehensive understanding of the specific mechanisms regulated by AP180 .

What approaches can researchers use to investigate AP180's role in neuronal function and synaptic vesicle recycling?

To investigate AP180's specific role in neuronal function and synaptic vesicle recycling, researchers can implement these specialized approaches:

• Neuron-Specific Manipulation:

  • Develop conditional knockout models with neuron-specific Cre drivers

  • Use AP180 siRNAs delivered via lentiviral vectors (sc-29698-V for human, sc-29699-V for mouse)

  • Create transgenic models expressing dominant-negative AP180 constructs

• Electrophysiological Analysis:

  • Perform patch-clamp recordings to assess synaptic transmission

  • Measure miniature excitatory postsynaptic currents (mEPSCs) frequency and amplitude

  • Analyze paired-pulse facilitation to assess presynaptic function

• Synaptic Vesicle Tracking:

  • Implement pH-sensitive fluorescent probes (pHluorins) to monitor vesicle exo-endocytosis

  • Use FM dyes to quantify vesicle recycling kinetics

  • Apply dual-color total internal reflection fluorescence (TIRF) microscopy to track AP180 and synaptic vesicle proteins simultaneously

• Ultrastructural Characterization:

  • Perform electron microscopy with immunogold labeling at 1:25 dilution

  • Quantify synaptic vesicle number, size, and distribution

  • Analyze clathrin-coated structures at presynaptic terminals

• Protein Interaction Analysis in Neuronal Context:

  • Conduct co-immunoprecipitation with brain lysates using 1-2 μg of antibody per 500 μg protein

  • Identify neuronal-specific binding partners

  • Compare AP180 interactions in different neuronal subtypes and developmental stages

These methods provide a comprehensive assessment of AP180's specific functions in neuronal systems, particularly its contributions to the specialized process of synaptic vesicle recycling, which is crucial for sustained neurotransmission .

What are emerging techniques that may enhance AP180 antibody applications in research?

Emerging techniques that promise to enhance AP180 antibody applications include advanced imaging modalities, innovative protein interaction analyses, and next-generation antibody engineering approaches. Super-resolution microscopy techniques such as Stimulated Emission Depletion (STED) microscopy and Stochastic Optical Reconstruction Microscopy (STORM) can now resolve AP180 localization with nanometer precision, enabling visualization of its distribution within clathrin-coated structures. Proximity-dependent biotinylation methods like BioID and TurboID allow researchers to identify transient or weak AP180 protein interactions in living cells, expanding our understanding of its protein interaction network. Additionally, the development of recombinant single-domain antibodies and nanobodies against AP180 may offer superior accessibility to epitopes within crowded molecular environments like clathrin-coated pits. These technological advances, combined with CRISPR-Cas9 genome editing for endogenous tagging of AP180, will provide unprecedented insights into its dynamic behavior and functional roles in various cellular processes .

How can researchers integrate AP180 antibody data with other experimental approaches to build comprehensive models of endocytic function?

Researchers can build comprehensive models of endocytic function by integrating AP180 antibody data with complementary experimental approaches in a multi-scale framework. This integration should span from molecular interactions to physiological consequences by combining:

• Structural Biology Data:

  • Correlate AP180 antibody epitope accessibility with protein conformation states

  • Integrate cryo-electron microscopy structures of clathrin assemblies with immunolocalization data

• Proteomics and Interactomics:

  • Compare immunoprecipitation results with proximity labeling datasets

  • Build protein interaction networks centered on AP180 under various cellular conditions

• Live Cell Dynamics:

  • Correlate fixed-cell antibody staining with live imaging of fluorescently-tagged AP180

  • Integrate data on protein turnover rates and post-translational modifications

• Systems Biology Approaches:

  • Develop mathematical models of endocytosis incorporating AP180 concentrations and stoichiometry

  • Validate model predictions using quantitative antibody-based measurements

• Physiological Relevance:

  • Connect molecular findings to cellular phenotypes and organism-level consequences

  • Integrate data across different model systems and cell types to identify conserved mechanisms

This integrative approach allows researchers to position AP180 antibody-derived data within a broader context, creating comprehensive models that span from molecular mechanisms to functional consequences in various physiological and pathological contexts .