HOMER1 Antibody

What is HOMER1 and why are antibodies against it important in neuroscience research?

HOMER1 is a postsynaptic density scaffolding protein enriched at excitatory synapses that binds to metabotropic glutamate receptors (mGluRs) . It plays crucial roles in synaptic plasticity, neuronal development, and cognitive functions including attention. HOMER1 has several isoforms (HOMER1a, HOMER1b, HOMER1c, HOMER1e, HOMER1h) with distinct functions .

Antibodies against HOMER1 allow researchers to visualize its subcellular distribution, quantify expression levels, investigate protein-protein interactions, and examine its role in neurological disorders. The short, activity-dependent isoforms (HOMER1a and Ania3) and the long, constitutively expressed isoforms (HOMER1b/c) have different functional implications, making isoform-specific detection particularly valuable .

What methodological approaches are optimal for Western blot applications using HOMER1 antibodies?

When performing Western blot with HOMER1 antibodies, researchers should consider the following methodological guidelines:

• Sample preparation:

  • Use appropriate tissue; cerebellum can serve as a negative control since it shows minimal HOMER1 expression

  • Extract proteins using buffers compatible with membrane proteins

  • Include protease inhibitors to prevent degradation

• Protein separation and transfer:

  • Use reducing conditions as specified in product documentation

  • Expect HOMER1 to appear at different molecular weights based on isoform:

    • HOMER1b/c: 46-52 kDa

    • HOMER1a: 40-43 kDa

    • Additional bands may appear at 20-26 kDa for other variants

• Antibody incubation:

  • Typical dilutions range from 1:1000 (common for many HOMER1 antibodies) to 1:4000

  • Primary antibody incubation is often performed overnight at 4°C for optimal results

• Detection controls:

  • Use positive controls such as brain lysates (except cerebellum)

  • PVDF membranes are commonly used with HOMER1 antibodies

  • For human samples, cerebellum tissue and IMR-32 neuroblastoma cells have been validated as showing specific bands

What are the optimal conditions for immunofluorescence using HOMER1 antibodies?

For immunofluorescence applications with HOMER1 antibodies:

• Sample preparation:

  • Paraformaldehyde fixation (4%) is commonly used

  • Both cultured neurons and brain slices can be used depending on research questions

• Antibody dilutions and conditions:

  • Dilutions typically range from 1:100 for immunocytochemistry to 1:2000 for tissue sections

  • Extended incubation periods (up to 3 days at 4°C) have been reported for certain applications

  • Secondary antibody selection should match the host species of the primary antibody (commonly mouse or rabbit)

• Co-staining markers for subcellular localization:

  • MAP-2 for dendrite identification

  • Tau-1 for axon identification

  • Synaptophysin for presynaptic terminals

  • GluR2 for postsynaptic sites

• Controls:

  • Cerebellar granule cells can serve as negative controls as they typically do not express Homer1

  • Pre-adsorption with immunizing peptide can verify specificity

How do I select the appropriate HOMER1 antibody for my specific research?

When selecting a HOMER1 antibody, consider:

• Isoform specificity:

  • Determine whether you need an antibody recognizing all HOMER1 isoforms or one specific to certain variants

  • Some antibodies target regions common to all Homer proteins (Homer 1, 2, and 3)

  • Others specifically recognize unique sequences in individual isoforms

• Species reactivity:

  • Verify compatibility with your model organism (human, mouse, rat)

  • Cross-reactivity information is typically provided in product documentation

• Application compatibility:

  • Confirm validation for your intended application (WB, ICC/IF, etc.)

  • Some antibodies may work well in multiple applications while others are application-specific

• Clonality considerations:

  • Monoclonal antibodies (e.g., clone 666241, clone 2G8) offer high specificity

  • Polyclonal antibodies might provide stronger signals but potentially higher background

• Immunogen information:

  • Antibodies raised against different epitopes may yield different results

  • Common immunogens include N-terminal regions or specific peptide sequences

What are the storage and handling recommendations for HOMER1 antibodies?

Proper storage and handling are crucial for maintaining antibody performance:

• Storage conditions:

  • Lyophilized antibodies should be stored at +4°C until reconstitution

  • Reconstituted antibodies typically require storage at -20°C to -80°C

  • Avoid repeated freeze-thaw cycles

• Reconstitution guidelines:

  • Follow manufacturer-specific protocols for reconstitution

  • Typical reconstitution involves adding specified volume of H₂O or buffer to reach 1mg/ml concentration

  • After reconstitution, aliquot before freezing to minimize freeze-thaw cycles

• Shelf-life considerations:

  • Most antibodies are stable for 12 months from date of receipt when stored properly

  • Once reconstituted, stability ranges from 1 month (at 2-8°C) to 6 months (at -20 to -70°C)

  • Working dilutions should be prepared fresh for each experiment

• Additional recommendations:

  • Store under sterile conditions after reconstitution

  • Do not freeze lyophilized antibodies before reconstitution

  • Some formulations contain stabilizers like albumin and azide

How can I distinguish between different HOMER1 isoforms using antibodies?

Distinguishing between HOMER1 isoforms is crucial as they serve distinct functional roles:

• Molecular characteristics for differentiation:

  • Short isoforms (HOMER1a, Ania-3): Activity-induced, lack coiled-coil domain, act as dominant negatives

  • Long isoforms (HOMER1b/c): Constitutively expressed, contain coiled-coil domains, form multimeric complexes

• Antibody-based approaches:

  • Western blot can distinguish isoforms by molecular weight:

    • HOMER1b/c: 46-52 kDa

    • HOMER1a: 40-43 kDa

  • Isoform-specific antibodies targeting unique C-terminal sequences can provide direct identification

• Complementary techniques:

  • RT-PCR with isoform-specific primers can verify isoform expression at mRNA level

  • Activity-induction protocols can help identify HOMER1a (increases after stimulation)

• Validation strategies:

  • Overexpression of tagged isoforms as positive controls

  • Knockdown of specific isoforms using targeted shRNAs

  • Comparison of expression patterns across brain regions with known isoform distributions

Research indicates that only the short, activity-dependent isoforms (HOMER1a and Ania3) showed differential expression between high and low attentional performers, highlighting the importance of isoform-specific detection .

What approaches can be used to study HOMER1 protein-protein interactions using antibodies?

Studying HOMER1 protein-protein interactions is essential for understanding synaptic organization:

• Co-immunoprecipitation (Co-IP):

  • Can be used to pull down HOMER1 and associated proteins

  • Effective for detecting interactions with mGluRs, as demonstrated in studies examining HOMER1-mGluR5 binding

  • Western blot analysis of precipitated complexes can reveal binding partners

• Immunocytochemistry co-localization:

  • Double-labeling with HOMER1 antibodies and antibodies against potential partners

  • Has been successfully used to show co-localization between HOMER1 and synaptophysin

  • Can reveal subcellular compartmentalization of interactions

• Proximity Ligation Assay (PLA):

  • Utilizes pairs of antibodies (HOMER1 and potential interacting partner)

  • Offers higher specificity than conventional co-localization

  • Useful for detecting endogenous interactions in intact tissue

• Experimental considerations:

  • The EVH1 domain of HOMER1 binds to proline-rich motifs (PPxxF) in partners

  • Long isoforms can multimerize through coiled-coil domains

  • HOMER1a disrupts interactions mediated by long isoforms

• Specific interactions to investigate:

  • mGluR5 targeting to dendrites or axons depends on HOMER1 interaction

  • HOMER1 clustering with postsynaptic density proteins

  • Differential interactions in excitatory versus inhibitory synapses

How can HOMER1 antibodies be used to study its role in neurological and psychiatric disorders?

HOMER1 has been implicated in various neurological and psychiatric conditions:

• Attention-related disorders:

  • Genetic mapping has identified HOMER1 as a developmental modifier of attention

  • Down-regulation of short isoforms (HOMER1a) in prefrontal cortex improved attention performance

  • Antibodies can track expression changes in animal models and potentially human samples

• Methodological approaches:

  • Western blot to quantify HOMER1 isoform expression changes

  • Immunohistochemistry to examine alterations in subcellular distribution

  • Co-labeling with markers for inhibitory receptors, as HOMER1 down-regulation is associated with GABAergic receptor up-regulation

• Circuit-level analysis:

  • Examine region-specific changes (PFC has been identified as particularly relevant)

  • Compare excitatory/inhibitory synapse ratios using HOMER1 and inhibitory synapse markers

  • Correlate molecular changes with electrophysiological and behavioral measurements

• Experimental considerations:

  • Developmental timing is critical—effects may differ between early postnatal and adult stages

  • Isoform-specific analysis is essential as short and long forms have opposing functions

  • Control for activity-dependent fluctuations in HOMER1a expression

Research shows that prefrontal HOMER1 down-regulation enhances inhibitory tone and signal-to-noise ratio, potentially offering therapeutic strategies for attention disorders beyond traditional stimulants .

What is the relationship between HOMER1 expression and excitatory synapse formation?

HOMER1 serves as a marker for excitatory synapses and plays a functional role in their formation:

• Marker utility:

  • HOMER1 immunostaining reveals the majority of excitatory synapses

  • Can be used to quantify excitatory synapse density in neuronal cultures and tissue sections

  • Particularly valuable when co-labeled with other synaptic markers

• Methodological approaches:

  • Quantitative immunocytochemistry to measure synapse number and size

  • Time-course analysis to track synaptogenesis during development

  • Co-labeling with pre- and postsynaptic markers to confirm functional synapses

• Technical considerations:

  • Dilution optimization is critical—recommendations range from 1:2000 to 1:20,000 depending on application

  • Different antibodies may preferentially label synapses at different developmental stages

  • Distinction between punctate synaptic HOMER1 and diffuse extrasynaptic HOMER1 requires high-resolution imaging

• Functional manipulations:

  • Knockdown/overexpression paired with antibody labeling to assess effects on synapse number

  • Activity-dependent changes in HOMER1 distribution following synaptic plasticity protocols

  • Correlation between HOMER1 levels and electrophysiological measures of synaptic strength

The relationship between HOMER1 and metabotropic glutamate receptors (mGluRs) plays a crucial role in excitatory synapse organization and function, with HOMER1 directing mGluR5 targeting to specific neuronal compartments .

What technical challenges exist in using HOMER1 antibodies for electron microscopy studies?

Electron microscopy (EM) with HOMER1 antibodies presents specific challenges:

• Sample preparation considerations:

  • Balance between ultrastructure preservation and epitope accessibility

  • Pre-embedding immunogold labeling may require specialized fixation protocols

  • Documented protocols have used extended antibody incubation (3 days at 4°C)

• Antibody dilution optimization:

  • EM applications may require different dilutions than light microscopy

  • Published protocols have used dilutions as high as 1:20,000 specifically for EM applications

  • Titration experiments are essential to minimize background while maintaining specific labeling

• HOMER1-specific challenges:

  • Postsynaptic density (PSD) is densely packed, potentially limiting antibody penetration

  • Distinguishing between different HOMER isoforms may require specialized probes

  • Quantification of immunogold particles requires standardized approaches

• Controls and validation:

  • Negative controls should include primary antibody omission

  • Cerebellar tissue can serve as a biological negative control

  • Pre-absorption with immunizing peptide can confirm specificity

• Data interpretation:

  • Requires correlation with functional or light microscopy data

  • Consider three-dimensional distribution within the PSD

  • Compare with other postsynaptic markers (e.g., GluR2, gephyrin)

How can HOMER1 antibodies help distinguish between excitatory and inhibitory synapses?

HOMER1 serves as a reliable marker for excitatory synapses, enabling differentiation from inhibitory contacts:

• Marker profile for synapse typing:

  • HOMER1: Localizes to excitatory postsynaptic sites

  • Can be paired with inhibitory synapse markers:

    • Gephyrin: Inhibitory postsynaptic marker

    • VGAT: Inhibitory presynaptic marker

  • Excitatory synapse verification with:

    • VGLUT1/VGLUT2: Excitatory presynaptic markers

    • GluR2: AMPA receptor subunit at excitatory synapses

• Methodological approach:

  • Double or triple immunofluorescence labeling

  • Quantification of puncta density, size, and colocalization

  • Comparison across brain regions or experimental conditions

• Technical considerations:

  • Dilution optimization for multiplexed staining

  • Selection of compatible secondary antibodies

  • Resolution requirements for distinguishing closely spaced synapses

The table below summarizes relevant antibody combinations for synapse typing based on search result :

What is the significance of HOMER1a induction in neuronal plasticity?

HOMER1a induction represents a crucial mechanism in activity-dependent synaptic plasticity:

• Functional significance:

  • HOMER1a acts as a dominant-negative regulator of long HOMER1 isoforms

  • Disrupts scaffolding complexes maintained by long isoforms

  • Uncouples mGluRs from intracellular signaling pathways

  • Short isoforms (HOMER1a, Ania3) are specifically implicated in attention processes

• Methodological detection approaches:

  • Western blot: Isoform-specific bands at different molecular weights

  • RT-PCR: Primers targeting unique 3' sequences of HOMER1a

  • Immunocytochemistry: Temporal analysis following stimulation

• Research applications:

  • Tracking neuronal activation patterns following behavioral tasks

  • Measuring homeostatic responses to prolonged activity changes

  • Assessing circuit-specific plasticity in learning paradigms

• Experimental design considerations:

  • Temporal dynamics: HOMER1a typically peaks 2-4 hours after stimulation

  • Regional specificity: Expression patterns differ across brain areas

  • Activity manipulations: Pharmacological, electrical, or behavioral

Research demonstrates that downregulation of short HOMER1 isoforms in prefrontal cortex during early postnatal development led to improvements in multiple measures of attention in adult mice, highlighting the critical role of these isoforms in cognitive function .

How can HOMER1 antibodies be used to investigate regional and developmental expression patterns?

HOMER1 expression varies across brain regions and developmental stages:

• Regional expression profile:

  • Strong expression in striatum and cortical regions

  • Notably absent in cerebellar granule cell layer

  • Present in excitatory synapses throughout various brain regions

• Developmental regulation:

  • Expression changes during postnatal development

  • Isoform-specific regulation at different developmental stages

  • Down-regulation of short isoforms during early postnatal development affects adult cognitive function

• Methodological approaches:

  • Western blot analysis of microdissected brain regions at different ages

  • Immunohistochemistry on developmental tissue series

  • Cell culture models comparing different neuronal populations:

    • Cultured striatal neurons express HOMER1b/c

    • Cerebellar granule cells lack HOMER1 expression

• Experimental designs:

  • Cross-sectional analysis across brain regions

  • Longitudinal developmental studies

  • Correlation with synaptogenesis and circuit maturation markers

  • Comparison between species using cross-reactive antibodies

• Technical considerations:

  • Consistent fixation and processing across developmental timepoints

  • Age-matched controls for comparative studies

  • Quantitative analysis methods for objective comparison

What approaches can be used to validate HOMER1 antibody specificity?

Validating HOMER1 antibody specificity is crucial for reliable research findings:

• Essential validation methods:

  • Western blot verification of expected molecular weight bands:

    • HOMER1b/c: 46-52 kDa

    • HOMER1a: 40-43 kDa

    • Other variants: 20-26 kDa

  • Testing in multiple applications to confirm consistent detection

  • Comparing results from antibodies targeting different epitopes

• Knockout/knockdown validation:

  • Use of HOMER1 knockout tissue as negative control

  • shRNA knockdown to reduce expression levels

  • Comparing staining patterns before and after knockdown

• Overexpression systems:

  • Transfection with tagged HOMER1 constructs

  • Colocalization of antibody signal with tag detection

  • Verification of epitope recognition in overexpression systems

• Peptide competition assays:

  • Pre-incubation with immunizing peptide/protein to block specific binding

  • Gradual reduction in signal with increasing blocking peptide concentration

• Cross-reactivity assessment:

  • Testing in tissues known to lack HOMER1 (e.g., cerebellar granule cells)

  • Evaluating potential cross-reactivity with other HOMER family members

  • Species cross-reactivity testing for comparative studies

How does HOMER1 contribute to metabotropic glutamate receptor function and localization?

HOMER1 plays a critical role in regulating mGluR function and distribution:

• Subcellular targeting mechanisms:

  • HOMER1b directs mGluR5 specifically to dendrites

  • HOMER1a allows mGluR5 localization in both axons and dendrites

  • This targeting affects synaptic vs. extrasynaptic receptor distribution

• Methodological approaches to study targeting:

  • Co-transfection of tagged mGluR5 with different HOMER1 isoforms

  • Immunolabeling with dendritic (MAP-2) and axonal (Tau-1) markers

  • Live imaging of receptor trafficking with surface-labeling antibodies

• Functional consequences:

  • Dendritically targeted mGluR5 colocalizes with synaptophysin, indicating synaptic localization

  • HOMER1-mGluR coupling affects calcium signaling from intracellular stores

  • Long isoforms promote receptor clustering while short isoforms disperse clusters

• Experimental design considerations:

  • Use of epitope-tagged constructs for tracking specific interactions

  • Pharmacological manipulation of mGluR activity

  • Electrophysiological correlation with receptor localization

Research demonstrates that HOMER1b restricts mGluR5 to dendrites, while HOMER1a allows distribution to both axons and dendrites, revealing how different isoforms uniquely regulate receptor compartmentalization .