NECAB1 Antibody

What is NECAB1 and why is it significant in neuroscience research?

NECAB1 (N-terminal EF-hand calcium binding protein 1), also known as EFCBP1 or STIP-1, is a brain-specifically expressed protein containing EF-hand and antibiotic biosynthesis monooxygenase domains. It has highest abundance in the temporal lobe . Its significance lies in its role in neuronal calcium signaling pathways and potential involvement in neurotransmitter release through interactions with synaptic vesicle-associated proteins. Research indicates NECAB1 may be involved in maintaining neuronal communication and plasticity, which are critical for cognitive functions and neural circuit adaptability .

What are the common applications for NECAB1 antibodies in research?

NECAB1 antibodies are primarily utilized in the following applications:

These applications allow researchers to detect and quantify NECAB1 protein expression and localization in various experimental contexts .

Which species reactivity should be considered when selecting a NECAB1 antibody?

Most commercial NECAB1 antibodies demonstrate reactivity with human, mouse, and rat samples . When selecting an antibody, consider the following:

• Confirm the specific reactivity in the product documentation

• For cross-species studies, select antibodies with validated multi-species reactivity

• Some antibodies show extended reactivity to rabbit, guinea pig, and hamster samples

• Sequence homology between species is high (e.g., 98% identity between human and mouse/rat), potentially enabling cross-reactivity

What are the optimal conditions for immunohistochemical detection of NECAB1 in brain tissue?

For optimal IHC detection of NECAB1 in brain tissue, follow these methodological guidelines:

• Tissue preparation: Use formalin-fixed, paraffin-embedded brain tissue sections

• Antigen retrieval:

  • Primary method: TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0

  • Heat treatment: Microwave treatment in citrate buffer for 1 minute at 900W

• Blocking and antibody incubation:

  • Block with 2% normal goat serum and 0.25% Triton in TBS for 1 hour

  • Primary antibody dilution: 1:50-1:500 in blocking solution

  • Incubation time: Overnight at room temperature for European mole tissue; 3 days for mouse tissue

• Detection system:

  • Secondary antibody (e.g., goat anti-rabbit, 1:300) in 2% normal goat serum and 0.1% BSA in TBS

  • ABC treatment followed by DAB staining

These conditions have been validated for detecting NECAB1 in multiple species including mouse, European mole, guinea pig, and sheep .

How should western blot protocols be optimized for NECAB1 detection?

For optimal western blot detection of NECAB1:

• Sample preparation:

  • For brain tissue: Homogenize in RIPA buffer with protease inhibitors

  • Expected molecular weight: ~40-40.6 kDa

• Gel electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels

  • Transfer to PVDF or nitrocellulose membranes

• Antibody incubation:

  • Blocking: 5% non-fat milk or BSA in TBST

  • Primary antibody: Dilute 1:2000 in blocking solution

  • Secondary antibody: HRP-conjugated anti-rabbit/mouse IgG (species matching primary antibody host)

• Detection considerations:

  • Standard ECL detection systems are suitable

  • Predicted band size: 40.4-40.6 kDa

  • Validate specificity with appropriate positive controls (brain tissue lysates)

The monoclonal antibody clone OTI2H5 has been specifically validated for western blot applications at 1:2000 dilution with human, mouse, and rat samples .

What controls should be included when validating NECAB1 antibody specificity?

To ensure experimental rigor when using NECAB1 antibodies, include these controls:

• Positive tissue controls:

  • Brain tissue sections (particularly cerebral cortex and temporal lobe)

  • Mouse brain tissue for IHC/IF applications

  • Human cerebral cortex for IHC

• Negative controls:

  • Primary antibody omission (secondary antibody only)

  • Non-expressing tissues (validate through literature)

  • Peptide competition assays (preincubation with immunizing peptide)

• Specificity validation methods:

  • Side-by-side comparison with alternative NECAB1 antibody clones

  • Knockout/knockdown validation where possible

  • Cross-reactivity testing with other NECAB family members (NECAB2, NECAB3)

• Expression pattern verification:

  • Confirm staining patterns align with published NECAB1 expression data

  • Verify subcellular localization (NECAB1 is primarily cytoplasmic)

How does NECAB1 expression pattern differ between brain regions, and what are the implications for research?

NECAB1 shows distinct expression patterns across brain regions:

• Hippocampus:

  • Strong expression in hilar polymorphic cells across multiple species

  • In European mole: Present in a subset of granule cells

  • In mice: Absent from dentate granule cells

  • Bipolar neurons in stratum radiatum show strong NECAB1 labeling

• Subiculum:

  • In mice: Staining in proximal superficial cells

  • In European mole: Staining in deep cells

• Species variations:

  • Expression patterns are largely conserved across taxonomically diverse species (mice, moles, guinea pigs)

  • Minor species-specific differences suggest evolutionary adaptations

Research implications include:

• NECAB1 can serve as a cell-type specific marker for certain neuronal populations

• Expression differences may correlate with functional specialization in different brain regions

• The conserved nature of expression patterns suggests NECAB1 serves critical neuronal functions

What is the relationship between NECAB1 and endocrine function, particularly in pancreatic β-cells?

Recent research has revealed an unexpected role for NECAB1 in pancreatic function:

• NECAB1 and insulin secretion:

  • NECAB1 functions as a negative regulator of insulin secretion in pancreatic β-cells

  • Mechanism: NECAB1 upregulation leads to reduced intracellular calcium levels, inhibiting insulin release

• Glucocorticoid regulation:

  • Cortisol and corticosterone increase NECAB1 expression via glucocorticoid receptor (GR) activation

  • GR binding to upstream regions of NECAB1 is essential for this regulatory effect

• Pathophysiological relevance:

  • NECAB1 expression is increased in pancreatic islets of db/db mice (a model of type 2 diabetes)

  • May represent a novel component of the adipoinsular axis

  • Potentially involved in obesity-related diabetes mellitus pathophysiology

This research opens new avenues for investigating NECAB1 antibodies beyond neuroscience applications, particularly in metabolic disease research.

How can researchers differentiate between NECAB family members (NECAB1, NECAB2, NECAB3) in experimental settings?

Distinguishing between NECAB family members requires careful antibody selection and experimental design:

• Antibody specificity:

  • Verify immunogen sequences to ensure they target unique regions of NECAB1

  • Many commercial antibodies are raised against full-length NECAB1 protein

  • Some target specific regions (N-terminal or C-terminal)

• Expression pattern differences:

  • NECAB1: Brain-specifically expressed with highest abundance in temporal lobe

  • NECAB2: Shows notable concentration in pyramidal cell layers on either side of the mossy fiber zone

  • NECAB3: Has distinct expression pattern from NECAB1/2

• Methodological approaches:

  • Parallel staining with specific antibodies against each family member

  • Multiplex immunofluorescence with spectrally distinct secondary antibodies

  • Sequential immunostaining on serial sections

• Molecular techniques for validation:

  • RT-PCR with isoform-specific primers

  • RNA-seq for transcriptional profiling

  • Mass spectrometry for protein identification

What are common issues when using NECAB1 antibodies in immunohistochemistry and how can they be resolved?

Researchers may encounter these challenges when using NECAB1 antibodies for IHC:

• Weak or absent signal:

  • Solution: Optimize antigen retrieval (try both TE buffer pH 9.0 and citrate buffer pH 6.0)

  • Increase antibody concentration (titrate between 1:50-1:500)

  • Extend primary antibody incubation time (overnight to 3 days)

• High background staining:

  • Solution: Increase blocking duration (2% normal serum for >1 hour)

  • Add 0.05% BSA to antibody diluent

  • Ensure proper washing between steps (multiple washes with TBS-Triton)

• Cross-reactivity concerns:

  • Solution: Pre-absorb antibody with blocking peptide if available

  • Compare staining pattern with alternative NECAB1 antibody clones

  • Include appropriate negative controls

• Species-specific optimization:

  • Solution: Adjust protocol parameters based on species (e.g., longer incubation for mouse tissue)

  • Use species-matched secondary antibodies

  • Consider tissue-specific fixation protocols

How can researchers ensure reproducibility when using different lots or sources of NECAB1 antibodies?

To ensure experimental reproducibility across antibody lots and sources:

• Antibody validation strategy:

  • Document antibody information (catalog number, lot number, clone ID)

  • Test new lots alongside previously validated lots

  • Maintain detailed protocol records with lot-specific optimization notes

• Standardization practices:

  • Use standardized positive controls (e.g., mouse brain tissue)

  • Include internal reference standards in each experiment

  • Quantify staining intensity using digital image analysis when possible

• Methodological considerations:

  • For polyclonal antibodies: Expect greater lot-to-lot variation

  • For monoclonal antibodies: More consistent but still verify each lot

  • Always titrate new antibody lots to determine optimal concentration

• Documentation requirements:

  • Record RRID (Research Resource Identifier) when available

  • Document complete antibody information in publications

  • Share detailed protocols in supplementary materials

What are the best practices for storing and handling NECAB1 antibodies to maintain optimal activity?

For maximum antibody stability and performance:

Additional handling recommendations:

• Allow antibody to reach room temperature before opening vial

• Centrifuge briefly before opening to collect liquid

• Always use clean pipette tips when handling antibody solutions

• Document date of first use and track number of freeze-thaw cycles

How is NECAB1 being studied in the context of neurological disorders and potential therapeutic targets?

Current research on NECAB1 in neurological contexts includes:

• Expression profiling in disease states:

  • Examining NECAB1 levels in neurodegenerative disease models

  • Comparing expression patterns between healthy and pathological tissue

  • Investigating potential biomarker applications

• Functional studies:

  • Role in calcium signaling dysregulation in neurological disorders

  • Interactions with synaptic proteins in various disease states

  • Effects on neuronal excitability and neurotransmitter release

• Methodological approaches:

  • CRISPR/Cas9-mediated gene editing to study NECAB1 function

  • Conditional knockout models in specific neuronal populations

  • High-resolution imaging of NECAB1 localization during disease progression

• Therapeutic implications:

  • Assessment of NECAB1 as a potential drug target

  • Screening compounds that modulate NECAB1 activity

  • Evaluating effects of existing neurological drugs on NECAB1 expression

What are the technical considerations when using NECAB1 antibodies in multiplexed immunofluorescence studies?

For successful multiplexed detection of NECAB1 with other markers:

• Antibody selection criteria:

  • Choose primary antibodies raised in different host species (e.g., rabbit anti-NECAB1 with mouse anti-other target)

  • Verify individual antibodies work under identical fixation conditions

  • Test for potential cross-reactivity between primary and secondary antibodies

• Protocol optimization:

  • Determine optimal antibody concentrations individually before multiplexing

  • Sequence primary antibody incubations (strongest signal first or most sensitive last)

  • Include appropriate blocking steps between primary antibody applications

• Detection strategies:

  • Use spectrally distinct fluorophores with minimal overlap

  • Consider tyramide signal amplification for weak signals

  • Include appropriate single-color controls

• Analysis considerations:

  • Apply spectral unmixing to separate overlapping signals

  • Use colocalization analysis tools with appropriate thresholding

  • Include quantification methods for signal intensity

How are quantitative approaches being applied to NECAB1 expression analysis in comparative neuroscience?

Advanced quantitative methods for NECAB1 analysis include:

• Digital pathology approaches:

  • Whole slide imaging with automated NECAB1-positive cell counting

  • Machine learning algorithms for pattern recognition

  • 3D reconstruction of NECAB1 expression in whole brain models

• Comparative analysis methods:

  • Standardized quantification across species (mole, mouse, rat, human)

  • Stereological sampling to estimate total NECAB1-positive cell populations

  • Cross-species normalization techniques accounting for brain size differences

• Integration with other data types:

  • Correlation of protein expression with transcriptomic data

  • Multimodal analysis combining electrophysiological and molecular data

  • Systems biology approaches to network analysis

• Reproducibility considerations:

  • Standardized analysis pipelines with open-source tools

  • Data sharing through neuroscience-specific repositories

  • Reporting guidelines for quantitative NECAB1 analysis