NECAB1 Antibody, Biotin conjugated

What is NECAB1 and why are biotin-conjugated antibodies useful for its detection?

NECAB1 (also known as EFCBP1, STIP-1) is a brain-specifically expressed protein with highest abundance in the temporal lobe. It contains EF-hand domains involved in calcium binding and antibiotic biosynthesis monooxygenase domains . The protein has an observed molecular weight of approximately 41 kDa in Western blot applications, although its calculated molecular weight is reported as 64 kDa .

Biotin-conjugated NECAB1 antibodies offer significant advantages for neuronal research due to:

• Strong binding affinity between biotin and streptavidin allowing for signal amplification

• Versatility across multiple detection platforms (fluorescence, enzymatic, etc.)

• Compatibility with complex multi-labeling experimental designs

• Enhanced sensitivity for detecting low-abundance proteins in neuronal tissues

What are the validated applications for biotin-conjugated NECAB1 antibodies?

Based on comprehensive validation studies, biotin-conjugated NECAB1 antibodies have been successfully used in:

These applications have been validated primarily in human, mouse, and rat samples .

What is the typical reactivity profile of biotin-conjugated NECAB1 antibodies?

Biotin-conjugated NECAB1 antibodies show reliable reactivity with:

• Human specimens (brain tissue, particularly cerebral cortex)

• Mouse tissue (brain regions including hippocampus, cerebral cortex)

• Rat neural tissue samples

Cross-reactivity testing has shown that most commercially available biotin-conjugated NECAB1 antibodies do not show significant cross-reactivity with other proteins, ensuring experimental specificity .

What is the optimal protocol for using biotin-conjugated NECAB1 antibodies in flow cytometry?

For optimal results in flow cytometry applications:

• Sample preparation:

  • Isolate target cells using appropriate methods (e.g., MACS for neuronal populations)

  • Fix cells with 4% paraformaldehyde (10 min, room temperature)

  • Permeabilize with 0.1% Triton X-100 if detecting intracellular NECAB1

• Staining protocol:

  • Block with PBS containing 1% BSA for 30 minutes

  • Incubate with biotin-conjugated NECAB1 antibody (1:100 dilution) for 45-60 minutes

  • Wash 3× with PBS + 0.1% Tween-20

  • Incubate with streptavidin-fluorophore conjugate (e.g., streptavidin-FITC)

  • Add 2 μL 7-AAD and incubate 2-3 minutes before final wash

• Analysis parameters:

  • Establish appropriate gating strategy based on cell size/granularity

  • After analysis, >80% of the population should be positive for your detection marker (FITC) and negative for 7-AAD

How should brain tissue samples be prepared for optimal NECAB1 detection using biotin-conjugated antibodies?

For immunohistochemistry and immunofluorescence applications:

• Tissue fixation and processing:

  • Fix brain tissue in 10% neutral buffered formalin

  • Process and embed in paraffin following standard protocols

  • Section at 4-6 μm thickness

• Antigen retrieval (critical step):

  • Heat-mediated antigen retrieval in EDTA buffer (pH 8.0) is strongly recommended

  • Alternative: TE buffer (pH 9.0) also yields good results

  • Citrate buffer (pH 6.0) may be used but generally gives weaker signal

• Blocking and antibody incubation:

  • Block with 10% goat serum (30 min, room temperature)

  • Incubate with biotin-conjugated NECAB1 antibody at 1:50-1:500 dilution overnight at 4°C

  • For detection, use either:

    • Streptavidin-HRP + DAB (for brightfield)

    • Streptavidin-fluorophore (for fluorescence microscopy)

Validation studies show strong and specific NECAB1 staining in cerebral cortex, hippocampus, and other brain regions using this protocol .

What are the recommended protocols for optimizing Western blot detection using biotin-conjugated NECAB1 antibodies?

For optimal Western blot results:

• Sample preparation:

  • Homogenize brain tissue in RIPA buffer with protease inhibitors

  • Load 30 μg protein per lane under reducing conditions

• Electrophoresis conditions:

  • 5-20% SDS-PAGE gel

  • Run at 70V (stacking)/90V (resolving) for 2-3 hours

• Transfer and detection:

  • Transfer to nitrocellulose membrane at 150 mA for 50-90 minutes

  • Block with 5% non-fat milk/TBS for 1.5 hours at room temperature

  • Incubate with biotin-conjugated NECAB1 antibody at 1:2000 dilution

  • Detect using streptavidin-HRP and ECL detection system

• Expected results:

  • Primary band at approximately 41 kDa

  • Validated in rat brain tissue lysates and mouse brain tissue lysates

How can biotin-conjugated NECAB1 antibodies be used to study intercellular protein transport in neuronal networks?

Biotin-conjugated NECAB1 antibodies provide a powerful tool for studying protein transport between neurons:

• Experimental approach:

  • Apply biotin-conjugated NECAB1 antibodies to specific neuronal populations (e.g., retinal ganglion cells)

  • Allow time for potential intercellular transport (typically 7-11 days)

  • Analyze recipient neurons for presence of biotinylated proteins

• Detection methods:

  • For light microscopy: Use streptavidin-conjugated fluorophores or enzymes

  • For electron microscopy: Use streptavidin-FluoroNanogold for ultrastructural localization

• Key findings from research:

  • Biotinylated proteins can be detected in postsynaptic compartments resembling endosomes and tubular recycling endosomes

  • Transfer appears selective for certain neuronal subtypes, with glutamatergic neurons showing preferential uptake compared to GABAergic neurons

  • Approximately 54% of retinogeniculate synapses show evidence of protein transfer

This approach allows researchers to investigate the hypothesis that direct interneuronal protein transfer occurs in both healthy and diseased brain states .

What are the emerging applications of biotin-conjugated NECAB1 antibodies in proteomics and mass spectrometry?

Biotin-conjugated NECAB1 antibodies enable advanced proteomic applications:

• Cell-surface biotinylation analysis:

  • Use magnetic cell isolation combined with surface biotinylation

  • Analyze biotinylated proteins using mass spectrometry to identify NECAB1 interacting partners

  • Identify potential membrane-associated functions of NECAB1

• Protein transfer proteomics:

  • Using DiDBiT (direct detection of biotin-containing tags) and NeutrAvidin protein enrichment strategies

  • MS/MS analysis has identified over 200 transferred proteins between neurons

  • Helps establish whether NECAB1 is among proteins transported between neurons

• Interactome mapping:

  • Biotin-conjugated antibodies can be used to pull down NECAB1 and associated proteins

  • When combined with proximity labeling techniques, can identify both stable and transient interaction partners

How can researchers validate the specificity of biotin-conjugated NECAB1 antibodies?

A comprehensive validation strategy should include:

• Western blot validation:

  • Confirm single band at expected molecular weight (~41 kDa)

  • Test in multiple species (human, mouse, rat brain lysates)

  • Compare to non-biotin conjugated antibodies against the same epitope

• Immunohistochemistry controls:

  • Positive tissue controls (cerebral cortex, hippocampus)

  • Negative tissue controls (tissues known not to express NECAB1)

  • Omission of primary antibody while maintaining streptavidin detection step

  • Pre-absorption with immunizing peptide (if available)

• Cross-reactivity assessment:

  • Test against recombinant NECAB family members (NECAB2, NECAB3)

  • Evaluate staining patterns in knockout models or following siRNA knockdown

• Epitope analysis:

  • Confirm antibody targets appropriate region (e.g., N-terminal EF-hand domain)

  • For monoclonal antibodies, epitope mapping can provide precise binding site information

What are common challenges when using biotin-conjugated NECAB1 antibodies and how can they be addressed?

Most researchers report successful detection when using proper antigen retrieval (particularly with EDTA buffer pH 8.0 or TE buffer pH 9.0) and extended antibody incubation times .

What are the optimal storage and handling conditions for maintaining biotin-conjugated NECAB1 antibody activity?

To maintain optimal antibody performance:

• Storage conditions:

  • Store at -20°C in manufacturer-provided buffer

  • Most formulations contain PBS (pH 7.3), 1% BSA, 50% glycerol, and 0.02% sodium azide

  • Stable for 12 months from date of receipt when properly stored

• Handling recommendations:

  • Aliquot upon receipt to avoid multiple freeze-thaw cycles

  • After thawing, can be stored at 4°C for up to one month

  • Avoid exposure to light (particularly important for dual-labeled antibodies)

  • Centrifuge briefly before opening vial

• Working solution preparation:

  • Dilute in appropriate buffer immediately before use

  • For reconstituted lyophilized antibodies, adding 0.2 ml distilled water yields approximately 500 μg/ml

How do different detection systems compare when using biotin-conjugated NECAB1 antibodies?

Research data indicates that for NECAB1 detection in brain tissue, streptavidin-HRP detection with DAB provides excellent cellular resolution, while fluorescence detection is superior for co-localization studies with other neuronal markers .

What is the pattern of NECAB1 expression across different brain regions as revealed by antibody studies?

Comprehensive immunohistochemical studies using NECAB1 antibodies have revealed specific regional and cellular expression patterns:

These expression patterns suggest NECAB1 may have region-specific functions in neuronal calcium signaling and potentially in neurodegenerative processes .

How can biotin-conjugated NECAB1 antibodies contribute to understanding neurodegeneration?

Biotin-conjugated NECAB1 antibodies offer unique advantages for neurodegeneration research:

• Protein transfer studies:

  • Intercellular transfer of toxic proteins between neurons is implicated in neurodegenerative disease progression

  • Biotin-conjugated antibodies allow tracking of NECAB1 and associated proteins between neurons

  • Research shows about 54% of retinogeniculate synapses demonstrate protein transfer

• Calcium signaling dysregulation:

  • As an EF-hand calcium-binding protein, NECAB1 may play roles in calcium homeostasis

  • Calcium dysregulation is implicated in Alzheimer's, Parkinson's, and other neurodegenerative conditions

  • Biotin-conjugated antibodies enable precise localization of NECAB1 in relation to calcium channels and other signaling proteins

• Proteomic identification of binding partners:

  • MS/MS studies using biotinylated proteins have identified over 200 transferred neuronal proteins

  • Understanding NECAB1's interaction network may reveal new therapeutic targets

How do monoclonal versus polyclonal biotin-conjugated NECAB1 antibodies compare in research applications?

For specific applications like flow cytometry, monoclonal biotin-conjugated antibodies like clone OTI2H5 provide excellent consistency and specificity . For applications requiring signal amplification across multiple species, polyclonal biotin-conjugated antibodies may be preferred .