CACNA1B Antibody, FITC conjugated

What is CACNA1B and why is it significant in neuroscience research?

CACNA1B (also known as Cav2.2) is the alpha-1B subunit of N-type voltage-dependent calcium channels. It belongs to the calcium channel alpha-1 subunit (TC 1.A.1.11) family and gives rise to N-type calcium currents . These channels are crucial mediators in calcium-dependent processes including:

• Neurotransmitter release

• Neuronal excitability

• Pain signal transmission

• Cell motility and migration

• Gene expression

N-type calcium channels belong to the 'high-voltage activated' (HVA) group and are blocked by omega-conotoxin-GVIA and omega-agatoxin-IIIA, while remaining insensitive to dihydropyridines and omega-agatoxin-IVA . CACNA1B plays a particularly important role in pain pathways, serving to regulate pain signals from the peripheral to central nervous system .

The alpha-1 subunit consists of 24 transmembrane segments and forms the pore through which calcium ions enter the cell. The complete calcium channel typically exists as a complex of alpha-1, alpha-2/delta, beta, and gamma subunits in a 1:1:1:1 ratio .

Proper storage is critical for maintaining antibody performance:

• Store at -20°C for long-term preservation

• Aliquot into multiple vials to avoid repeated freeze-thaw cycles

• Protect from light to prevent photobleaching of the FITC fluorophore

• Some formulations contain glycerol (typically 50%) which prevents freezing at -20°C

• Standard buffer components include:

  • PBS or TBS (pH 7.2-7.4)

  • Preservatives (0.01% thimerosal, 0.03% Proclin300)

  • Protein stabilizers (1% BSA)

When working with the antibody, minimize exposure to light, keep cold, and avoid contamination of the stock solution.

What protocol is recommended for immunofluorescence using FITC-conjugated CACNA1B antibodies?

Basic Protocol for FITC-CACNA1B Immunofluorescence:

• Fixation:

  • For tissues: 4% paraformaldehyde (PFA) in PBS (pH 7.4) for 24 hours at 4°C

  • For cells: 4% PFA for 10-15 minutes at room temperature

• Antigen Retrieval:

  • For neural tissues: TE buffer pH 9.0 is recommended

  • Alternative: citrate buffer pH 6.0

  • Heat-induced epitope retrieval (HIER) for 15-20 minutes

• Blocking:

  • 5-10% normal serum (from species unrelated to primary antibody)

  • 0.1-0.3% Triton X-100 for permeabilization

  • 1 hour at room temperature

• Primary Antibody Incubation:

  • Dilute FITC-conjugated CACNA1B antibody 1:50-1:200 in blocking buffer

  • Incubate overnight at 4°C in a humidified chamber

  • Protect from light

• Nuclear Counterstain:

  • DAPI or other non-green fluorescent nuclear stain

  • Follow manufacturer's recommended dilution

• Mounting:

  • Use anti-fade mounting medium

  • Seal edges with nail polish for long-term storage

• Imaging:

  • Use FITC filter set (Ex: 490nm, Em: 525nm)

  • Minimize exposure times to prevent photobleaching

Note: When performing double or triple immunofluorescence, select secondary antibodies with non-overlapping spectra (e.g., Cy3, Cy5) for other primary antibodies.

How do I optimize immunofluorescence signal when working with CACNA1B antibodies?

Signal optimization requires attention to several variables:

• Antibody Dilution Optimization:

  • Perform a dilution series (e.g., 1:50, 1:100, 1:200, 1:500)

  • Evaluate signal-to-noise ratio at each dilution

  • The recommended starting range for FITC-conjugated CACNA1B antibodies is 1:50-1:200

• Antigen Retrieval Methods:

  • For CACNA1B detection in brain tissue, TE buffer (pH 9.0) is recommended

  • Alternative: citrate buffer (pH 6.0)

• Fixation Optimization:

  • Over-fixation can mask epitopes

  • Under-fixation can compromise tissue morphology

  • For most neuronal applications, 4% PFA for 24-48 hours provides good results

• Background Reduction:

  • Increase blocking time/concentration

  • Add 0.1-0.3% Triton X-100 to enhance antibody penetration

  • Include 0.1% Tween-20 in wash buffers

  • Consider tissue autofluorescence quenching steps

• Control Experiments:

  • Include a negative control (omit primary antibody)

  • If possible, include a CACNA1B knockout control or peptide-blocked control

  • Use tissues known to express CACNA1B (brain, cerebellum) as positive controls

What tissues and subcellular locations show positive CACNA1B immunoreactivity?

CACNA1B shows specific expression patterns across tissues and subcellular compartments:

Tissue Distribution:

• Brain tissue: Strong positive detection

• Cerebellum: Strong positive detection

• Ciliary body and iris: Positive immunoreactivity

• Anterior lens epithelium: Positive staining

• Optic nerve glia and vascular endothelial cells: Positive immunoreactivity

• Retina: Strong diffuse staining in specific layers

Retinal Layer-Specific Localization:

• Photoreceptor inner segments (IS)

• Inner nuclear layer (INL)

• Outer nuclear layer (ONL)

• Nerve fiber layer (NFL)

Subcellular Localization:

• Primarily on nerve terminals and dendrites

• Cell membrane of neuroendocrine cells

• Synaptic sites

Interestingly, research has shown that CACNA1B expression is higher in human ocular tissue-derived cells than in cells of non-ocular origin , suggesting tissue-specific regulatory mechanisms.

How can I validate the specificity of CACNA1B antibodies in my experimental system?

Thorough validation is essential for antibody-based studies:

• Western Blot Analysis:

  • Expected molecular weight: 262 kDa (calculated)

  • Observed molecular weight: May appear around 200 kDa

  • Positive control cell lines: SH-SY5Y cells, Y79 cells

• RNA Knockdown/Knockout Controls:

  • siRNA or shRNA knockdown of CACNA1B

  • CRISPR/Cas9 knockout models

  • Several published studies have utilized KD/KO approaches for validation

• Peptide Competition Assay:

  • Pre-incubate antibody with immunizing peptide

  • Should eliminate specific staining

  • For FITC-conjugated antibodies derived from synthetic peptides (e.g., AA 101-200/2339) , contact the manufacturer for the blocking peptide

• Reactivity Verification:

  • CACNA1B antibodies have been verified for reactivity with human, mouse, and rat samples

  • When using in other species, homology analysis of the immunogen sequence is recommended

• Comparison with Alternative Antibodies:

  • Use antibodies targeting different epitopes of CACNA1B

  • Similar staining patterns increase confidence in specificity

• Correlation with mRNA Expression:

  • Compare antibody staining with in situ hybridization or RNA-seq data

  • Should show similar tissue/cellular distribution patterns

What considerations should be made when studying CACNA1B in relation to disease states?

CACNA1B has been implicated in several pathological conditions:

• Exfoliation Syndrome (XFS):

  • A common variant mapping to CACNA1A is associated with XFS

  • Research has shown differences in CACNA1B staining patterns between normal and XFS globes:

    • Non-XFS: Strong diffuse staining in retinal layers

    • XFS globes: Focal and patchy immunostaining in the same layers

• Pain Pathways:

  • CACNA1B (Cav2.2) is a critical target for pain management

  • N-type calcium channels are blocked by omega-conotoxins

  • Consider studying interaction with opioid receptors, as research shows DAMGO (μ-opioid agonist) affects CACNA1B-mediated currents

• Alternative Splicing:

  • CACNA1B undergoes alternative splicing that affects function

  • This can impact antibody binding depending on epitope location

  • Studies have shown that alternative splicing in the Cacna1b gene affects opioid inhibition of N-type Ca2+ channels and spinal analgesia

• Neuronal Migration:

  • CACNA1B may play a role in directed migration of immature neurons

  • Consider co-staining with neuronal migration markers when studying development

When studying disease states, it's important to include appropriate disease and control samples, and to consider how disease-associated modifications might affect antibody binding.

How do different CACNA1B antibody clones compare in research applications?

Various antibodies targeting different epitopes of CACNA1B exist with distinct characteristics:

When selecting an antibody:

• Consider the epitope location and its conservation across species

• Choose an antibody validated for your specific application

• Select an antibody targeting epitopes away from regions subject to alternative splicing if studying all isoforms

• For FITC-conjugated antibodies, consider potential interference with other fluorophores in multiplexed experiments

What are best practices for multiplexed immunofluorescence including CACNA1B detection?

When combining FITC-conjugated CACNA1B antibodies with other markers:

• Fluorophore Selection:

  • Pair FITC (green) with fluorophores that have minimal spectral overlap:

    • Cy3/TRITC (red)

    • Cy5/AlexaFluor647 (far-red)

    • Pacific Blue (blue)

• Sequential Staining:

  • For complex multi-labeling experiments, consider sequential rather than simultaneous staining

  • Especially important when using multiple antibodies raised in the same host species

• Cross-Reactivity Prevention:

  • When using multiple rabbit antibodies, consider directly conjugated antibodies

  • Alternatively, use Fab fragments to block before adding the second primary

• Image Acquisition:

  • Use sequential scanning to minimize bleed-through

  • Establish single-color controls to set acquisition parameters

  • Consider spectral unmixing for closely overlapping fluorophores

• Recommended Combinations:

  • FITC-CACNA1B + Cy3-labeled synaptic markers (e.g., synaptophysin)

  • FITC-CACNA1B + Cy5-labeled voltage-gated sodium channels

  • FITC-CACNA1B + DAPI (nuclear stain) + Cy3-labeled neuronal markers

Remember that FITC is relatively sensitive to photobleaching, so minimize exposure during imaging and consider using anti-fade mounting media with protective agents.

What are common issues when working with CACNA1B antibodies and how can they be resolved?

Problem: Weak or No Signal

Potential Causes and Solutions:

• Insufficient antibody concentration: Increase primary antibody concentration (start with 1:50 dilution for FITC-conjugated CACNA1B)

• Inadequate antigen retrieval: For IHC, use TE buffer pH 9.0 as recommended

• Overfixation masking epitopes: Reduce fixation time or try different fixatives

• Antibody degradation: Ensure proper storage at -20°C and avoid repeated freeze-thaw cycles

• Low target expression: Confirm CACNA1B expression in your sample type; use positive controls (brain tissue, SH-SY5Y cells)

Problem: High Background

Potential Causes and Solutions:

• Insufficient blocking: Increase blocking time or concentration; consider using different blocking agents

• Antibody concentration too high: Perform a dilution series to optimize signal-to-noise ratio

• Insufficient washing: Increase number and duration of washes; add 0.1% Tween-20 to wash buffer

• Tissue autofluorescence: Use autofluorescence quenching treatments appropriate for your tissue

• FITC photobleaching: Minimize exposure to light during all steps of the protocol

Problem: Non-specific Binding

Potential Causes and Solutions:

• Cross-reactivity: Validate antibody specificity using controls (peptide blocking, KO tissues)

• Hydrophobic interactions: Add 0.1-0.3% Triton X-100 to antibody dilution buffer

• Fc receptor binding: Add species-appropriate serum to blocking buffer

• Endogenous biotin: If using biotin-based detection systems, block endogenous biotin

How should I approach experimental design for quantitative analysis of CACNA1B expression?

Quantitative analysis requires rigorous experimental design:

• Sample Preparation Consistency:

  • Use consistent fixation protocols across all samples

  • Process control and experimental samples in parallel

  • Use the same antibody lot for all samples in a study

• Imaging Parameters:

  • Establish settings on positive controls

  • Maintain identical acquisition parameters across all samples

  • Avoid saturated pixels which prevent accurate quantification

  • Include exposure/gain settings in methods documentation

• Quantification Methods:

  • For Western Blot: Use appropriate loading controls and normalization

  • For IF/IHC:

    • Define consistent ROIs for analysis

    • Consider mean fluorescence intensity, area fraction, or integrated density

    • Use automated analysis when possible to reduce bias

• Statistical Considerations:

  • Determine appropriate sample size through power analysis

  • Blind the analyst to experimental conditions during quantification

  • Use appropriate statistical tests based on data distribution

  • Consider technical vs. biological replicates in analysis

• Controls for Quantification:

  • Include calibration standards when possible

  • Use reference samples across multiple experiments for normalization

  • Include negative controls to establish background threshold

When publishing, report detailed methods including antibody catalog numbers, dilutions, exposure times, and quantification parameters to ensure reproducibility.

How can FITC-conjugated CACNA1B antibodies be utilized in advanced imaging techniques?

FITC-conjugated CACNA1B antibodies can be leveraged in several cutting-edge imaging approaches:

• Super-Resolution Microscopy:

  • While FITC isn't optimal for some super-resolution techniques due to photobleaching, it can be used with:

    • Structured Illumination Microscopy (SIM)

    • Stimulated Emission Depletion (STED) microscopy with careful optimization

  • These techniques provide ~100nm resolution, allowing detailed analysis of CACNA1B clustering and co-localization with synaptic proteins

• Live-Cell Imaging Applications:

  • For cell-surface epitopes, consider non-permeabilizing labeling of live neurons

  • Monitor CACNA1B trafficking in response to stimuli

  • Note: For intracellular epitopes (such as AA 851-867) , live imaging is not possible with conventional antibodies

• Expansion Microscopy:

  • Physical expansion of specimens can provide improved resolution with standard confocal microscopy

  • FITC-conjugated antibodies are compatible with many expansion protocols

  • Allows visualization of nanoscale organization of calcium channels

• Correlative Light and Electron Microscopy (CLEM):

  • Identify FITC-CACNA1B positive structures by fluorescence

  • Follow with electron microscopy of the same sample

  • Provides ultrastructural context of CACNA1B localization

• Tissue Clearing Techniques:

  • Methods like CLARITY, iDISCO, and CUBIC allow whole-organ imaging

  • Verify compatibility of your specific FITC-CACNA1B antibody with the clearing protocol

  • Enables 3D mapping of CACNA1B distribution throughout intact neural structures

These advanced techniques require careful optimization and may require higher antibody concentrations than conventional methods.

What is known about the relationship between CACNA1B alternative splicing and functional diversity?

Alternative splicing of CACNA1B creates functionally diverse channels with important research implications:

• Functional Consequences of Splicing:

  • Alternative splicing in the Cacna1b gene affects:

    • Voltage-dependent vs. voltage-independent inhibition by G-protein coupled receptors

    • Opioid inhibition of N-type Ca2+ channels

    • Sensitivity to specific blockers

    • Kinetics of channel activation and inactivation

• Experimental Considerations:

  • Antibody epitope selection should consider known splice variants

  • For AA 101-200 epitopes (as in bs-10490R-FITC) , verify whether this region undergoes alternative splicing

  • When studying specific variants, select antibodies targeting invariant regions or splice-specific sequences

• Disease Relevance:

  • Research has demonstrated that alternative splicing affects spinal analgesia

  • Consider splice variant-specific expression when studying:

    • Pain processing

    • Synaptic transmission

    • Neuronal development

    • Response to therapeutic agents

• Genetically Modified Models:

  • Studies have utilized Cacna1b gene modifications to study splicing effects:

    • Cacna1b aa/aa mice

    • Cacna1b bb/bb mice

  • These models show differences in DAMGO (μ-opioid agonist) inhibition of N-type currents

For comprehensive study of CACNA1B splice variants, consider combining antibody-based detection with RT-PCR or RNA-seq to correlate protein expression with specific transcript variants.