CALB2 Antibody

What is CALB2/Calretinin and why is it important in neuroscience research?

CALB2 (Calbindin 2), commonly known as Calretinin, is an intracellular calcium-binding protein belonging to the troponin C superfamily. It has a molecular weight of approximately 29 kDa and plays crucial roles in calcium signaling and homeostasis within neurons . The importance of CALB2 in neuroscience research stems from its specific expression pattern in distinct neuronal populations, making it an excellent marker for neuronal subtyping in both the central and peripheral nervous systems. The rat and human calretinin sequences exhibit 98% homology, with 91% homology to many other species, making it a well-conserved protein across mammals . Calretinin is particularly abundant in Purkinje cells and other cell types in the cerebellum, allowing researchers to identify and study these specific neuronal populations in normal development and pathological conditions.

What structural and functional characteristics make CALB2 antibodies valuable research tools?

CALB2 antibodies recognize specific epitopes on the calretinin protein, often targeting sequences at the N-terminus, which are highly conserved across species . These antibodies are valuable because calretinin has six EF-hand calcium-binding motifs that undergo conformational changes upon calcium binding, allowing researchers to study calcium-dependent processes in neurons. Most commercially available antibodies are developed against synthetic peptides corresponding to human CALB2 sequences, with the immunogen sequence typically including amino acids that form the calcium-binding domains . This structural targeting makes CALB2 antibodies particularly useful for distinguishing calcium-binding states in experimental contexts. The high specificity and cross-reactivity with human, mouse, and rat CALB2 allow for comparative studies across species models .

How do polyclonal and monoclonal CALB2 antibodies differ in research applications?

Most available CALB2 antibodies are polyclonal (typically raised in rabbits), which recognize multiple epitopes on the calretinin protein . This multi-epitope recognition provides stronger signal amplification and better tolerance to protein denaturation, making polyclonal antibodies particularly effective for western blot and immunohistochemistry applications where proteins may undergo conformational changes during sample processing. Polyclonal antibodies like those from Proteintech (12278-1-AP) and Boster Bio (PA1015) demonstrate high sensitivity across various applications including Western blot (1:5000-1:50000 dilution range), immunohistochemistry (1:500-1:2000), and immunofluorescence . While monoclonal antibodies offer higher specificity for a single epitope, polyclonal options are more prevalent in CALB2 research due to their versatility across multiple experimental conditions and their robust performance in fixed tissue preparations.

What are the optimal antigen retrieval methods for CALB2 immunohistochemistry?

For optimal CALB2 detection in paraffin-embedded tissues, heat-mediated antigen retrieval is essential. The recommended protocol includes:

• Primary antigen retrieval using TE buffer at pH 9.0, which provides superior epitope exposure for most CALB2 antibodies

• Alternatively, EDTA buffer (pH 8.0) has shown excellent results, particularly with antibodies like Boster's PA1015

• For tissues with high background, citrate buffer at pH 6.0 can be used as an alternative method

The antigen retrieval process typically involves:

• Deparaffinization and rehydration of sections

• Immersion in the selected buffer

• Heating in a pressure cooker or microwave for 15-20 minutes

• Gradual cooling to room temperature before blocking steps

The choice between these methods depends on tissue fixation history and specific antibody requirements. For CALB2 detection in brain tissues, the more alkaline buffers (pH 8.0-9.0) typically yield stronger signal intensity with lower background .

What are the recommended dilution ranges and detection systems for different CALB2 antibody applications?

CALB2 antibodies require specific dilution ranges for optimal results across different applications:

For IHC applications, Strepavidin-Biotin-Complex (SABC) systems have shown excellent sensitivity with DAB as the chromogen . For immunofluorescence, DyLight®488 or similar fluorophores provide good signal with low background when combined with DAPI counterstaining . Western blot detection is most effective with enhanced chemiluminescence (ECL) systems, requiring secondary antibody dilutions around 1:5000 . These parameters should be optimized for each specific experimental system, as tissue fixation and processing variables can significantly impact antibody performance.

How can multiplexed immunofluorescence be performed using CALB2 antibodies?

For multiplexed detection of CALB2 alongside other neuronal markers:

• Select compatible primary antibodies raised in different host species (e.g., rabbit anti-CALB2 with mouse anti-NeuN)

• Use a sequential staining approach when multiple rabbit antibodies are needed:

  • First primary antibody incubation (anti-CALB2, 5 μg/mL) overnight at 4°C

  • Detection with species-specific secondary antibody

  • Microwave treatment (10 minutes in citrate buffer) to denature remaining primary antibodies

  • Second primary antibody incubation with different fluorophore-conjugated secondary

The specific protocol for brain tissue sections includes:

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

• Blocking with 10% goat serum to reduce non-specific binding

• Primary antibody incubation at 5 μg/mL overnight at 4°C

• Secondary detection with appropriate fluorophore conjugates

• DAPI counterstaining for nuclear visualization

This approach allows for simultaneous visualization of CALB2 with other markers such as GABAergic interneuron markers or calcium-binding proteins like parvalbumin, providing comprehensive characterization of neuronal subtypes.

What is the expression pattern of CALB2 across different brain regions and tissue types?

CALB2 exhibits distinct expression patterns that vary by brain region and tissue type:

Immunohistochemical studies have confirmed CALB2 expression in human brain tissue, human cerebellum tissue, mouse brain tissue, and rat brain tissue with consistent staining patterns . Western blot analyses have detected CALB2 in human ileum tissue, human colon tissue, and fetal human brain tissue . This distinct distribution pattern makes CALB2 a valuable marker for identifying specific neuronal subpopulations in both central and enteric nervous systems. The antibody can be used to track developmental changes in CALB2 expression or alterations in neurological disease states.

How reliable is CALB2 immunoreactivity for distinguishing neuronal subtypes in the cerebellum?

CALB2 immunoreactivity is highly reliable for distinguishing specific neuronal subtypes in the cerebellum due to several factors:

• Consistent expression in Purkinje cells with clear demarcation of cellular morphology

• Distinct staining pattern that differentiates from other calcium-binding proteins like calbindin D28k

• Reproducible results across multiple antibody sources when using optimized protocols

The antibody validation data demonstrates specific staining in cerebellar tissue sections, with clear discrimination between CALB2-positive and negative cells . For maximum reliability, researchers should:

• Use positive control tissues (cerebellum sections) alongside experimental samples

• Maintain consistent antigen retrieval methods (preferably EDTA buffer, pH 8.0)

• Employ appropriate dilution ranges (1:500-1:2000 for IHC)

• Consider double-labeling with other neuronal markers for confirmation

When proper protocols are followed, CALB2 antibodies consistently label specific neuronal populations, making them valuable tools for cerebellar cytoarchitecture studies and for identifying abnormalities in neurodevelopmental or neurodegenerative conditions.

Can CALB2 antibodies effectively distinguish between neuronal and non-neuronal cells?

• In pathological conditions, aberrant expression may occur in typically CALB2-negative cells

• During development, transient expression patterns may differ from adult patterns

• In certain tissues like mesothelioma, CALB2 can be expressed in non-neuronal cells

For definitive cell type identification, it is recommended to:

• Use dual-labeling with established neuronal markers (NeuN, MAP2) and glial markers (GFAP, IBA1)

• Compare staining patterns with known anatomical distributions

• Employ appropriate morphological criteria alongside immunoreactivity

Experimental data from immunohistochemistry and immunofluorescence studies show that CALB2 antibodies provide clear discrimination between neuronal populations and surrounding glial cells in brain tissue sections from human, mouse, and rat samples .

What are common causes of false negative results when detecting CALB2 in Western blots?

False negative results in CALB2 Western blots can stem from several technical issues:

• Inadequate protein extraction: CALB2 requires proper solubilization; use lysis buffers containing 1% Triton X-100 or RIPA buffer with protease inhibitors.

• Insufficient transfer efficiency: Given CALB2's molecular weight (29 kDa), use semi-dry transfer systems at 150 mA for 50-90 minutes or wet transfer at 100V for 60 minutes with methanol-containing transfer buffer.

• Inappropriate antibody dilution: While the recommended range is 1:5000-1:50000, start with 1:5000 for unknown samples and optimize accordingly . For difficult samples, use 0.5-1 μg/mL concentration.

• Inadequate blocking: Use 5% non-fat milk in TBS for 1.5 hours at room temperature before primary antibody incubation .

• Inappropriate secondary antibody: Ensure secondary antibody (typically goat anti-rabbit IgG-HRP) is used at 1:5000 dilution and is compatible with the detection system .

Validation data from Boster Bio shows successful detection of CALB2 in human Hela whole cell lysates using their standardized protocol, which includes overnight incubation at 4°C with the primary antibody, followed by appropriate washing steps and secondary antibody incubation .

How can background staining be reduced when using CALB2 antibodies in immunohistochemistry?

To reduce background staining in CALB2 immunohistochemistry:

• Optimize blocking conditions: Use 10% serum from the same species as the secondary antibody (typically goat serum) for 1-2 hours at room temperature .

• Adjust antibody concentration: Titrate the antibody; start with 1:500 for IHC and increase dilution if background persists .

• Modify antigen retrieval: If background is high with EDTA buffer (pH 8.0), switch to citrate buffer (pH 6.0), which can provide more controlled epitope exposure .

• Include washing detergents: Add 0.1% Tween-20 to PBS or TBS washing buffers to reduce non-specific binding.

• Consider endogenous peroxidase and biotin blocking: For HRP-based detection systems, include a 3% hydrogen peroxide treatment step; for biotin-based systems, use avidin/biotin blocking kits.

• Employ Sudan Black B treatment: For tissues with high autofluorescence (especially brain), treat with 0.1% Sudan Black B in 70% ethanol for 20 minutes after secondary antibody incubation.

Experimental evidence shows that these approaches effectively reduce background while maintaining specific CALB2 staining in brain tissues, colon tissues, and human appendicitis samples .

How should antibody storage and handling be optimized to maintain CALB2 antibody activity?

Proper storage and handling of CALB2 antibodies are crucial for maintaining activity:

• Storage temperature: Store at -20°C in the original container. CALB2 antibodies are typically stable for one year after shipment when stored properly .

• Aliquoting considerations: For 100 μL size antibodies, aliquoting is generally unnecessary for -20°C storage, though it may be beneficial for larger volumes to avoid freeze-thaw cycles .

• Buffer composition: Most CALB2 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which provides stability during storage . Some products (20 μL sizes) may contain 0.1% BSA for additional stability .

• Thawing procedure: Thaw antibodies on ice or at 4°C rather than at room temperature to preserve activity.

• Working dilution preparation: When preparing working dilutions, use freshly prepared buffers (PBS or TBS) containing 1-5% BSA or normal serum.

• Freeze-thaw cycles: Limit to 5 or fewer cycles; document any signs of activity loss.

Following these guidelines will help maintain antibody reactivity and ensure consistent results across experiments, particularly important for quantitative analyses of CALB2 expression .

How can CALB2 antibodies be incorporated into multiplexed imaging systems with other neuronal markers?

CALB2 antibodies can be effectively incorporated into multiplexed imaging systems through several strategic approaches:

• Sequential immunostaining: For multiple rabbit-derived antibodies, employ sequential staining with intermediate stripping steps using 0.1M glycine (pH 2.5) or mild microwave treatment.

• Spectral unmixing techniques: When using fluorophores with overlapping spectra, incorporate spectral imaging microscopy with computational unmixing algorithms.

• Tyramide signal amplification (TSA): This approach allows multiple antibodies from the same species to be used sequentially:

  • Apply first primary antibody at high dilution (1:2000-1:5000)

  • Detect with HRP-conjugated secondary and tyramide-fluorophore

  • Heat-inactivate HRP (microwave 2-3 minutes in citrate buffer)

  • Repeat with next primary antibody and different tyramide-fluorophore

• Compatible marker combinations: Effective combinations with CALB2 include:

  • CALB2 + parvalbumin (PV) for interneuron subtyping

  • CALB2 + GAD67 for inhibitory neuron identification

  • CALB2 + NeuN + GFAP for distinguishing neurons from glia

Validated protocols show successful multiplexing in mouse brain tissue using CALB2 antibody (5 μg/mL) with DyLight®488 secondary antibody visualization, counterstained with DAPI for nuclear identification .

What considerations are important when using CALB2 antibodies for quantitative protein expression analysis?

When employing CALB2 antibodies for quantitative protein expression analysis, several critical factors must be addressed:

• Standard curve calibration: Use recombinant CALB2 protein at known concentrations (10-500 ng) to generate a standard curve for absolute quantification.

• Sample normalization strategies:

  • For Western blots: Normalize to loading controls (β-actin, GAPDH, or total protein)

  • For IHC/IF: Use internal reference structures or stereological counting methods

• Antibody saturation assessment: Test multiple antibody concentrations to ensure operation within the linear detection range.

• Technical replication requirements: Perform at least 3 independent experiments with duplicate or triplicate technical replicates.

• Image acquisition parameters: For fluorescence quantification:

  • Use identical exposure settings across all samples

  • Ensure signal is below pixel saturation

  • Employ flat-field correction for uneven illumination

• Data normalization approaches:

  • Area-based normalization (signal intensity per unit area)

  • Cell-based normalization (signal intensity per cell)

  • Relative expression (comparison to control samples)

The observed molecular weight of CALB2 is 29 kDa, matching the calculated value, which is important for accurate band identification in quantitative Western blot applications .

How reliable are CALB2 antibodies for studying neurodevelopmental processes and disease pathology?

CALB2 antibodies demonstrate high reliability for neurodevelopmental and pathological studies, with several important considerations:

• Developmental expression patterns: CALB2 expression changes during development, requiring:

  • Age-matched controls for developmental studies

  • Awareness of transient expression in certain cell populations

  • Verification with RNA expression data when possible

• Disease-specific alterations:

  • In neurodegenerative diseases: Monitor potential loss of CALB2-positive neurons

  • In inflammatory conditions: Be aware of potential upregulation in typically negative cells

  • In neoplastic tissues: Consider aberrant expression patterns

• Reproducibility considerations:

  • Use antibodies with established RRID numbers (e.g., AB_2228338) to ensure reproducibility

  • Employ enhanced validation approaches like RNAi knockdown

  • Include appropriate positive controls (cerebellum) and negative controls in each experiment

• Species-specific validation:

  • Confirm reactivity in target species (human, mouse, rat confirmed for most antibodies)

  • Be aware of potential species-specific differences in staining patterns

Experimental evidence from multiple sources confirms that CALB2 antibodies reliably detect the protein in both normal and pathological tissues, including human rectal cancer tissue and human appendicitis tissue, making them valuable tools for both basic research and clinical studies .

How should discrepancies between CALB2 mRNA and protein expression levels be interpreted?

Discrepancies between CALB2 mRNA and protein expression levels should be interpreted with consideration of several biological and technical factors:

• Post-transcriptional regulation mechanisms:

  • microRNA-mediated repression may suppress translation despite high mRNA levels

  • RNA-binding proteins might affect mRNA stability or translation efficiency

  • Alternative splicing could generate transcript variants detected at the mRNA level but not by protein-specific antibodies

• Protein stability and turnover rates:

  • CALB2 protein may have tissue-specific half-lives affecting steady-state levels

  • Calcium-binding status might influence protein stability and detection

  • Cellular stress responses could alter protein degradation pathways

• Technical considerations:

  • Different detection sensitivities between qPCR and immunodetection methods

  • Antibody epitope accessibility might be affected by protein conformation or interactions

  • Fixation or extraction methods may influence protein detection efficiency

• Analytical approaches:

  • Perform time-course studies to assess temporal relationships between mRNA and protein expression

  • Use multiple antibodies targeting different epitopes to confirm protein expression patterns

  • Implement proteomic approaches (mass spectrometry) as an antibody-independent validation

These considerations are particularly important when studying developmental changes in CALB2 expression or disease-related alterations in calcium-binding protein networks.

What are best practices for quantifying CALB2 immunoreactivity in tissue sections?

Quantification of CALB2 immunoreactivity in tissue sections requires rigorous methodology:

• Stereological approaches:

  • Use unbiased sampling methods (optical fractionator, physical disector)

  • Implement systematic random sampling across tissue sections

  • Maintain consistent counting criteria (cell size, staining intensity thresholds)

• Intensity-based measurements:

  • Define intensity thresholds consistently across samples

  • Use calibration standards or internal controls to normalize between batches

  • Apply background subtraction appropriate to tissue type

• Morphological analyses:

  • Measure dendritic complexity of CALB2-positive neurons (Sholl analysis)

  • Quantify cell body size and shape parameters

  • Assess colocalization with other markers (Manders' coefficient, Pearson's correlation)

• Software and image acquisition standards:

  • Use consistent microscope settings (exposure, gain, offset)

  • Implement flat-field correction to account for illumination variability

  • Employ automated analysis pipelines to reduce observer bias

• Statistical considerations:

  • Determine appropriate sample sizes through power analysis

  • Account for biological and technical replicates in statistical models

  • Consider hierarchical statistical approaches for nested data structures

These approaches ensure reproducible quantification of CALB2 expression patterns in both normal tissues and pathological conditions, enabling reliable comparisons across experimental groups.

How do post-translational modifications affect CALB2 antibody recognition and experimental interpretation?

Post-translational modifications (PTMs) can significantly influence CALB2 antibody recognition and experimental interpretation:

• Calcium-binding status:

  • CALB2 undergoes conformational changes upon calcium binding

  • Some antibodies may preferentially recognize calcium-bound or calcium-free forms

  • Experimental conditions (fixatives, buffers) may alter calcium-binding status

• Phosphorylation:

  • Potential phosphorylation sites exist near calcium-binding domains

  • Phosphorylation could alter epitope accessibility or antibody affinity

  • Consider phosphatase treatment controls for variable results

• Proteolytic processing:

  • Partial degradation may generate fragments with altered antibody recognition

  • C-terminal or N-terminal epitopes may be differentially affected

  • Western blot analysis can help identify potential proteolytic fragments

• Experimental strategies:

  • Use multiple antibodies targeting different regions of CALB2

  • Compare native versus denaturing conditions to assess conformation-dependent recognition

  • Consider mass spectrometry approaches to identify specific PTMs affecting recognition

• Interpretation guidelines:

  • Document exact experimental conditions (fixation, buffers, calcium concentration)

  • Be cautious when comparing quantitative results across different protocols

  • Consider physiological state of tissue (activity levels, pathological conditions) when interpreting variability

Understanding these factors is crucial for accurate interpretation of CALB2 immunoreactivity patterns, particularly in comparative studies across different physiological or pathological states.