CALB2 Antibody

What is CALB2 and why is it important in research?

CALB2 (Calbindin 2), also known as Calretinin, is a calcium-binding protein encoded by the CALB2 gene. It is a 271 amino acid protein with a molecular mass of approximately 31,540 daltons and belongs to the Calbindin family. Calretinin contains 6 EF-hand domains which bind to calcium and functions as a vitamin D-dependent calcium-binding protein involved in calcium signaling pathways .

The protein is particularly important in research for several reasons:

• It serves as a neuronal marker, being present in subsets of neurons throughout the brain and spinal cord, including sensory ganglia

• It functions as a documented cancer marker for differential diagnosis

• It plays a role in calcium-dependent cellular processes

• Recent studies have implicated it in drug sensitivity mechanisms, particularly in cancer treatment contexts

How do I select the appropriate CALB2 antibody for my specific experimental needs?

When selecting a CALB2 antibody, researchers should consider multiple factors based on their experimental requirements:

Application compatibility: Different antibodies are optimized for specific applications such as Western Blot (WB), Immunohistochemistry (IHC), Immunocytochemistry (ICC), Immunofluorescence (IF), or ELISA. Review the validated applications listed by manufacturers to ensure compatibility with your methodology .

Species reactivity: Verify that the antibody recognizes CALB2 in your species of interest. Many antibodies are validated for human (Hu), mouse (Ms), and rat (Rt) samples, but reactivity with other species varies considerably .

Clonality considerations:

• Monoclonal antibodies offer high specificity and reproducibility for a single epitope

• Polyclonal antibodies recognize multiple epitopes, potentially providing stronger signals but with possible increased background

Detection method: Consider whether you need unconjugated antibodies or those directly conjugated to fluorophores, enzymes, or other tags based on your detection system .

For fluorescence applications, consider the spectral properties of conjugated fluorophores:

Note that blue fluorescent dyes (e.g., CF®405S) may not be optimal for low-abundance targets due to lower fluorescence intensity and potentially higher background .

What are the primary research applications of CALB2 antibodies?

CALB2 antibodies have several important research applications:

Diagnostic pathology: CALB2 antibodies are valuable tools for differentiating mesothelioma from adenocarcinomas of the lung and for distinguishing adrenal cortical neoplasms from pheochromocytomas .

Neuroscience research: Given the expression of CALB2 in specific neuronal populations, these antibodies are useful for characterizing neuronal subtypes and studying calcium-dependent neuronal processes.

Cancer research: CALB2 has been identified as a potential regulator of cancer drug sensitivity, particularly in colorectal cancer response to 5-Fluorouracil (5-FU) .

Developmental biology: CALB2 antibodies can be used to study the development of specific neuronal populations during embryogenesis.

Calcium signaling studies: As a calcium-binding protein, CALB2 antibodies are useful in investigating calcium homeostasis and signaling pathways.

Each application requires specific optimization of antibody dilution, incubation conditions, and detection methods to achieve optimal results while minimizing background signal.

What are the recommended protocols for using CALB2 antibodies in Western blot applications?

Sample preparation for Western blot:

• Extract proteins from your samples using an appropriate lysis buffer containing protease inhibitors

• Determine protein concentration using Bradford or BCA assay

• Prepare samples containing 20-50 μg of total protein in loading buffer with reducing agent

• Heat samples at 95°C for 5 minutes

Western blot procedure:

• Separate proteins using SDS-PAGE (10-12% gel recommended for CALB2 detection)

• Transfer proteins to PVDF or nitrocellulose membrane

• Block membrane with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Incubate with primary CALB2 antibody at 1:1000 dilution overnight at 4°C

• Wash membrane with TBST (3 × 5-10 minutes)

• Incubate with appropriate HRP-conjugated secondary antibody (e.g., HRP-conjugated Goat Anti-Rabbit IgG) at 1:2000 dilution for 1 hour at room temperature

• Wash membrane with TBST (3 × 5-10 minutes)

• Apply ECL substrate and capture chemiluminescent signal

Critical considerations:

• Include positive controls (tissues/cells known to express CALB2)

• Use beta-actin (ACTB) or similar loading controls

• Expected molecular weight for CALB2 is approximately 31.5 kDa

• Optimize antibody concentration if signal strength or background is suboptimal

This protocol has been successfully used to detect CALB2 in various samples, with optimal results achieved when using freshly prepared samples .

How should I optimize immunohistochemistry protocols for CALB2 detection in tissue samples?

Immunohistochemistry protocol for CALB2 detection:

• Tissue preparation:

  • Fix tissues in 10% neutral buffered formalin

  • Process and embed in paraffin

  • Section at 4-5 μm thickness

  • Mount on positively charged slides

• Deparaffinization and rehydration:

  • Xylene: 2 × 5 minutes

  • 100% ethanol: 2 × 3 minutes

  • 95% ethanol: 1 × 3 minutes

  • 70% ethanol: 1 × 3 minutes

  • Wash in PBS: 3 × 5 minutes

• Antigen retrieval (critical step):

  • Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) for 10 minutes

  • Allow to cool to room temperature

  • Wash with PBS: 2 × 10 minutes

• Peroxidase blocking:

  • Incubate sections with 3% hydrogen peroxide (H₂O₂) for 10 minutes

  • Wash with PBS: 3 × 5 minutes

• Blocking:

  • Incubate with normal goat serum for 30 minutes at room temperature

• Primary antibody incubation:

  • Apply CALB2 polyclonal antibody at 1:100 dilution

  • Incubate overnight at 4°C

  • For negative control, substitute primary antibody with PBS

• Secondary antibody:

  • Wash with PBS: 3 × 5 minutes

  • Incubate with biotin-labeled goat anti-rabbit IgG (1:1000) for 30 minutes

• Signal amplification:

  • Apply streptavidin-biotin complex (SABC) for 30 minutes

  • Wash with PBS: 3 × 10 minutes

• Visualization:

  • Develop with DAB substrate

  • Monitor color development under microscope

  • Stop reaction by rinsing with water for 5-10 minutes

• Counterstaining and mounting:

  • Counterstain with hematoxylin

  • Dehydrate, clear, and mount with permanent mounting medium

Optimization considerations:

• Antibody dilution should be empirically determined for each tissue type

• Antigen retrieval methods may need adjustment (try EDTA buffer pH 9.0 as alternative)

• Incubation times may need optimization based on tissue type and fixation conditions

• For multiplex IHC, careful selection of primary antibodies from different host species is essential

What controls should I include when using CALB2 antibodies in my experiments?

Proper controls are essential for validating results with CALB2 antibodies:

Positive tissue controls:

• Brain tissue (cerebellar granule cells)

• Mesothelioma samples

• Other tissues known to express CALB2

Positive cell line controls:

• HCT116 colorectal cancer cells (known to express CALB2)

• Neuronal cell lines with documented CALB2 expression

Negative controls:

• Antibody diluent only (omit primary antibody)

• Isotype control (irrelevant antibody of same isotype)

• Tissues/cells known not to express CALB2

Knockdown/knockout controls:

• siRNA-mediated CALB2 silencing to confirm antibody specificity

• CRISPR/Cas9 knockout cell lines where available

Peptide blocking:

• Pre-incubation of antibody with CALB2 peptide to verify specificity

• Should abolish or significantly reduce positive staining

Loading/processing controls:

• Housekeeping proteins (ACTB, GAPDH) for Western blots

• Tissue processing controls to ensure technique consistency

The inclusion of these controls helps validate antibody specificity, confirms technical success, and provides confidence in the interpretation of experimental results.

How can CALB2 antibodies be used to study cancer drug sensitivity mechanisms?

Recent research has revealed that CALB2 plays an important role in cancer drug sensitivity, particularly in colorectal cancer response to 5-Fluorouracil (5-FU). CALB2 antibodies can be instrumental in elucidating these mechanisms:

Monitoring CALB2 expression changes:

• Western blot and immunohistochemistry with CALB2 antibodies can track expression changes in response to drug treatment

• Research has shown that CALB2 mRNA and protein expression are down-regulated in p53 wild-type and p53 null isogenic HCT116 colorectal cancer cell lines following 48h and 72h 5-FU treatment

Subcellular localization studies:

• Immunofluorescence with CALB2 antibodies can reveal translocation events

• Following 5-FU treatment, CALB2 translocates to the mitochondria, suggesting involvement in the intrinsic apoptotic pathway

Mechanistic investigations:

• Combine CALB2 antibodies with other markers of apoptosis (cytochrome c, smac)

• Research demonstrates that CALB2 silencing decreases 5-FU-induced cytochrome c and smac release from mitochondria and reduces caspase 9 and 3/7 activation

Experimental approach:

• Treat cancer cells with drug of interest (e.g., 5-FU)

• Harvest cells at various timepoints

• Perform protein fractionation to separate mitochondrial and cytosolic components

• Use CALB2 antibodies for Western blot analysis of each fraction

• Correlate CALB2 localization with apoptotic markers and cellular outcomes

This approach has revealed that CALB2 modulation significantly impacts drug sensitivity, with CALB2 silencing conferring resistance to 5-FU through impaired mitochondrial apoptosis pathway activation .

What are the optimal approaches for quantifying CALB2 expression levels in different experimental systems?

Accurate quantification of CALB2 expression requires selection of appropriate methods based on experimental goals:

Protein-level quantification:

• Western blot densitometry:

  • Separate proteins by SDS-PAGE and transfer to membrane

  • Probe with CALB2 antibody and housekeeping protein control

  • Capture images using a digital imaging system (e.g., ImageQuantTM LAS 4000)

  • Use densitometry software to normalize CALB2 band intensity to loading control

  • Include calibration standards when absolute quantification is needed

• Flow cytometry:

  • Fix and permeabilize cells

  • Stain with fluorophore-conjugated CALB2 antibody

  • Analyze signal intensity distribution across cell population

  • Include isotype control to set negative gates

  • Particularly useful for heterogeneous populations

• ELISA:

  • Use sandwich ELISA with capture and detection CALB2 antibodies

  • Generate standard curve with recombinant CALB2 protein

  • Interpolate unknown sample concentrations

  • Provides absolute quantification in ng/ml or similar units

mRNA-level quantification:

• Quantitative RT-PCR:

  • Extract total RNA from samples

  • Perform reverse transcription to generate cDNA

  • Use CALB2-specific primers for qPCR

  • Normalize to appropriate reference genes

  • Calculate relative expression using 2^(-ΔΔCt) method

• RNA-Seq:

  • Perform RNA sequencing

  • Map reads to reference genome

  • Quantify CALB2 transcript abundance as TPM or FPKM

  • Use for genome-wide expression context

Single-cell analysis:

• Single-cell RNA-Seq for transcriptional heterogeneity

• Imaging mass cytometry with CALB2 antibodies for spatial context

• Multiplex immunofluorescence for co-expression patterns

Comparative sensitivity of detection methods:

Selection of the appropriate quantification method depends on required sensitivity, sample type, and whether cellular localization information is needed.

How can I effectively use CALB2 antibodies in multiplex immunofluorescence studies?

Multiplex immunofluorescence with CALB2 antibodies requires careful planning to avoid spectral overlap and antibody cross-reactivity:

Protocol optimization:

• Antibody panel design:

  • Select CALB2 antibody conjugated to appropriate fluorophore based on microscope capabilities

  • Choose additional antibodies from different host species

  • Ensure spectral separation between fluorophores

  • Consider signal intensity of each target (pair abundant targets with dimmer fluorophores)

• Sequential staining approach:

  • For primary antibodies from the same species, use tyramide signal amplification (TSA)

  • Apply first primary antibody, detect with HRP-conjugated secondary

  • Develop with TSA-fluorophore

  • Perform heat-mediated stripping of antibodies but not fluorophores

  • Repeat with next primary antibody

  • This allows use of multiple antibodies from the same species

• Direct conjugate approach:

  • Use directly conjugated primary antibodies

  • CALB2 antibodies are available with various CF® dye conjugations

  • Reduces protocol complexity and background

Fluorophore selection considerations:

Sample preparation considerations:

• Optimize fixation to preserve antigenicity while maintaining morphology

• For formalin-fixed tissues, test multiple antigen retrieval methods

• Consider tissue autofluorescence quenching (Sudan Black B or commercial quenchers)

• Include single-stained controls for compensation/spectral unmixing

Analysis approaches:

• Use multispectral imaging systems for improved spectral separation

• Apply automated cell segmentation and phenotyping algorithms

• Quantify co-localization using Pearson's or Mander's coefficients

• Consider spatial distribution analyses (nearest neighbor, etc.)

This approach enables simultaneous visualization of CALB2 with other proteins of interest, providing insights into co-expression patterns and subcellular localization in complex tissues.

What are common technical issues when working with CALB2 antibodies and how can they be resolved?

Researchers frequently encounter several technical challenges when using CALB2 antibodies. Here are solutions to common problems:

High background in immunohistochemistry:

• Increase blocking time/concentration (try 5% BSA or 10% normal serum)

• Reduce primary antibody concentration (try 1:200 instead of 1:100)

• Include 0.1-0.3% Triton X-100 in wash buffers

• Ensure complete deparaffinization of FFPE sections

• Try different antigen retrieval methods (citrate pH 6.0 vs. EDTA pH 9.0)

• Include 0.3% H₂O₂ treatment to block endogenous peroxidases

Weak or no signal in Western blot:

• Increase protein loading (50-100 μg total protein)

• Reduce transfer time/voltage for small proteins like CALB2

• Try different membrane types (PVDF often better than nitrocellulose for small proteins)

• Extend primary antibody incubation (overnight at 4°C)

• Use signal enhancement systems (biotin-streptavidin amplification)

• Verify sample preparation (include protease inhibitors)

• Check that CALB2 is expressed in your experimental system

Non-specific bands in Western blot:

• Increase blocking time/concentration

• Optimize antibody dilution (typically 1:1000)

• Include 0.1% Tween-20 in all buffers

• Perform peptide competition assay to identify specific band

• Try monoclonal antibody if using polyclonal

Inconsistent immunofluorescence results:

• Optimize fixation conditions (4% PFA for 10-15 minutes for cells)

• Include permeabilization step (0.1-0.3% Triton X-100 for 5-10 minutes)

• Use mounting media with anti-fade to prevent photobleaching

• Avoid blue fluorescent conjugates for low abundance targets due to higher background

Tissue-specific optimization:

• Different tissues may require specific conditions

• For brain tissue, perfusion fixation often yields better results

• For gonads, optimize fixation time to preserve morphology

• Epitope accessibility may vary between species requiring protocol adjustments

Antibody validation approaches:

• Test antibody on known positive and negative controls

• Verify results with multiple antibodies targeting different epitopes

• Use siRNA knockdown to confirm specificity

• Include recombinant protein standards where possible

Implementing these troubleshooting strategies will help ensure reliable and reproducible results when working with CALB2 antibodies.

How do I properly interpret changes in CALB2 expression in response to experimental treatments?

Interpreting changes in CALB2 expression requires careful consideration of multiple factors:

Establish proper baseline and controls:

• Include time-matched untreated controls, as CALB2 expression can change over time naturally

• Use appropriate normalization (housekeeping genes/proteins) to account for loading differences

• Consider cell density effects on baseline expression

Quantitative analysis approaches:

• Use at least three biological replicates for statistical validity

• Apply appropriate statistical tests (t-test for two conditions, ANOVA for multiple conditions)

• Report both magnitude (fold change) and statistical significance (p-value)

• Consider both mRNA and protein levels, as they may not correlate perfectly

Time-course considerations:

• CALB2 expression changes can be time-dependent

• In drug treatment studies, perform time-course experiments (e.g., 24h, 48h, 72h post-treatment)

• Distinguish between early regulatory events and later compensatory responses

Context-dependent interpretation:

• Down-regulation of CALB2 following 5-FU treatment in colorectal cancer cells has been observed, contrary to initial expectations

• This highlights the importance of time-matched controls and careful experimental design

• Consider both expression level and subcellular localization changes

Functional validation:

• Gene silencing experiments can reveal the functional importance of observed changes

• In colorectal cancer cells, CALB2 silencing reduced 5-FU-induced apoptosis, indicating its role in drug response

• Consider rescue experiments to confirm specificity of observed effects

Multi-omics integration:

• Correlate protein expression with mRNA levels

• Consider post-translational modifications that may affect function without changing expression

• Examine downstream pathway activation/inhibition

By following these guidelines, researchers can properly interpret CALB2 expression changes and their biological significance in various experimental contexts.

What are the critical considerations for studying CALB2 localization and translocation in response to stimuli?

CALB2 localization and translocation studies require specialized approaches to capture the dynamic nature of this protein:

Subcellular fractionation approach:

• Prepare cytosolic, nuclear, and mitochondrial fractions using differential centrifugation

• Verify fraction purity using marker proteins (e.g., GAPDH for cytosol, COX IV for mitochondria)

• Analyze CALB2 distribution by Western blot across fractions

• Compare treated vs. untreated samples to detect translocation

• Normalize to fraction-specific loading controls

This approach revealed that CALB2 translocates to mitochondria following 5-FU treatment in colorectal cancer cells .

Live-cell imaging considerations:

• Create fluorescent protein-tagged CALB2 constructs (e.g., GFP-CALB2)

• Verify that tagging doesn't interfere with localization using antibody validation

• Use spinning disk or light sheet microscopy for reduced phototoxicity

• Capture images at appropriate intervals to track dynamic changes

• Include co-localization markers for specific organelles

Immunofluorescence approach for fixed cells/tissues:

• Fix samples at various timepoints after treatment

• Perform immunofluorescence with CALB2 antibody

• Co-stain with organelle markers:

  • Mitochondria: MitoTracker or TOMM20 antibody

  • Nucleus: DAPI or Hoechst

  • ER: Calnexin antibody

  • Golgi: GM130 antibody

• Analyze co-localization using confocal microscopy

• Quantify using co-localization coefficients or intensity correlation

Critical analysis parameters:

Functional correlation:

• Combine localization studies with functional assays

• For mitochondrial translocation, measure parameters like membrane potential (Δψm)

• Determine if translocation correlates with downstream events (e.g., cytochrome c release)

• Research has shown that 5-FU-induced loss of mitochondrial membrane potential is abrogated in CALB2-silenced cells

Advanced approaches:

• Proximity labeling techniques (BioID, APEX) to identify proteins near CALB2

• FRET-based assays to detect direct interactions with binding partners

• Super-resolution microscopy (STORM, PALM) for precise localization

These approaches collectively provide comprehensive insights into the dynamic localization patterns of CALB2 and their functional significance in response to various stimuli.

How is CALB2 research contributing to our understanding of cancer biology and treatment resistance?

Recent studies have revealed significant roles for CALB2 in cancer biology and treatment response mechanisms:

CALB2 in drug response pathways:

• CALB2 has been identified as a regulator of 5-Fluorouracil (5-FU) sensitivity in colorectal cancer

• Down-regulation of CALB2 mRNA and protein occurs following 5-FU treatment in both p53 wild-type and p53 null isogenic HCT116 colorectal cancer cell lines

• CALB2 silencing significantly reduces 5-FU-induced apoptosis in multiple colorectal cancer cell lines, indicating its functional importance in drug response

Mechanistic insights:

• Following 5-FU treatment, CALB2 translocates to mitochondria

• This translocation appears critical for maintaining mitochondrial membrane potential during drug treatment

• CALB2 facilitates cytochrome c and smac release from mitochondria

• CALB2 silencing decreases 5-FU-induced activation of caspases 9 and 3/7

• Co-silencing of XIAP can overcome 5-FU resistance in CALB2-silenced cells, suggesting potential combination therapy approaches

Clinical correlations:

• Analysis of public microarray datasets has revealed associations between CALB2 expression and clinical outcomes

• CALB2 expression patterns vary across different cancer types and stages

• The protein serves as a diagnostic marker in distinguishing mesothelioma from adenocarcinomas

Future research directions:

• Investigation of CALB2's role in resistance to other chemotherapeutic agents

• Exploration of CALB2 as a predictive biomarker for treatment response

• Development of strategies to modulate CALB2 activity to enhance drug sensitivity

• Characterization of CALB2 interaction partners in cancer-specific contexts

These findings collectively suggest that CALB2 functions as an important mediator of cancer drug response through the intrinsic mitochondrial apoptotic pathway, and its down-regulation may represent an intrinsic mechanism of resistance to anticancer treatments.

What novel techniques are being developed for studying CALB2 function in complex biological systems?

Research into CALB2 function is benefiting from several cutting-edge methodological approaches:

CRISPR-based functional genomics:

• CRISPR/Cas9 knockout models for precise elimination of CALB2

• CRISPR interference (CRISPRi) for tunable repression of CALB2 expression

• CRISPR activation (CRISPRa) for controlled upregulation

• CRISPR base editing for introducing specific mutations to study structure-function relationships

• These approaches allow more precise manipulation than traditional siRNA methods

Advanced imaging technologies:

• Super-resolution microscopy (STORM, PALM, STED) for nanoscale localization

• Lattice light-sheet microscopy for long-term live imaging with reduced phototoxicity

• Expansion microscopy for physical magnification of specimens

• Correlative light and electron microscopy (CLEM) to combine functional and ultrastructural information

• These methods provide unprecedented spatial resolution for studying CALB2 localization

Proximity labeling proteomics:

• BioID or TurboID fusion proteins to identify proximal interacting partners

• APEX2-based proximity labeling for temporal resolution of interactions

• Split-BioID for detecting conditional interactions

• These approaches identify context-specific protein interactions in living cells

Single-cell technologies:

• Single-cell RNA-seq to reveal cell-to-cell variation in CALB2 expression

• Single-cell proteomics for protein-level heterogeneity assessment

• Spatial transcriptomics to map CALB2 expression in tissue context

• CODEX or Imaging Mass Cytometry for multiplexed protein detection in tissues

• These methods address cellular heterogeneity often masked in bulk analyses

Organoid and patient-derived models:

• Patient-derived organoids for studying CALB2 in near-physiological contexts

• Organ-on-chip platforms incorporating multiple cell types

• These systems better recapitulate in vivo complexity than traditional cell lines

Computational approaches:

• Molecular dynamics simulations of CALB2 structure and calcium binding

• Network analysis to position CALB2 in broader signaling contexts

• AI-based image analysis for quantitative assessment of localization patterns

These innovative techniques are expanding our understanding of CALB2 beyond traditional methods, enabling researchers to address previously intractable questions about its roles in complex biological processes and disease states.