CIB1 Antibody

What is CIB1 and why are CIB1 antibodies important in research?

CIB1 is a 22 kDa calcium-binding protein that belongs to the calcium-binding protein family with significant homology to calmodulin and calcineurin B. It plays crucial roles in numerous cellular processes including cell differentiation, division, proliferation, migration, thrombosis, angiogenesis, cardiac hypertrophy, and apoptosis. CIB1 is expressed in platelets and binds to heterodimeric integrin αIIb/β3 (GPIIb/IIIa) as a potential intracellular regulatory molecule .

CIB1 antibodies are vital research tools because they enable scientists to:

• Detect and quantify CIB1 expression in different tissues

• Study CIB1's interactions with binding partners such as FAK and PAK1

• Investigate CIB1's role in various signaling pathways

• Examine changes in CIB1 localization under different cellular conditions

What are the main applications for CIB1 antibodies in laboratory research?

CIB1 antibodies have been successfully employed in multiple research applications:

The versatility of these applications makes CIB1 antibodies valuable for investigating both expression levels and functional aspects of this protein in different experimental contexts.

How can researchers verify the specificity of a CIB1 antibody?

To ensure antibody specificity, researchers should implement several validation approaches:

• Western blot analysis: Confirm a single band at the expected molecular weight (22 kDa) in tissues known to express CIB1, such as human kidney, spleen, or platelets .

• Knockout/knockdown controls: Compare antibody reactivity between wild-type samples and those where CIB1 has been depleted. For example, examining CIB1-deficient DT40 cells as done in studies of antibody gene diversification .

• Epitope mapping: Verify that the antibody recognizes the expected region. Some CIB1 antibodies specifically target the C-terminal half, while others may target different epitopes .

• Cross-reactivity assessment: Test reactivity across species if working with non-human models. Some antibodies recognize both human and mouse CIB1, while others may be species-specific .

• Immunoprecipitation followed by mass spectrometry: Confirm that the immunoprecipitated protein is indeed CIB1.

How can CIB1 antibodies be used to study protein-protein interactions in calcium signaling pathways?

CIB1 antibodies have proven invaluable for investigating calcium-dependent protein interactions through several sophisticated approaches:

• Co-immunoprecipitation with calcium manipulation: By varying calcium concentrations during immunoprecipitation experiments, researchers can determine whether CIB1's interactions with partners like ASK1 are calcium-dependent. Studies have shown that the binding of CIB1 to ASK1 was abolished by the Ca²⁺ ionophore ionomycin, suggesting calcium regulation of this interaction .

• Proximity ligation assays: These can detect in situ protein interactions when using CIB1 antibodies in combination with antibodies against suspected binding partners, providing spatial information about where these interactions occur within cells.

• Calcium chelation experiments: Comparing CIB1 immunoprecipitation results in the presence and absence of calcium chelators (like EGTA) can reveal which interactions require calcium. For example, research has demonstrated that Ca²⁺ binding to CIB1 alters its ability to modulate stress-induced signaling pathways .

• Structural immunology approaches: Using CIB1 antibodies that recognize specific conformational states can help determine how calcium binding affects CIB1's structure and subsequent protein interactions.

The choice of methodology should reflect whether you're investigating established or novel CIB1 binding partners in calcium signaling networks.

What approaches enable using CIB1 antibodies to investigate its role in cancer biology?

CIB1 has emerging significance in cancer research, and antibodies against this protein are being employed in increasingly sophisticated ways:

• Tissue microarray analysis: CIB1 antibodies have been used to analyze expression patterns across multiple tumor samples simultaneously. Immunohistochemistry studies have detected specific CIB1 staining in the cytoplasm of breast cancer cells .

• Xenograft tumor models: Research has employed CIB1 antibodies to monitor protein levels following CIB1 depletion in xenograft models, revealing that CIB1 inhibition induces triple-negative breast cancer (TNBC) cell death in culture and tumor regression in vivo .

• Signaling pathway dissection: CIB1 antibodies can help elucidate how this protein affects oncogenic pathways:

  • CIB1 supports PI3K/AKT and MEK/ERK pathways by directly modulating enzymes in these cascades

  • Detecting phosphorylation states of downstream effectors after CIB1 manipulation

• Combination with survival analysis: Correlating CIB1 expression levels (detected by antibodies) with patient outcomes and treatment responses.

When designing such studies, researchers should consider tissue-specific CIB1 expression patterns and potential isoforms that might affect antibody recognition.

How are CIB1 antibodies being utilized to study the protein's role in stress response pathways?

Advanced research has revealed CIB1's function as a Ca²⁺-sensitive modulator of stress-induced signaling:

• Apoptosis pathway mapping: CIB1 antibodies have helped demonstrate that CIB1 physically associates with ASK1 (apoptosis signal-regulating kinase 1), inhibiting its activity and affecting downstream stress-activated MAPK signaling pathways .

• Mechanistic studies: Immunoprecipitation experiments with CIB1 antibodies revealed that:

  • CIB1 interferes with the recruitment of TRAF2 to ASK1

  • CIB1 inhibits the autophosphorylation of ASK1 on threonine-838

  • CIB1 mitigates apoptotic cell death initiated by TNF-α or 6-hydroxydopamine

• Stress response induction experiments: Using CIB1 antibodies to track the protein's behavior during:

  • Oxidative stress (H₂O₂ treatment)

  • TNF-α stimulation

  • ER stress (tunicamycin treatment)

  • Antimicrotubule agent exposure (paclitaxel)

• Calcium influx modulation: Research has shown that Ca²⁺ influx induced by membrane depolarization reverses CIB1's inhibitory effect on stress-induced ASK1 activation and cell death in dopaminergic neurons .

These approaches highlight how CIB1 antibodies enable detailed investigation of stress response mechanisms.

What are the optimal conditions for using CIB1 antibodies in immunoprecipitation experiments?

Successful immunoprecipitation with CIB1 antibodies requires careful optimization:

• Antibody selection: Choose antibodies specifically validated for immunoprecipitation. For example, some studies have successfully used 5 μg/ml of CIB1 antibody for immunoprecipitation experiments .

• Lysis buffer considerations:

  • Include calcium (typically 1-2 mM) if studying calcium-dependent interactions

  • Consider mild detergents (0.5-1% NP-40 or Triton X-100) to preserve protein-protein interactions

  • Add protease and phosphatase inhibitors to prevent degradation

• Pre-clearing protocols: Pre-clear lysates with appropriate control IgG and protein A/G beads to reduce non-specific binding.

• Controls to implement:

  • IgG control immunoprecipitation

  • DNase/RNase treatment to confirm direct protein-protein interactions are not mediated by nucleic acids (as demonstrated in AID-CIB1 interaction studies)

  • CIB1-depleted lysates as negative controls

• Validation of interactions: Consider reciprocal co-IPs where both CIB1 and its binding partner are immunoprecipitated in separate experiments.

Many studies have successfully demonstrated CIB1 interactions with proteins like ASK1, integrin αIIb, and AID using carefully optimized immunoprecipitation conditions .

How should researchers approach epitope selection when choosing CIB1 antibodies for different applications?

The effectiveness of CIB1 antibodies varies by application based on epitope recognition:

• Epitope mapping considerations:

  • C-terminal antibodies: Some antibodies like clone 3C5 specifically recognize the C-terminal half of CIB1 , which may affect binding to partners that interact with this region

  • N-terminal antibodies: May be preferable when studying interactions involving CIB1's C-terminus

  • Conformational epitopes: Some antibodies may recognize calcium-bound versus calcium-free conformations

• Application-specific epitope selection:

  Application	Preferred Epitope Region	Rationale
  Western blot	Linear epitopes	Denatured protein exposes linear sequences
  IP	Surface-accessible epitopes	Need to bind native protein in solution
  IHC/ICC	Accessible in fixed tissues	Must penetrate and recognize in fixed context

• Protein interaction site awareness: CIB1 interacts with various partners through specific regions:

  • The N-terminal half contains two nonfunctional EF-hand-like motifs

  • The hydrophobic pocket contains residues (Ile73, Ile114, Leu131, Ile153, Ile168, Ile177) that contact the αIIb cytoplasmic domain

  • Choosing antibodies that don't interfere with binding sites of interest is crucial for interaction studies

• Validation across applications: An antibody that works well for Western blotting may not perform optimally for immunohistochemistry due to epitope accessibility differences.

Understanding the structural biology of CIB1 can guide more informed antibody selection for specific experimental questions.

What controls are essential when using CIB1 antibodies for localization studies?

When investigating CIB1 subcellular localization, implement these critical controls:

• Expression controls:

  • CIB1 knockdown/knockout samples to establish baseline and antibody specificity

  • Overexpression systems (tagged CIB1) to validate antibody recognition patterns

• Compartment markers: Co-stain with established subcellular markers to confirm localization:

  • Nuclear markers (DAPI, lamin)

  • Cytoplasmic markers (tubulin, actin)

  • Membrane markers (integrin subunits, Na⁺/K⁺ ATPase)

• Treatment-specific controls:

  • Calcium modulation: CIB1 localization may change with calcium levels

  • Cellular stress responses: H₂O₂ or TNF-α treatments have been shown not to affect the CIB1-ASK1 interaction extent

• Cross-validation techniques:

  • Subcellular fractionation followed by Western blotting

  • Live-cell imaging with fluorescently tagged CIB1

  • Electron microscopy with immunogold labeling

• Fixation method comparison: Different fixation protocols may affect epitope accessibility and apparent localization patterns.

Research has revealed that CIB1 can be detected in multiple cellular compartments, including the cytoplasm, plasma membrane, and in complex with nuclear proteins, highlighting the importance of rigorous localization controls .

How should researchers interpret varying CIB1 expression patterns across different tissue types?

Interpreting CIB1 expression variations requires systematic analysis:

• Baseline expression normalization: Establish reference expression levels across normal tissues:

  • CIB1 is ubiquitously expressed but may have tissue-specific expression patterns

  • Western blot data shows specific CIB1 detection in human kidney, spleen, and other tissues

• Standardized quantification approaches:

  • Use multiple housekeeping genes/proteins appropriate for each tissue type

  • Apply digital image analysis for immunohistochemistry quantification

  • Consider relative rather than absolute quantification when comparing across tissues

• Biological context considerations:

  • Correlate CIB1 levels with binding partners in each tissue

  • Consider tissue-specific functions (e.g., platelet function vs. neuronal signaling)

  • Assess calcium signaling demands of different tissues

• Pathological state comparisons: Several studies have examined CIB1 in disease contexts:

  • CIB1 expression in breast cancer tissue shows specific cytoplasmic localization

  • Altered CIB1 levels may correlate with disease progression

• Multi-method validation: Confirm expression patterns using complementary approaches (qPCR, proteomics, immunoblotting) to rule out antibody-specific artifacts.

Understanding tissue-specific CIB1 regulation provides valuable context for interpreting experimental results and identifying potential therapeutic opportunities.

How can researchers address contradictory results obtained with different CIB1 antibodies?

When faced with contradictory results from different CIB1 antibodies, implement this systematic approach:

• Epitope mapping and competition assays:

  • Determine exact binding sites of each antibody

  • Assess whether antibodies compete for binding or recognize distinct epitopes

  • Some CIB1 antibodies specifically target the C-terminal half, while others may recognize different regions

• Structural context analysis:

  • Consider whether protein modifications (phosphorylation, calcium binding) affect epitope recognition

  • The hydrophobic pocket and C-terminal helix (H10) of CIB1 undergo conformational changes that might affect antibody binding

• Validation in knockout/knockdown systems:

  • Test all antibodies against CIB1-depleted samples

  • Reintroduce CIB1 expression to confirm specificity

  • CIB1-deficient DT40 cells have been used for such validation

• Protocol optimization comparison:

  Potential Issue	Troubleshooting Approach
  Fixation artifacts	Test multiple fixation protocols
  Blocking inefficiency	Optimize blocking conditions
  Antibody concentration	Perform titration experiments
  Detection system	Compare different visualization methods

• Independent confirmation: Use non-antibody-based methods (mass spectrometry, RNA analysis) to resolve discrepancies.

Studies have demonstrated that seemingly contradictory results can occur due to context-dependent CIB1 functions rather than antibody issues, such as CIB1's dual role in both activating and inhibiting integrin αIIb .

What statistical approaches are recommended when quantifying CIB1 levels across experimental conditions?

Robust statistical analysis of CIB1 expression data requires:

• Appropriate normalization strategies:

  • Use multiple reference proteins when possible

  • Apply normalization formulas that account for loading variations:

    • For Western blot analysis: [(CIB1_ZT - b_ZT)/(C_ZT - b_ZT)]/[CIB1_ZT0W - b_ZT0W)/(C_ZT0W - b_ZT0W)]

    • Where ZT represents time points, b represents background signal, and C represents loading control

• Statistical test selection:

  • For comparing two conditions: paired t-tests for within-sample comparisons

  • For multiple conditions: ANOVA with appropriate post-hoc tests

  • For non-normally distributed data: Non-parametric alternatives

• Experimental design considerations:

  • Include sufficient biological replicates (minimum n=3, preferably more)

  • Account for batch effects in multi-day experiments

  • Consider power analysis to determine required sample sizes

• Visualization approaches:

  • Use box plots to show distribution of data points

  • Include individual data points alongside means/medians

  • Clearly indicate statistical significance and P-values

• Correlation analyses: When examining CIB1 in relation to binding partners or functional outcomes, apply correlation statistics (Pearson's or Spearman's) based on data distribution.

Studies tracking CIB1 levels in response to treatments or across time points benefit particularly from careful statistical design and analysis .

What are the most effective approaches for resolving weak or non-specific CIB1 antibody signals?

When encountering weak or non-specific signals with CIB1 antibodies, consider these methodological solutions:

• Antibody optimization strategies:

  Issue	Solution Approach
  Weak signal	Increase antibody concentration, extend incubation time, enhance detection system sensitivity
  High background	Optimize blocking (BSA vs. milk), increase washing stringency, reduce antibody concentration
  Multiple bands	Use gradient gels, optimize sample preparation, consider protein modifications or degradation

• Sample preparation refinements:

  • Include phosphatase inhibitors to preserve modification states

  • Optimize protein extraction based on CIB1's subcellular localization

  • Consider native vs. denaturing conditions based on application

  • Fresh vs. frozen sample comparison

• Protocol modification considerations:

  • For Western blotting: Test different membrane types (PVDF vs. nitrocellulose)

  • For IHC/ICC: Compare different antigen retrieval methods (as used for CIB1 detection in breast cancer tissue)

  • For IP: Test different lysis buffers and immunoprecipitation techniques

• Epitope accessibility enhancement:

  • For fixed samples: Optimize permeabilization conditions

  • For membrane proteins: Consider mild detergents to expose epitopes

• Positive control inclusion: Always run samples known to express CIB1 (e.g., platelets, human kidney tissue) alongside experimental samples.

Publications have shown successful CIB1 detection using specific conditions, such as reducing conditions for Western blotting and heat-induced epitope retrieval for IHC .

How can researchers determine if their experimental conditions are affecting CIB1 antibody recognition?

Experimental conditions can significantly impact CIB1 antibody performance:

• Calcium-dependent recognition assessment:

  • Compare samples with and without calcium chelators (EGTA/EDTA)

  • Test fixed vs. calcium-variable conditions

  • CIB1's conformation changes with calcium binding, potentially affecting epitope accessibility

• Treatment effect evaluation:

  • Include untreated controls alongside experimental conditions

  • Consider whether treatments (H₂O₂, TNF-α) might alter post-translational modifications

  • Research has shown that H₂O₂ or TNF-α treatments do not affect the extent of the CIB1-ASK1 interaction

• Protein-protein interaction impact:

  • Test whether protein binding partners might mask antibody epitopes

  • CIB1 interacts with many partners including ASK1, integrin αIIb, and others that could affect recognition

• Fixation/extraction method comparison:

  Method	Potential Impact on CIB1 Recognition
  Paraformaldehyde	May preserve structure but cross-link epitopes
  Methanol	Denatures proteins but may improve some epitope access
  Triton X-100	Solubilizes membranes, potentially releasing membrane-associated CIB1

• Recombinant protein controls: Use purified CIB1 protein treated with experimental conditions to isolate direct effects on antibody recognition.

Understanding how experimental manipulations affect CIB1 structure and interactions is crucial for accurate interpretation of antibody-based data .

What approaches can resolve discrepancies between CIB1 protein detection and functional studies?

When protein detection and functional results don't align, consider these resolution strategies:

• Activity vs. abundance distinction:

  • CIB1 may be present but functionally inactive (or vice versa)

  • Post-translational modifications may alter function without changing detection

  • Calcium binding states may affect function but not antibody recognition

• Localization-specific function assessment:

  • CIB1 has different roles in different cellular compartments

  • Combine fractionation with functional assays

  • CIB1 interacts with proteins in multiple locations: plasma membrane (integrins), cytoplasm (ASK1), and nucleus (AID)

• Binding partner competition analysis:

  • Test if experimental conditions affect CIB1's interaction with key partners

  • CIB1 competes with TRAF2 for binding to ASK1, affecting downstream signaling

• Functional redundancy evaluation:

  • Research has shown that CIB1 is not required for some processes despite physical interaction

  • Studies demonstrated CIB1 is nonessential for antibody gene diversification despite interacting with AID

• Temporal dynamics consideration:

  • Assess whether time-dependent changes in CIB1 function occur that might be missed in endpoint assays

  • Design time-course experiments to capture transient effects

When interpreting such discrepancies, remember that CIB1 has multifaceted roles, and some interactions may be context-dependent rather than constitutive .