KIDINS220 Antibody

What is KIDINS220 and what cellular functions is it involved in?

KIDINS220, also known as Ankyrin Repeat-rich Membrane Spanning (ARMS) protein, functions as a transmembrane scaffold protein implicated in multiple cellular processes. Originally identified in neuronal cells, KIDINS220 plays crucial roles in:

• B cell antigen receptor (BCR) signaling as a novel interaction partner

• Regulation of B cell development, particularly cells expressing λ light chain

• Enhancement of developing B cell survival

• Pre-BCR and BCR signaling to facilitate λ light chain locus opening and gene recombination

• Neural stem cell survival in adult neurogenic niches through AKT pathway regulation

• Setting molecular thresholds for neural stem cell responsiveness to growth factors

The protein was first characterized in neuronal tissues but has since been identified as a multifunctional regulator across various cell types. Mutations in the KIDINS220 gene have been associated with several neurological disorders, including schizophrenia and SINO syndrome (spastic paraplegia, intellectual disability, nystagmus, and obesity) .

What types of KIDINS220 antibodies are available for research applications?

Multiple KIDINS220 antibodies are available targeting different regions of the protein:

Most commercially available antibodies are rabbit-derived polyclonals, with some targeting specific amino acid sequences or post-translational modifications like phosphorylation at Ser918 .

How should researchers select the appropriate KIDINS220 antibody for their experiments?

Selection should be guided by the specific research question, target species, and application requirements. Consider the following factors:

• Experimental application: Different antibodies perform optimally in specific applications (WB, IF, IHC, IP). Review validation data for your intended application.

• Region specificity: Choose antibodies targeting internal regions for total KIDINS220 detection or specific domains for studying structure-function relationships.

• Post-translational modifications: For signaling studies, phospho-specific antibodies (e.g., pSer918) can provide valuable insights into activation states.

• Species cross-reactivity: Verify reactivity with your experimental model organism. Most available antibodies react with human, mouse, and rat KIDINS220 .

• Validation evidence: Review literature where the antibody has been successfully used in similar experimental contexts.

When investigating KIDINS220's role in B cell development, antibodies detecting total KIDINS220 in mouse tissues would be appropriate for tracking expression patterns during developmental stages .

What are the best practices for using KIDINS220 antibodies in Western blot analysis?

Western blotting for KIDINS220 requires specific considerations due to its high molecular weight (~220 kDa):

• Sample preparation:

  • Use fresh tissue/cells and include protease inhibitors

  • For membrane proteins like KIDINS220, consider specialized lysis buffers containing 1% Triton X-100 or NP-40

  • Heat samples at 70°C instead of 95°C to prevent aggregation of large proteins

• Gel electrophoresis:

  • Use low percentage (6-8%) or gradient gels to resolve high molecular weight proteins

  • Extend running time at lower voltage (80-100V) for better separation

• Transfer conditions:

  • Employ wet transfer systems for large proteins

  • Use 0.2 μm PVDF membranes (rather than 0.45 μm)

  • Consider extended transfer times (overnight at 30V at 4°C)

• Antibody conditions:

  • Optimal dilution range: typically 1:500-1:1000 for commercial KIDINS220 antibodies

  • Include BSA (3-5%) in blocking and antibody solutions to reduce background

  • Consider extended primary antibody incubation (overnight at 4°C)

• Controls:

  • Include KIDINS220 knockout or knockdown samples as negative controls

  • Use brain tissue lysate as a positive control (high KIDINS220 expression)

  • Consider using an antibody targeting a different epitope to confirm specificity

Studies examining KIDINS220's role in B cell development have successfully employed Western blotting to confirm deletion efficiency in conditional knockout models by comparing protein levels in control versus knockout tissues .

What controls are recommended when using KIDINS220 antibodies for immunostaining?

When performing immunohistochemistry (IHC) or immunofluorescence (IF) with KIDINS220 antibodies, appropriate controls are critical:

• Primary antibody controls:

  • Negative control: Omit primary antibody but include all other steps

  • Isotype control: Use matched concentration of non-specific IgG from same species

  • Absorption control: Pre-incubate antibody with immunizing peptide

  • Genetic control: Use tissues/cells from KIDINS220 knockout models

• Sample-specific controls:

  • Positive control tissue: Include samples known to express KIDINS220 (brain tissue, B cell-rich tissues)

  • Developmental controls: For B cell studies, include samples from different developmental stages

• Multi-color staining controls:

  • Single-color controls: Run each fluorophore separately to assess bleed-through

  • Co-localization validation: Include markers with known relation to KIDINS220

• Signal validation approaches:

  • Confirm specificity with a second antibody targeting a different epitope

  • Verify expression pattern correlates with known distribution (e.g., KIDINS220 in B cell subpopulations or neuronal tissues)

Research examining KIDINS220's role in B cells successfully used immunostaining to visualize its expression in specific cell populations, particularly in bone marrow and SGZ (subgranular zone) neurogenic niches .

How can KIDINS220 antibodies be used to study B cell development and receptor editing?

KIDINS220 has been identified as a critical regulator of B cells bearing the λ light chain. Researchers can employ multiple antibody-based approaches to investigate this regulatory role:

• Developmental stage analysis:

  • Use flow cytometry with KIDINS220 antibodies alongside developmental markers (B220, CD19, IgM, etc.)

  • Track KIDINS220 expression through B cell maturation stages: pro-B, pre-B, immature B, and mature B cells

  • Correlate expression with κ versus λ light chain expression

• Receptor editing investigation:

  • Employ KIDINS220 antibodies in combination with autoreactive BCR models

  • Monitor receptor editing through simultaneous detection of RAG proteins, KIDINS220, and light chain expression

  • Investigate KIDINS220's role in extending B cell survival during receptor editing

• Mechanistic studies:

  • Use phospho-specific antibodies to track KIDINS220-dependent signaling during B cell selection

  • Combine with proximity ligation assays to visualize KIDINS220-BCR interactions

  • Track chromatin accessibility at the Igλ locus in relation to KIDINS220 expression

Research has demonstrated that KIDINS220 knockout B cells fail to open and recombine genes of the Igλ locus, even under conditions where Igκ genes cannot be rearranged or where κLC confers autoreactivity. This suggests KIDINS220 plays a critical role in extending the developmental window for λ light chain gene rearrangement .

What approaches exist to study KIDINS220 interactions with the B cell antigen receptor?

Several antibody-based techniques can investigate the physical and functional relationship between KIDINS220 and BCR:

• Co-immunoprecipitation (Co-IP):

  • Use anti-KIDINS220 antibodies to pull down protein complexes and blot for BCR components

  • Alternatively, use anti-BCR antibodies and probe for KIDINS220

  • Compare interaction under resting versus activated conditions

• Proximity-based methods:

  • Proximity ligation assay (PLA) to visualize KIDINS220-BCR interactions in situ

  • FRET/FLIM using fluorescently-tagged antibodies to measure distance between proteins

  • BioID or APEX2 proximity labeling with KIDINS220 as the bait protein

• Functional interaction studies:

  • Combine KIDINS220 antibody staining with calcium flux assays

  • Phospho-flow analysis of BCR signaling components in presence/absence of KIDINS220

  • Analyze BCR clustering and internalization in relation to KIDINS220 localization

• Domain-specific interactions:

  • Use antibodies targeting specific domains of KIDINS220 to map interaction regions

  • Competitive binding assays with domain-specific antibodies or peptides

Research has identified KIDINS220 as an interaction partner of the BCR, demonstrating that it plays a crucial role in optimal pre-BCR and BCR signaling required for proper B cell development, particularly for cells expressing the λ light chain .

How can researchers address non-specific binding when using KIDINS220 antibodies?

Non-specific binding is a common challenge when working with KIDINS220 antibodies. Here are methodological approaches to minimize and address this issue:

• Antibody optimization:

  • Titrate antibody concentrations (typically 1:500-1:2000 for Western blots)

  • Optimize blocking conditions (5% BSA often performs better than milk for phospho-epitopes)

  • Test different incubation times and temperatures

  • For IF/IHC, consider antigen retrieval optimization

• Cross-reactivity reduction:

  • Pre-absorb antibody with tissues/lysates from KIDINS220 knockout models

  • Use more stringent washing conditions (increase salt concentration or detergent)

  • For secondary antibodies, use highly cross-adsorbed versions

• Validation approaches:

  • Compare multiple antibodies targeting different KIDINS220 epitopes

  • Include genetic models (knockdown, knockout) as negative controls

  • Use peptide competition assays with the immunizing peptide

• Data interpretation considerations:

  • Be cautious with bands/signals significantly different from the expected 220 kDa size

  • For closely related protein families, verify specificity with mass spectrometry

  • Consider developmental or tissue-specific isoforms when interpreting unexpected patterns

Research using KIDINS220 antibodies for immunodetection has successfully employed these approaches to confirm specificity, particularly when examining expression in specific cell populations like B cells or neural stem cells .

How should researchers interpret differences in KIDINS220 detection between various cell lines and tissues?

Variation in KIDINS220 detection across different experimental systems requires careful interpretation:

• Expression level variations:

  • KIDINS220 expression varies naturally between tissues (high in neural tissues, variable in immune cells)

  • Developmental stage influences expression (e.g., different levels in developing versus mature B cells)

  • Cellular activation state affects expression (e.g., resting versus activated B cells)

• Technical considerations:

  • Different antibodies may have varying affinities for species-specific or tissue-specific isoforms

  • Sample preparation methods affect membrane protein detection (lysis conditions, detergents)

  • Post-translational modifications may mask epitopes in certain contexts

• Biological interpretation framework:

  • Correlate expression patterns with functional outcomes

  • Consider isoform-specific expression or tissue-specific processing

  • Examine expression in context of known KIDINS220 regulatory mechanisms

• Quantification approaches:

  • Use multiple normalization strategies, especially for tissue comparisons

  • Consider absolute quantification methods when comparing across tissues

  • For complex tissues, complement with single-cell approaches (IF, flow cytometry)

Research has demonstrated that KIDINS220 shows differential expression in B cell subpopulations, particularly those bearing λ versus κ light chains, highlighting the importance of considering cellular context when interpreting detection patterns .

How can KIDINS220 antibodies be utilized in neurological research?

Recent findings highlight KIDINS220's critical role in neurological processes, opening several research avenues using KIDINS220 antibodies:

• Neural stem cell (NSC) regulation:

  • Track KIDINS220 expression in neurogenic niches (SGZ, SEZ)

  • Investigate co-localization with NSC markers (Sox2, GFAP) in adult brain

  • Examine relationship between KIDINS220 expression and NSC survival/proliferation

• Neurodevelopmental disorder research:

  • Study KIDINS220 expression in models of SINO syndrome and schizophrenia

  • Examine altered signaling pathways in disease models using phospho-specific antibodies

  • Investigate effects of disease-associated mutations on KIDINS220 localization and function

• Neuronal survival and plasticity:

  • Track KIDINS220-dependent AKT activation in response to growth factors

  • Examine KIDINS220's role in GSK3 pathway regulation

  • Investigate memory-related functions through hippocampal studies

• Methodological approaches:

  • Use KIDINS220 antibodies for brain region-specific expression analysis

  • Employ live imaging with non-perturbing antibody fragments

  • Combine with electrophysiology to correlate expression with neuronal function

Research has shown that Kidins220 deficiency provokes severe neurogenic deficits and hippocampal-based spatial memory defects. Mechanistically, Kidins220-dependent activation of AKT in response to epidermal growth factor restrains GSK3 activity, preventing NSC apoptosis. This identifies KIDINS220 as a key player for sensing growth factor availability to sustain adult neurogenesis .

What are the promising approaches for using KIDINS220 antibodies in studies related to human diseases?

KIDINS220 has been implicated in multiple human pathologies, offering several research opportunities:

• Neurodevelopmental disorders:

  • KIDINS220 mutations are associated with SINO syndrome and schizophrenia

  • Use antibodies to examine expression/localization patterns in patient-derived samples

  • Study signaling alterations in iPSC-derived neurons from affected individuals

• Immune system dysregulation:

  • Given KIDINS220's role in B cell development, investigate its expression in autoimmune conditions

  • Examine λ:κ light chain ratios in relation to KIDINS220 expression in disease models

  • Study BCR signaling strength in autoimmune conditions in relation to KIDINS220 levels

• Translational research applications:

  • Develop tissue microarray analysis of KIDINS220 expression across disease states

  • Use phospho-specific antibodies as potential biomarkers for pathway activation

  • Investigate KIDINS220 as a potential therapeutic target using antibody-based approaches

• Methodological considerations:

  • Compare antibody-based detection with genetic approaches (RNA-seq, single-cell analysis)

  • Validate findings across multiple patient cohorts

  • Consider species differences when translating animal model findings

The ability of KIDINS220 to regulate B cell development, particularly λ light chain-expressing cells, suggests potential involvement in B cell-mediated pathologies. Additionally, its critical role in neural development makes it relevant for understanding neurological disorders .