NCKIPSD Antibody

What is NCKIPSD and why is it important in research?

NCKIPSD (also known as AF3P21, DIP, DIP1, SPIN90, WASLBP, or WISH) is a protein localized exclusively in the cell nucleus. It plays critical roles in:

• Signal transduction pathways

• Maintenance of sarcomeres

• Assembly of myofibrils into sarcomeres

• Stress fiber formation

• Involvement in therapy-related leukemia through chromosomal translocation t(3;11)(p21;q23)

Research on NCKIPSD provides insights into cellular structural organization and potential therapeutic targets for leukemia and other disorders of cytoskeletal regulation.

What types of NCKIPSD antibodies are available for research?

NCKIPSD antibodies are available in several formats with varying specifications:

When selecting an antibody, consider your experimental application, target species, and the specific domain of NCKIPSD you wish to detect.

How do I validate a new NCKIPSD antibody for my research?

Methodological validation of NCKIPSD antibodies should include:

• Positive and negative controls: Use cell lines known to express NCKIPSD (positive control) and those with low/no expression (negative control)

• Knockdown validation: Compare antibody reactivity in wild-type versus NCKIPSD-knockdown samples

• Cross-reactivity assessment: Test antibody against related proteins, particularly in multi-species studies

• Application-specific validation:

  • For Western blotting: Confirm band at expected molecular weight (~54 kDa)

  • For immunohistochemistry: Compare staining pattern with known expression profiles

  • For ELISA: Generate standard curves using recombinant NCKIPSD protein

Remember that validation procedures should be reported in publications to ensure reproducibility .

How can I design custom antibodies targeting specific epitopes within NCKIPSD?

Custom antibody design for NCKIPSD follows several strategic approaches:

• Epitope selection: For disordered regions within NCKIPSD, employ rational design methods utilizing complementary peptides . Key considerations include:

  • Secondary structure prediction

  • Hydrophilicity/hydrophobicity analysis

  • Antigenic index calculation

  • Conservation analysis across species if cross-reactivity is desired

• Antibody scaffold selection: Choose between:

  • Full antibody formats

  • Single-domain antibodies

  • Recombinant antibody fragments

• Complementary peptide design: As demonstrated in comparable research on disordered proteins:

  • Design peptides complementary to target epitope

  • Graft peptides onto CDR regions of antibody scaffold

  • Express and purify antibody constructs

This rational design approach enables targeting of specific functional domains within NCKIPSD for more precise experimental applications.

What are the optimal conditions for using NCKIPSD antibodies in different cellular compartments?

NCKIPSD localizes primarily to the nucleus but may have functional roles in other cellular compartments. Optimization strategies include:

• Nuclear detection:

  • Adequate nuclear permeabilization (0.1-0.5% Triton X-100)

  • Nuclear extraction buffers for biochemical analyses

  • Low formaldehyde fixation (2-4%) to preserve epitope accessibility

• Cytoskeletal interactions:

  • Preservation of cytoskeletal structures using specialized fixatives

  • Dual staining with cytoskeletal markers to assess colocalization

  • Detergent selection to maintain structural integrity

• Membrane-associated fractions:

  • Subcellular fractionation protocols

  • Membrane-specific extraction methods

  • Crosslinking approaches for transient interactions

Each compartment requires specific optimization of antibody concentration, incubation times, and buffer compositions.

How can single-cell RNA-seq data inform NCKIPSD antibody panel design?

Recent advances in integrating single-cell RNA-seq with antibody panel design offer powerful approaches:

• Expression-guided selection: Identify cell populations with differential NCKIPSD expression to:

  • Select appropriate positive and negative control populations

  • Design targeted antibody panels for specific cell types

  • Validate antibody performance in heterogeneous samples

• Validation strategy:

  • Compare RNA expression with protein detection at single-cell level

  • Account for RNA-protein correlation variability

  • Incorporate orthogonal data from paired proteomic and RNA analyses

• Limitations:

  • RNA-protein correlations vary across genes and tissues

  • scRNA-seq typically profiles only highly expressed transcripts

  • Low expression transcripts (like some transcription factors) may be missed

This integrated approach ensures antibody panels detect NCKIPSD across relevant cellular contexts and expression levels.

Why might NCKIPSD antibodies show unexpected cross-reactivity or non-specific binding?

Troubleshooting cross-reactivity requires systematic analysis:

• Common causes of non-specific binding:

  • Sequence homology with related proteins (check sequence alignments)

  • Post-translational modifications altering epitope recognition

  • Antibody concentration too high

  • Insufficient blocking

  • Sample preparation artifacts

• Resolution strategies:

  • Titrate antibody concentration

  • Increase blocking agent concentration (5% BSA or 5-10% normal serum)

  • Pre-adsorption with related proteins

  • Use alternative antibodies targeting different epitopes

  • Validate with knockout/knockdown controls

• Species-specific considerations:

  • Human NCKIPSD shows 72% sequence identity with mouse and 69% with rat orthologs in some regions

  • Select antibodies validated for your species of interest

  • Consider epitope conservation when using antibodies across species

What are the best practices for optimizing NCKIPSD antibody use in immunohistochemistry?

Optimization protocol for NCKIPSD immunohistochemistry:

• Antigen retrieval methods comparison:

  • Heat-induced epitope retrieval (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

  • Enzymatic retrieval (proteinase K vs. trypsin)

  • No retrieval

• Blocking optimization:

  • Test different blocking solutions (normal serum, BSA, commercial blockers)

  • Evaluate endogenous peroxidase quenching methods

  • Include avidin/biotin blocking for biotin-based detection systems

• Antibody dilution series:

  • Prepare serial dilutions (typically 1:100 to 1:2000)

  • Determine optimal signal-to-noise ratio

  • Adjust incubation times (1 hour at room temperature vs. overnight at 4°C)

• Detection system selection:

  • DAB vs. AEC chromogen

  • Polymer-based vs. traditional ABC methods

  • Consider signal amplification for low abundance targets

Each parameter should be systematically optimized while keeping others constant to identify optimal conditions.

How should researchers interpret differences in NCKIPSD detection between antibody clones?

Interpreting variant results between antibody clones requires careful analysis:

• Epitope mapping analysis:

  • Different antibodies recognize distinct regions of NCKIPSD

  • Clone 2E4 targets N-terminal region (AA 1-99)

  • Other antibodies may target internal regions or C-terminus

  • Alternative splicing of NCKIPSD produces two distinct isoforms that may not be recognized by all antibodies

• Interpretation framework:

  • Discrepancies may reveal biological insights about protein conformation, complexes, or modifications

  • Differential accessibility of epitopes in various cellular contexts

  • Potential detection of specific isoforms or modified forms

• Validation approach:

  • Use multiple antibodies targeting different epitopes

  • Compare with genetic approaches (tagged constructs, CRISPR-edited cells)

  • Correlate with mRNA expression data

This analytical framework converts apparent discrepancies into biological insights about NCKIPSD function and regulation.

How can researchers accurately quantify NCKIPSD expression levels across different experimental conditions?

Quantification of NCKIPSD requires rigorous methodological approaches:

• Western blot quantification:

  • Include standard curve of recombinant NCKIPSD protein

  • Use appropriate housekeeping controls (GAPDH, β-actin, or β-tubulin)

  • Apply linear range detection methods

  • Normalize to total protein (Ponceau S or Stain-Free technology)

• ELISA-based quantification:

  • Develop sandwich ELISA using validated antibody pairs

  • Generate standard curves with purified protein

  • Apply appropriate statistical analysis for replicate samples

• Image-based quantification (IHC/ICC):

  • Define consistent regions of interest

  • Apply automated image analysis algorithms

  • Report quantification parameters (intensity thresholds, background subtraction)

  • Use appropriate statistical tests for comparison

These approaches ensure reliable quantitative analysis of NCKIPSD across experimental conditions.

What considerations are important when interpreting NCKIPSD antibody results in disease models?

Interpretation in disease contexts requires specialized considerations:

• Expression pattern analysis:

  • Compare subcellular localization in normal vs. disease tissues

  • Assess co-localization with disease-related markers

  • Evaluate expression changes across disease progression

• Post-translational modification assessment:

  • Use modification-specific antibodies where available

  • Consider how disease states might alter protein processing

  • Correlate with functional assays

• Mutation-specific considerations:

  • In leukemia models with t(3;11)(p21;q23) translocation, consider whether antibodies detect fusion proteins

  • Epitope accessibility may change in disease contexts

  • Validate antibody recognition of mutant forms

• Technical validation in disease samples:

  • Include appropriate disease and normal control tissues

  • Account for disease-specific artifacts (increased background, altered protein expression)

  • Consider tissue-specific optimization for each disease model

These analytical frameworks ensure robust interpretation of NCKIPSD antibody results in complex disease models.