NKAPL Antibody, FITC conjugated

What is NKAPL and what are its key functions in cellular processes?

NKAPL (NF-κB-activating protein-like) is a novel transcriptional factor that functions primarily as a transcriptional suppressor in Notch signaling. Research has established that NKAPL is associated with several molecules of the Notch corepressor complex such as CIR, HDAC3, and CSL . The protein is predominantly expressed in spermatogonia and early spermatocytes after 3 weeks of age, with particularly robust expression in differentiating spermatogonia .

Key functional characteristics include:

• Nuclear localization via specific amino acid sequences (GSQKRRRFSE at position 15, HSTKKKRKKK at 180, and KPSKRKHKKYY at 189)

• Weak activation capability of NF-κB signaling in a dose-dependent manner

• Critical role in spermatogenesis through transcriptional regulation

• Involvement in cognitive function, particularly in relation to schizophrenia pathology

• Potential tumor suppressor role in hepatocellular carcinoma

Notably, research has identified that NKAPL T152N polymorphism (rs1635) affects NKAPL phosphorylation levels, with the phosphorylation level of NKAPL-152N being significantly decreased compared to NKAPL-152T. This alteration appears to influence neuronal migration during embryonic development .

What are the typical applications for NKAPL antibody, FITC conjugated in research?

NKAPL antibody, FITC conjugated can be employed in several key research applications:

• Flow Cytometry: The FITC conjugation makes it particularly suitable for flow cytometric analysis of NKAPL expression in various cell populations, especially in studies involving spermatogenic cells or neuronal cells .

• Immunofluorescence Microscopy: For visualization of NKAPL localization in tissue sections or cell cultures, particularly useful for studying its nuclear localization and expression patterns during development .

• ELISA-based Assays: The antibody has been validated for ELISA applications, allowing quantitative measurement of NKAPL levels in biological samples .

• Mechanistic Studies: Particularly valuable for investigating:

  • Notch signaling pathway regulation

  • Neuronal migration and cognitive development

  • Transcriptional suppression mechanisms

  • Cancer-related methylation studies

When designing experiments, researchers should note that NKAPL shows tissue-specific expression, being restricted primarily to testis while its homolog NKAP is ubiquitously expressed . This specificity should be considered when selecting appropriate experimental models.

How does site-specific conjugation affect the functionality of NKAPL antibodies compared to traditional methods?

Site-specific conjugation offers significant advantages over traditional random conjugation methods when working with antibodies like NKAPL:

Traditional Conjugation Limitations:

• Random conjugation to amine/thiol groups often results in heterogeneous antibody display

• Can lead to hindered biological activity

• May cause aggregation due to multivalent interactions

• Often requires displaying high numbers of antibodies, causing steric inhibition of antigen recognition

Site-Specific Conjugation Benefits:

• Preserves antibody functionality by targeting specific sites away from the antigen-binding region

• Provides homogeneous antibody-conjugate products

• Reduces aggregation risks

• Allows controlled display of antibodies with optimal orientation

A two-step enzymatic reaction approach has proven particularly effective:

• Use of PNGase F to remove N-linked glycans (particularly from Asn297 in the Fc region)

• Use of microbial transglutaminase (MTGase) to catalyze reaction between deglycosylated antibody and azide-functional linker

• Subsequent conjugation via strain-promoted azide-alkyne cycloaddition

This site-specific approach has been successfully applied across multiple antibody isotypes (including human IgG, rat IgG2a, and humanized IgG1) and maintains antibody functionality post-conjugation .

What are the recommended protocols for using NKAPL antibody, FITC conjugated in flow cytometry?

Protocol for Flow Cytometric Analysis Using NKAPL Antibody, FITC Conjugated:

Sample Preparation:

• Harvest cells of interest (spermatogonia, neuronal cells, or HCC cells depending on research focus)

• Wash cells in cold PBS containing 1% BSA (2-5 × 10^6 cells/mL)

• Fix cells with 4% paraformaldehyde for 10-15 minutes at room temperature

• Permeabilize with 0.1% Triton X-100 in PBS for 5-10 minutes (NKAPL is primarily nuclear)

Staining Procedure:

• Block with 5% normal serum in PBS for 30 minutes

• Add NKAPL antibody, FITC conjugated at recommended dilution (typically 1:100-1:500; validate concentration empirically)

• Incubate for 30-60 minutes at room temperature or 4°C in the dark

• Wash twice with PBS containing 1% BSA

• Resuspend cells in appropriate buffer for flow cytometry analysis

Critical Controls:

• Isotype control antibody, FITC conjugated (rabbit IgG-FITC)

• Unstained cells

• Single-stained controls if performing multicolor flow cytometry

• Known positive control (testicular cells for NKAPL)

Analysis Considerations:

• Use 488 nm laser for FITC excitation

• Collect emission at 530/30 nm

• Compensate for spectral overlap if using multiple fluorophores

• Analyze nuclear expression pattern, particularly in developmental studies

Note: When studying NKAPL variants (such as T152N polymorphism), additional controls comparing expression between variant carriers may be necessary, as peripheral blood mRNA expression levels of NKAPL in 152N carriers of EOS patients were shown to be significantly lower than in 152T carriers .

What methods are recommended for studying NKAPL phosphorylation using antibody techniques?

Based on research demonstrating that NKAPL T152N polymorphism affects phosphorylation levels , the following methodology is recommended for investigating NKAPL phosphorylation:

Immunoprecipitation and Western Blot Method:

• Cell Lysate Preparation:

  • Transfect cells with expression vectors for NKAPL variants (e.g., NKAPL-152T and NKAPL-152N)

  • After 48 hours, lyse cells in buffer containing:

    • 25 mM HEPES (pH 7.5)

    • 150 mM NaCl

    • 0.1% Triton X-100

    • 10 mM MgCl₂

    • 1 mM EDTA

    • Protease inhibitor mixture

    • Phosphatase inhibitor mixture

• Immunoprecipitation:

  • Immunoprecipitate phosphorylated NKAPL using phosphothreonine antibodies conjugated to Dynabeads Protein G

  • Wash immunoprecipitates thoroughly with lysis buffer

• Western Blot Analysis:

  • Separate immunoprecipitated proteins on NuPAGE 10% BT Gel

  • Transfer to nitrocellulose membranes

  • Incubate with primary antibody (anti-HA or anti-NKAPL)

  • Detect using appropriate secondary antibodies and imaging system (infrared or chemiluminescent)

• Quantification:

  • Perform densitometric analysis to compare phosphorylation levels between variants

  • Normalize to total NKAPL protein expression

Phosphorylation Site Prediction:

• Use computational tools such as NetPhos 3.1 to predict potential phosphorylation sites within NKAPL

• Focus analysis on threonine residues, particularly around position 152

Additional Considerations:

• Include wild-type NKAPL as control

• Consider performing mass spectrometry to identify and confirm specific phosphorylation sites

• For in vivo studies, tissue-specific extraction protocols may be necessary given NKAPL's restricted expression pattern

Research has shown that phosphorylation level of NKAPL-152N is significantly decreased compared to NKAPL-152T, which may affect NKAPL mRNA expression levels and embryonic cortical neuronal migration by regulating NKAPL protein phosphorylation .

How can NKAPL antibody, FITC conjugated be used to study NKAPL's role in neuropsychiatric disorders?

Research has established an association between NKAPL rs1635 (T152N) and cognitive function in early-onset schizophrenia (EOS) . The following methodological approaches can be employed when using NKAPL antibody, FITC conjugated to investigate this connection:

1. Flow Cytometric Analysis of Patient Samples:

• Isolate peripheral blood mononuclear cells from patients with different NKAPL genotypes

• Perform intracellular staining with NKAPL antibody, FITC conjugated

• Compare NKAPL expression levels between genotype groups

• Correlate with cognitive performance metrics (e.g., MATRICS Consensus Cognitive Battery)

2. Immunofluorescence Studies in Neuronal Models:

• Utilize neural progenitor cells or neuronal cultures from induced pluripotent stem cells

• Apply NKAPL antibody, FITC conjugated to visualize NKAPL localization and expression

• Compare cultures with different NKAPL variants (152T vs. 152N)

• Combine with markers for neuronal development and migration

3. Correlation Analysis with Cognitive Parameters:
Research has shown that among EOS patients:

• CC genotype carriers (encoding NKAPL-152T) performed better in:

  • Speed of processing (t = 2.644, p = 0.009)

  • Trail making test (t = 2.221, p = 0.028)

  • Category fluency (t = 2.578, p = 0.011)

• NKAPL mRNA expression in 152N carriers was significantly lower than in 152T carriers

4. Neuronal Migration Studies:

• Use in utero electroporation techniques to investigate NKAPL's role in neuronal migration

• Combine with FITC-labeled NKAPL antibody to track protein localization

• Compare wild-type NKAPL with variant forms

5. Mechanistic Investigations:

• Examine NKAPL's interaction with Notch signaling components in neuronal systems

• Investigate phosphorylation differences between NKAPL variants in neural cells

• Study effects on downstream gene expression related to cognitive function

Research suggests that NKAPL T152N may affect NKAPL mRNA expression level and embryonic cortical neuronal migration by regulating NKAPL protein phosphorylation, potentially explaining the association with cognitive function in EOS patients .

What methodologies are used to investigate NKAPL methylation in cancer using antibody-based approaches?

Research has identified NKAPL hypermethylation as a common event in hepatocellular carcinoma (HCC) with potential prognostic value . The following methodological approaches can be applied when using NKAPL antibody, FITC conjugated for methylation studies in cancer:

1. Combined Methylation and Expression Analysis:

• Perform methylation-specific PCR to determine NKAPL promoter methylation status

• Use NKAPL antibody, FITC conjugated in flow cytometry or immunofluorescence to assess protein expression

• Correlate methylation status with protein expression levels

• Compare tumor tissues with adjacent non-tumor tissues

2. Demethylation Studies:

• Treat cancer cell lines with demethylating agents (e.g., 5-aza-2'-deoxycytidine)

• Monitor changes in NKAPL expression using FITC-conjugated antibody

• Quantify expression changes via flow cytometry or microscopy

• Validate that NKAPL expression is regulated by promoter methylation

3. Prognostic Correlation Analysis:

• Stratify patient samples based on NKAPL methylation status

• Use NKAPL antibody, FITC conjugated to assess protein expression

• Correlate methylation and expression with patient outcomes

• Research has shown that high methylation level of NKAPL and low expression predict poor outcome in HCC

4. Functional Studies:

• Perform ectopic expression of NKAPL in HCC cells

• Monitor effects on cell growth and cell cycle

• Use FITC-conjugated NKAPL antibody to confirm expression

• Research has demonstrated that ectopic expression of NKAPL in HCC cells inhibits cell growth

5. Methylation Pattern Analysis:

• Combine immunofluorescence using NKAPL antibody, FITC conjugated with methylation-specific in situ hybridization

• Create methylation pattern maps across tumor tissues

• Correlate with histopathological features

Technical Considerations:

• Include both tumor and adjacent non-tumor tissues for comparative analysis

• Use appropriate methylation-specific and non-methylation-specific primers

• Include controls for antibody specificity

• Consider laser capture microdissection for tissue-specific analysis

This integrated approach allows researchers to establish connections between NKAPL methylation, expression levels, and cancer progression, potentially identifying NKAPL as a valuable prognostic biomarker and therapeutic target .

Can NKAPL antibody, FITC conjugated be used for intracellular staining, and what protocol is recommended?

Yes, NKAPL antibody, FITC conjugated can be used for intracellular staining, which is essential given that NKAPL is primarily a nuclear protein functioning as a transcriptional suppressor . The following protocol is recommended for effective intracellular staining:

Protocol for Intracellular Staining with NKAPL Antibody, FITC Conjugated:

Cell Preparation:

• Harvest cells (1-5 × 10^6 cells) and wash twice with PBS

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

• Wash twice with PBS containing 1% BSA

Permeabilization Options:

• For Flow Cytometry:

  • Permeabilize with 0.1% Triton X-100 in PBS for 10 minutes at room temperature, or

  • Use commercial permeabilization buffer (e.g., containing saponin)

• For Microscopy:

  • Permeabilize with 0.2% Triton X-100 in PBS for 10 minutes at room temperature

Blocking and Staining:

• Block with 5% normal serum in PBS for 30-60 minutes at room temperature

• Dilute NKAPL antibody, FITC conjugated in blocking buffer (1:100-1:500, optimal concentration should be determined empirically)

• Incubate cells with diluted antibody for 1-2 hours at room temperature or overnight at 4°C in the dark

• Wash three times with PBS containing 0.1% Tween-20

For Microscopy - Additional Steps:

• Counterstain nuclei with DAPI (1 μg/mL) for 5 minutes

• Mount slides with anti-fade mounting medium

• Seal with nail polish and store at 4°C protected from light

Critical Considerations:

• NKAPL contains specific nuclear localization signals (particularly GSQKRRRFSE at position 15), which should result in nuclear staining patterns

• Include appropriate controls:

  • Isotype control antibody, FITC conjugated

  • Blocking with unlabeled NKAPL antibody before adding FITC-conjugated antibody

  • Cells known to be negative for NKAPL expression

• For co-localization studies, combine with antibodies against:

  • Other Notch signaling components

  • Nuclear markers

  • Cell type-specific markers (e.g., TRA98 for germ cells, c-kit for differentiating spermatogonia)

Optimization Tips:

• Adjust fixation time based on cell type

• Test different permeabilization reagents and times

• Optimize antibody concentration using titration

• Consider signal amplification for low-abundance detection

This protocol should enable effective visualization and quantification of NKAPL in various experimental systems, particularly in studies investigating its role in transcriptional regulation, neuronal development, and cancer biology.

What methods can be used to deliver NKAPL antibodies into living cells for functional studies?

Delivering antibodies into living cells presents unique challenges, but several methods have been developed that can be applied to NKAPL antibodies for functional studies:

1. Electroporation:

• Most robust approach with 90-99% delivery efficiency and 80-90% cell viability

• Protocol:

  • Harvest cells and wash in electroporation buffer

  • Mix cells (1-5 × 10^6) with NKAPL antibody (10-50 μg/mL)

  • Apply electrical pulse according to cell type-specific parameters

  • Allow cells to recover in complete medium

  • Analyze 24-72 hours post-electroporation

2. Cell Squeezing/Microfluidic Cell Deformation:

• Cells are mechanically deformed through microfluidic channels

• Creates transient membrane disruption allowing antibody entry

• Gentler than electroporation but requires specialized equipment

3. Protein Transfection Reagents:

• Commercial reagents (e.g., protein transfection reagents) form complexes with antibodies

• Allow endocytosis followed by endosomal escape

• Generally lower efficiency than physical methods

4. Cell-Penetrating Peptides:

• Conjugate antibodies with cell-penetrating peptides

• Enables transport across plasma membrane

• May require chemical modification of the antibody

5. Microinjection:

• Direct injection of antibodies into individual cells

• Highest precision but lowest throughput

• Suitable for specific applications requiring single-cell analysis

Functional Validation Approaches:

• Co-delivery with fluorescent markers to confirm successful internalization

• Immunostaining post-delivery to verify antibody localization to nuclear compartment

• Assessing transcriptional changes in Notch signaling genes to confirm functional impact

• Co-immunoprecipitation to verify disruption of NKAPL's interaction with Notch corepressor complex components

Considerations for NKAPL-Specific Applications:

• Given NKAPL's nuclear localization, delivered antibodies must reach the nucleus

• Nuclear localization signals on NKAPL (particularly GSQKRRRFSE at position 15) may be blocked by antibody binding

• Consider coupling with nuclear localization peptides if nuclear targeting is inefficient

• Include controls to distinguish between effects of antibody delivery and functional inhibition

Research has shown that delivered antibodies remain functional in the cytosol despite the reducing environment, with a long half-life allowing binding to targets even 3-4 days after delivery .

What are common causes of weak signal when using NKAPL antibody, FITC conjugated?

When experiencing weak signal issues with NKAPL antibody, FITC conjugated, several factors may be contributing to the problem. Understanding these potential causes and their solutions is crucial for optimizing experimental outcomes:

1. Insufficient NKAPL Expression:

• Cause: NKAPL expression is tissue-specific, being primarily restricted to testis with robust expression in spermatogonia and early spermatocytes after 3 weeks of age .

• Solution: Confirm NKAPL expression in your experimental system using RT-PCR before antibody studies. For developmental studies, ensure appropriate age of samples (expression increases significantly after 3 weeks in mice).

2. Inadequate Fixation/Permeabilization:

• Cause: NKAPL is a nuclear protein requiring effective nuclear permeabilization.

• Solution: Optimize fixation (4% paraformaldehyde, 15 min) and increase permeabilization stringency (try 0.2-0.5% Triton X-100 instead of 0.1%).

3. FITC Photobleaching:

• Cause: FITC is susceptible to photobleaching under prolonged exposure to light.

• Solution: Minimize light exposure during all steps, use anti-fade mounting media, consider using photobleaching inhibitors, and acquire images promptly after staining.

4. Phosphorylation State Interference:

• Cause: Research shows differences in phosphorylation between NKAPL variants (152T vs. 152N) , which might affect antibody recognition.

• Solution: Use phosphatase inhibitors during sample preparation, and consider the target epitope in relation to known phosphorylation sites.

5. Genetic Variation Effects:

• Cause: NKAPL variants (e.g., T152N) show different expression levels; 152N carriers have lower NKAPL mRNA levels than 152T carriers .

• Solution: Genotype samples when possible and adjust exposure/acquisition settings accordingly.

6. Buffer Compatibility Issues:

• Cause: The antibody's buffer (50% Glycerol, 0.01M PBS, pH 7.4 with 0.03% Proclin 300) may interact with certain fixatives or permeabilization agents.

• Solution: Test different buffer conditions and consider dialyzing the antibody into a more compatible buffer if necessary.

7. Antibody Concentration:

• Cause: Suboptimal antibody concentration for your specific application.

• Solution: Perform titration experiments (try 1:50, 1:100, 1:200, 1:500 dilutions) to determine optimal concentration.

8. Technical Troubleshooting Table:

9. Optimization Strategies:

• Use positive control tissues (testis for NKAPL)

• Include isotype control (rabbit IgG-FITC) at same concentration

• Consider signal amplification methods if protein is expressed at low levels

• Compare results with non-conjugated primary NKAPL antibody plus FITC-secondary antibody

These comprehensive troubleshooting approaches should help resolve weak signal issues when working with NKAPL antibody, FITC conjugated in various experimental applications.

How can I validate the specificity of NKAPL antibody, FITC conjugated in my experimental system?

Validating antibody specificity is crucial for ensuring reliable experimental results, particularly for targets like NKAPL with tissue-specific expression patterns. The following comprehensive approach will help establish the specificity of NKAPL antibody, FITC conjugated:

1. Positive and Negative Control Tissues/Cells:

• Positive Controls: Use testicular tissue/cells, particularly spermatogonia and early spermatocytes after 3 weeks of age, where NKAPL is known to be expressed robustly .

• Negative Controls: Examine tissues where NKAPL is not expressed (most non-testicular tissues), as NKAPL expression is restricted to testis while its homolog NKAP is ubiquitously expressed .

2. Genetic Validation Approaches:

• NKAPL Knockout/Knockdown: Use CRISPR-Cas9 or siRNA to reduce NKAPL expression, which should result in reduced antibody signal.

• Overexpression Studies: Transfect cells with NKAPL expression vectors and confirm increased antibody signal.

• Variant Expression: Compare staining patterns between cells expressing different NKAPL variants (e.g., 152T vs. 152N) .

3. Peptide Competition Assay:

• Pre-incubate NKAPL antibody with excess recombinant NKAPL protein (114-123AA immunogen) or synthetic peptide

• Apply to samples in parallel with non-blocked antibody

• Specific signal should be significantly reduced in the peptide-blocked condition

4. Orthogonal Detection Methods:

• Western Blot Validation: Confirm antibody recognizes a band of appropriate size (~52 kDa)

• mRNA Correlation: Perform RT-PCR or RNA-seq and correlate transcript levels with protein signal intensity

• Multiple Antibody Validation: Compare staining patterns with different antibodies targeting distinct NKAPL epitopes

5. Cross-Reactivity Assessment:

• Test antibody on closely related proteins, particularly NKAP (67% identity in mice, 70% in humans)

• Examine staining in species where NKAPL is highly conserved vs. less conserved

6. Methodological Controls:

• Isotype Control: Use rabbit IgG-FITC at the same concentration to assess non-specific binding

• Secondary-Only Control: If using indirect detection method, include secondary antibody-only control

• Autofluorescence Control: Include unstained samples to assess natural autofluorescence

7. Subcellular Localization Verification:

• NKAPL should demonstrate nuclear localization due to specific nuclear localization signals

• Co-stain with nuclear markers and verify co-localization

• Verify staining pattern consistent with known NKAPL biology (nuclear transcriptional suppressor)

8. Validation Documentation:

9. Developmental Validation:

• If studying developmental systems, verify age-appropriate expression

• NKAPL expression is low until 2 weeks of age, but significantly up-regulated from 3 weeks onward in mice