NUMA1 Antibody, HRP conjugated

Basic Research Questions

• What is NUMA1 and what cellular functions should researchers consider when selecting detection methods?

NUMA1 (Nuclear Mitotic Apparatus Protein 1) is a large protein (~238 kDa) with diverse cellular functions. It plays critical roles in mitotic spindle organization during cell division but also has non-mitotic functions. Recent research has identified NUMA1 as significantly upregulated in esophageal squamous cell carcinoma (ESCC), where it promotes tumor progression and inhibits apoptosis . NUMA1 is also transiently located at the axon initial segment (AIS) during neuronal development, where it promotes AIS assembly .

When selecting detection methods, researchers should consider NUMA1's dynamic subcellular localization: primarily nuclear during interphase, relocated to spindle poles during mitosis, and transiently at the AIS in developing neurons. Sample preparation techniques must preserve these localization patterns while maintaining epitope accessibility for antibody binding.

• How should researchers optimize immunoprecipitation protocols when using NUMA1 antibodies?

Successful immunoprecipitation (IP) of NUMA1 requires attention to several methodological details:

• Lysis conditions: Use gentle lysis buffers (e.g., NP-40 or CHAPS-based) that preserve protein-protein interactions while effectively releasing NUMA1 from nuclear and cytoskeletal associations.

• Pre-clearing: Pre-clear lysates with protein A/G beads to reduce nonspecific binding.

• Antibody amounts: Titrate antibody concentration (typically 2-5 μg per 500 μg total protein) for optimal results.

• Incubation conditions: Perform IP incubation overnight at 4°C with gentle rotation.

• Controls: Include IgG controls and input samples (5-10% of starting material).

• Validation: Confirm successful IP using western blot with a second NUMA1 antibody targeting a different epitope.

For studying NUMA1's interactions with partners like GSTP1 and TRAF2, researchers should consider sequential IPs or crosslinking approaches to capture transient complexes .

• What validation strategies confirm NUMA1 antibody specificity for experimental applications?

Comprehensive validation should include:

• Knockout controls: Test antibodies on NUMA1 knockout samples, such as CRISPR-Cas9-derived NUMA1 knockout HAP1 cells as described in recent literature .

• RNAi validation: Compare signals in cells treated with NUMA1-specific siRNA/shRNA versus scrambled controls.

• Recombinant protein detection: Test antibody against purified recombinant NUMA1 or specific domains.

• Multiple detection methods: Confirm consistent results across western blot, immunofluorescence, and flow cytometry.

• Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding.

• Co-localization: In immunofluorescence applications, verify that staining patterns match known NUMA1 distributions (nuclear in interphase, spindle poles in mitosis, or transient AIS localization in neurons).

These validation approaches ensure that experimental observations genuinely reflect NUMA1 biology rather than antibody artifacts.

• How can researchers troubleshoot weak or non-specific signals when using NUMA1 antibodies?

When experiencing detection challenges with NUMA1 antibodies:

For weak signals:

• Sample preparation: Ensure complete protein extraction, particularly from nuclear fractions.

• Antigen retrieval: For fixed samples, optimize antigen retrieval methods using heat or enzymatic approaches.

• Signal amplification: Utilize biotin-streptavidin systems or tyramide signal amplification.

• Loading amount: Increase protein loading (considering NUMA1's expression level in your samples).

• Exposure time: For HRP-conjugated antibodies, optimize exposure times for chemiluminescent detection.

For non-specific signals:

• Blocking optimization: Increase blocking reagent concentration or time.

• Antibody dilution: Test a range of dilutions to find optimal signal-to-noise ratio.

• Washing stringency: Increase number or duration of wash steps.

• Secondary antibody controls: Include controls omitting primary antibody.

• Cross-reactivity assessment: Validate specificity using NUMA1 knockout samples .

For neuronal studies focusing on AIS localization, consider detergent extraction methods to remove soluble NUMA1 while retaining the AIS-associated pool .

• What are the optimal sample preparation conditions for detecting NUMA1 in different cellular compartments?

Sample preparation should be tailored to the specific NUMA1 compartment being studied:

For nuclear NUMA1:

• Use nuclear extraction buffers containing DNase to release NUMA1 from chromatin

• Consider subcellular fractionation to separate nuclear and cytoplasmic components

• Avoid harsh detergents that might disrupt nuclear membrane integrity prematurely

For mitotic spindle-associated NUMA1:

• Synchronize cells in mitosis using nocodazole or other cell cycle inhibitors

• Use microtubule-stabilizing buffers during fixation

• Consider methanol fixation to better preserve spindle structures

For AIS-associated NUMA1 in neurons:

• Time experiments appropriately, as NUMA1's presence at the AIS is developmentally regulated

• Use detergent extraction to reveal the AIS-specific pool of NUMA1, as demonstrated with 4.1B protein retention at the AIS after extraction

• Co-stain with established AIS markers like ankyrinG for colocalization confirmation

Advanced Research Questions

• How can NUMA1 antibodies be used to investigate the ASK1-MKK4-SAPK/JNK signaling pathway in cancer studies?

Recent research has revealed NUMA1's role in regulating the ASK1-MKK4-SAPK/JNK signaling pathway through interactions with GSTP1 and TRAF2 . Methodological approaches include:

Co-immunoprecipitation (Co-IP) strategy:

• Use NUMA1 antibody for IP followed by immunoblotting for GSTP1, TRAF2, and ASK1

• Perform reciprocal IPs using antibodies against pathway components

• Include appropriate IgG controls and input samples

• Use NUMA1 knockout cells as negative controls

Pathway activation assessment:

• Monitor phosphorylation states of MKK4, JNK, and c-Jun using phospho-specific antibodies

• Compare phosphorylation levels in NUMA1 knockdown versus control cells

• Analyze the effects of NUMA1 overexpression on pathway activation

Data from ESCC research shows that knocking down NUMA1 increases phosphorylation of MKK4, JNK, and c-Jun, while overexpression inhibits this phosphorylation . This provides a useful functional readout for NUMA1 activity.

When designing experiments, researchers should consider sequential Co-IPs to detect trimeric complexes and competition assays to assess how NUMA1 affects the TRAF2-ASK1 interaction.

• What methodological approaches can detect changes in NUMA1 phosphorylation status and its impact on protein interactions?

NUMA1 phosphorylation, particularly at residue T2041, affects its membrane association and interactions with proteins like 4.1B . To study this:

Phosphorylation detection methods:

• Phospho-specific antibodies: Use antibodies that recognize phosphorylated NUMA1 at key sites

• Phos-tag SDS-PAGE: Separate phosphorylated from non-phosphorylated NUMA1 forms

• Mass spectrometry: Perform phosphoproteomic analysis after NUMA1 immunoprecipitation

• In vitro kinase assays: Test specific kinases like Cdk1/Cdk2

Functional impact assessment:

• Phosphomimetic mutants: Create phosphomimetic mutants (e.g., T2041E) to simulate constitutive phosphorylation

• Phospho-null mutants: Create T2041A mutants to prevent phosphorylation

• Compare binding properties of these mutants in Co-IP experiments

Research has shown that phosphomimetic mutations of NUMA1 dramatically reduce its interaction with 4.1B , demonstrating how phosphorylation regulates NUMA1's protein interactions. When designing experiments, consider cell cycle synchronization since NUMA1 phosphorylation varies across cell cycle stages.

• What controls and experimental design are necessary for studying NUMA1's role in apoptotic pathways using HRP-conjugated antibodies?

Based on recent findings that NUMA1 modulates apoptosis in ESCC , rigorous experimental design should include:

Essential controls:

• Positive apoptosis controls: Cells treated with known apoptosis inducers

• Negative controls: NUMA1 knockout or knockdown cells

• Antibody controls: IgG controls for immunoprecipitation

• Loading controls: Housekeeping proteins for western blots

Experimental design:

• Multiple apoptosis detection methods:

  • Annexin V/PI staining for flow cytometry

  • Caspase activity assays (caspase-3/7)

  • TUNEL assay for DNA fragmentation

  • Western blot for cleaved PARP and cleaved caspases

NUMA1 manipulation approaches should include:

• siRNA or shRNA knockdown of NUMA1 as described in research

• CRISPR-Cas9 knockout of NUMA1

• Overexpression of wildtype NUMA1

• Expression of specific NUMA1 domains to identify regions critical for apoptosis regulation

Published research demonstrates that knockdown of NUMA1 promotes cell apoptosis while suppressing cell proliferation and colony formation , providing clear functional readouts for experimental verification.

• How can researchers distinguish between different functional domains of NUMA1 in co-immunoprecipitation experiments?

NUMA1 has distinct functional domains mediating different protein interactions. Based on recent research:

Domain-specific approaches:

• Generate a series of NUMA1 truncation or deletion constructs targeting specific domains

• Create epitope-tagged constructs for easier detection and immunoprecipitation

• Use domain-specific antibodies when available

Key domain information from research:

• C-terminal domain: Important for interaction with GSTP1

• Globular domain, C-terminal domain, and part of coiled domain: Involved in TRAF2 interaction

• 4.1-binding domain: Mediates interaction with 4.1B

• T2041 phosphorylation site: Affects membrane association and protein interactions

Experimental approaches should include:

• Competitive binding assays using purified domain fragments

• Sequential Co-IPs to identify multi-protein complexes

• Systematic mutation of key residues within interaction domains

Recent studies have successfully mapped domain interactions using tagged domain deletion mutants, revealing that the C-terminal domain of NUMA1 is responsible for its interaction with GSTP1, while its interaction with TRAF2 involves multiple domains .

• What technical considerations are important when studying NUMA1's role in neuronal axon initial segment (AIS) development?

Recent research has revealed NUMA1's transient location at the AIS during development, where it promotes AIS assembly by inhibiting the endocytosis of neurofascin-186 . Technical considerations include:

Developmental timing:

• Ensure appropriate developmental stages are examined (NUMA1 is transiently at the AIS)

• Use DIV-specific neuronal cultures (DIV7 was mentioned in research)

• Include age-matched controls for in vivo studies

Subcellular localization techniques:

• Detergent extraction methods to retain AIS-associated NUMA1 while removing soluble proteins

• Co-localization with established AIS markers (ankyrinG, neurofascin-186)

• Super-resolution microscopy for precise localization within the AIS structure

Functional studies:

• shRNA silencing of NUMA1 disrupts AIS assembly but not maintenance

• Investigate NUMA1's interaction with 4.1B and Lis1 at the AIS

• Examine neurofascin-186 endocytosis in relation to NUMA1 expression

Research demonstrates that NUMA1 interacts with the scaffolding protein 4.1B and the dynein regulator Lis1 at the AIS , suggesting important experimental targets for researchers investigating NUMA1's neuronal functions.