NPM1 Antibody

What applications have been validated for NPM1 antibodies?

Anti-Nucleophosmin/NPM1 antibodies have been extensively validated for multiple applications including Western blotting (WB) and immunohistochemistry (IHC). When designing experiments, researchers should consider that antibodies like PB9341 have demonstrated effectiveness in both applications across human, mouse, and rat tissues . For optimal results in immunohistochemistry, follow standardized protocols that include proper antigen retrieval steps and optimal antibody dilutions based on your specific tissue type. Western blotting applications typically yield a band at approximately 37 kDa corresponding to the NPM1 protein.

Which species show confirmed reactivity with commercial NPM1 antibodies?

Most commercial NPM1 antibodies, including the PB9341 antibody, demonstrate confirmed reactivity with human, mouse, and rat samples . This cross-reactivity is supported by the high conservation of NPM1 protein sequence between species, with human Nucleophosmin sharing approximately 95% amino acid sequence identity with both mouse and rat Nucleophosmin . When working with other species, researchers should first conduct validation experiments, as cross-reactivity may occur but has not always been specifically tested. For example, when asked about goat tissues, suppliers have indicated that while not specifically validated, there is a reasonable probability of cross-reactivity due to sequence homology .

In which tissues is NPM1 normally expressed?

NPM1 demonstrates widespread expression across multiple tissue types. According to compiled research evidence, NPM1 expression has been documented in:

• Hematopoietic tissues: Bone marrow, lymphoblasts, B-cell and T-cell lymphoma tissues

• Reproductive tissues: Testis, placenta, amnion

• Major organs: Brain, kidney, lung, liver, prostate

• Pathological samples: Various carcinomas (cervix, colon), leukemic cells

This extensive expression profile is supported by multiple published studies, with specific PubMed references confirming expression in these tissues . When designing tissue-specific experiments, researchers should account for this broad expression pattern and include appropriate positive controls.

How can NPM1 antibodies be used to investigate NPM1 mutations in AML?

For investigating NPM1 mutations in AML, researchers should implement a multi-technique approach:

• Immunohistochemistry method: Use NPM1 antibodies to detect aberrant cytoplasmic localization characteristic of NPM1c mutations. This requires careful optimization of fixation protocols to preserve subcellular localization patterns.

• Western blot analysis: NPM1 antibodies can detect both wildtype and mutant proteins, though their close molecular weights may require high-resolution gels (10-12% polyacrylamide) for proper separation.

• Immunofluorescence approach: For detailed subcellular localization studies, combine NPM1 antibodies with nuclear/nucleolar markers to quantify cytoplasmic versus nuclear distribution ratios in patient samples.

Research shows that NPM1 mutations result in increased export of NPM1 to the cytoplasm (NPM1c) which is associated with multiple transforming events, including aberrant upregulation of MEIS1 . This characteristic localization pattern serves as a useful diagnostic and research indicator when using NPM1 antibodies.

What controls should be included when studying NPM1 haploinsufficiency?

When investigating NPM1 haploinsufficiency, comprehensive controls must include:

• Wildtype NPM1 expression controls: Include samples with confirmed normal NPM1 expression levels to establish baseline expression patterns.

• Heterozygous NPM1+/- models: Utilize validated Npm1+/- mouse models as described in literature to accurately represent haploinsufficiency.

• NPM1c mutation models: Include Npm1flox-cA/+ models to distinguish between effects of haploinsufficiency alone versus those combined with cytoplasmic NPM1 mutation.

• Protein quantification controls: Implement precise quantitative Western blotting with loading controls to accurately measure NPM1 protein levels.

Research demonstrates that NPM1 haploinsufficiency paired with MEIS1 overexpression is sufficient to induce fully penetrant AML in mice that transcriptionally resembles human NPM1c AML . This indicates haploinsufficiency itself contributes significantly to leukemogenesis independent of cytoplasmic localization.

How can NPM1 antibodies be used to study the MEIS1-SMC4 axis in AML?

To investigate the MEIS1-SMC4 axis in AML using NPM1 antibodies, researchers should implement this methodological workflow:

• Co-immunoprecipitation approach: Use NPM1 antibodies for protein complex isolation followed by MEIS1 and SMC4 detection to investigate physical interactions.

• Chromatin immunoprecipitation (ChIP) analysis: Implement sequential ChIP with NPM1 and MEIS1 antibodies to identify co-regulated genomic regions, particularly at the SMC4 promoter.

• Proximity ligation assays: Apply this technique to visualize potential NPM1-MEIS1 protein interactions in situ with single-molecule resolution.

• Comparative analysis between models: Compare binding patterns between NPM1 wildtype, NPM1 haploinsufficient, and NPM1c AML cells.

Research has revealed that NPM1 haploinsufficiency alters MEIS1-binding occupancies such that it binds the promoter of the oncogene structural maintenance of chromosome protein 4 (SMC4) in NPM1 haploinsufficient AML cells but not in NPM1 wildtype cells . This axis represents a potential therapeutic target in NPM1-mutated AML.

What tissue-specific protocols are recommended for IHC with NPM1 antibodies?

When performing immunohistochemistry with NPM1 antibodies across different tissue types, researchers should implement these tissue-specific optimizations:

• Kidney tissues: For kidney samples, use mild antigen retrieval methods (citrate buffer pH 6.0, 95°C for 20 minutes) to preserve tissue morphology while ensuring adequate antigen exposure. NPM1 is highly expressed in kidney tissue , making it essential to optimize antibody dilutions (typically 1:200-1:500) to avoid background while maintaining specific signal.

• Bone marrow and hematopoietic tissues: These require special consideration for fixation. Brief fixation (4-6 hours) in 10% neutral buffered formalin followed by decalcification in EDTA-based solutions (rather than acid-based) better preserves NPM1 antigenicity. For bone marrow biopsies, reduced antibody concentration (1:300-1:500) often yields optimal results.

• Frozen tissue sections: For frozen tissue applications, researchers should use acetone or methanol fixation (10 minutes at -20°C) rather than paraformaldehyde to maintain NPM1 antibody reactivity . This approach is particularly useful for sensitive applications or when rapid processing is required.

Each tissue type may require empirical optimization of these parameters to achieve optimal signal-to-noise ratios.

What are the critical factors for successful Western blotting with NPM1 antibodies?

Successful Western blotting with NPM1 antibodies depends on several critical technical factors:

• Sample preparation: Nuclear proteins require efficient extraction. Use specialized nuclear extraction buffers containing 10-20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, and 1 mM DTT with protease inhibitors. For detecting cytoplasmic NPM1c forms, separate fractionation of nuclear and cytoplasmic components is essential.

• Gel percentage optimization: As NPM1 protein has a molecular weight of approximately 37 kDa, 10-12% polyacrylamide gels provide optimal resolution. For detecting post-translational modifications or distinguishing between wildtype and mutant forms, gradient gels (4-15%) may offer better separation.

• Blocking conditions: BSA-free formulations of NPM1 antibodies are available and recommended when using BSA as a blocking agent to prevent potential cross-reactivity . Alternatively, 5% non-fat dry milk in TBST provides effective blocking with standard antibody formulations.

• Detection sensitivity: For low-abundance NPM1 forms or in samples with limited material (like clinical specimens), enhanced chemiluminescence detection systems with longer exposure times (2-5 minutes) may be necessary for visualizing weaker signals while maintaining specificity.

Researchers have reported successful Western blotting results with anti-NPM1 antibody PB9341 across multiple tissue types including kidney and testis .

How should NPM1 antibodies be validated for novel research applications?

When validating NPM1 antibodies for novel research applications, implement this comprehensive validation strategy:

• Positive and negative control selection: Include:

  • Positive controls: Cell lines with confirmed NPM1 expression (OCI-AML3 for NPM1-mutated cells, HL-60 for wildtype expression)

  • Negative controls: NPM1-knockout models or siRNA-depleted samples

  • Isotype controls: Matched antibody isotypes without NPM1 specificity

• Antibody specificity verification:

  • Blocking peptide competition assays: Pre-incubate antibody with NPM1-specific blocking peptide before application to confirm signal specificity

  • Multiple antibody comparison: Use at least two antibodies targeting different NPM1 epitopes to verify concordant results

  • Western blot molecular weight verification: Confirm detection at expected 37 kDa size

• Cross-application validation:

  • When adapting from validated applications (like IHC or WB) to novel techniques (like ChIP-seq or mass cytometry), perform parallel validations using established methods alongside the new application

• Statistical quantification:

  • Implement quantitative image analysis for immunohistochemistry signals

  • Calculate signal-to-noise ratios across technical replicates

  • Establish thresholds for positive signal determination based on control samples

Suppliers indicate that for novel applications outside the validated uses, researchers can participate in innovator award programs that recognize successful validation of new applications .

How should researchers interpret contradictory NPM1 localization patterns?

When encountering contradictory NPM1 localization patterns, researchers should implement this systematic analysis approach:

• Technical vs. biological variation assessment:

  • Repeat experiments with alternative fixation methods (paraformaldehyde vs. methanol)

  • Test multiple antibody clones targeting different NPM1 epitopes

  • Quantify nuclear:cytoplasmic ratios across multiple fields and samples

• Context-specific interpretation:

  • In NPM1c AML samples, expect significant cytoplasmic localization due to mutation-driven nuclear export

  • In stress conditions (oxidative stress, nucleolar stress), temporary relocalization from nucleolus to nucleoplasm may occur

  • During mitosis, normal NPM1 dispersal throughout the cell can be mistaken for aberrant localization

• Mutation status correlation:

  • Sequence samples showing unexpected localization to identify potential novel NPM1 mutations

  • Correlate localization patterns with specific NPM1 mutation variants (Type A/B/C) which may exhibit different degrees of cytoplasmic localization

• Subcellular fractionation verification:

  • Perform complementary biochemical fractionation (nuclear/cytoplasmic) followed by Western blotting to quantitatively validate microscopy observations

What are common pitfalls when using NPM1 antibodies in leukemia research?

Researchers using NPM1 antibodies in leukemia research should be aware of these common pitfalls and their solutions:

• Mutant vs. wildtype NPM1 detection challenges:

  • Pitfall: Standard antibodies may not distinguish between wildtype and mutant NPM1 proteins by size alone

  • Solution: Combine with subcellular fractionation or use immunofluorescence to assess localization differences; consider mutation-specific antibodies for certain NPM1 mutations

• Misinterpretation of haploinsufficiency effects:

  • Pitfall: Attributing all phenotypes to cytoplasmic NPM1 when haploinsufficiency itself contributes significantly

  • Solution: Include Npm1+/- models without cytoplasmic mutation to isolate haploinsufficiency effects

• Cell fixation artifacts:

  • Pitfall: Overfixation can mask cytoplasmic NPM1 or create artificial patterns

  • Solution: Optimize fixation conditions with time-course experiments; compare multiple fixation methods

• Heterogeneity in patient samples:

  • Pitfall: Bulk analysis obscuring subpopulation differences in NPM1 expression/localization

  • Solution: Implement single-cell techniques or microdissection of specific regions; quantify cell-to-cell variation

• MEIS1 expression confounding:

  • Pitfall: Not accounting for MEIS1 levels when interpreting NPM1 antibody results

  • Solution: Include parallel MEIS1 antibody staining as MEIS1 overexpression collaborates with NPM1 haploinsufficiency in leukemogenesis

Research indicates that NPM1 haploinsufficiency paired with MEIS1 overexpression is sufficient to induce AML in mice, highlighting the importance of considering multiple factors in interpreting NPM1 antibody results .

How can researchers detect NPM1 mutations in clinical samples using antibodies?

For detecting NPM1 mutations in clinical samples using antibodies, researchers should implement this multi-layered approach:

• Immunohistochemical detection strategy:

  • Utilize NPM1 antibodies for immunohistochemistry focusing on subcellular localization patterns

  • Employ standardized scoring systems: 0 (nuclear only), 1+ (<25% cytoplasmic), 2+ (25-50% cytoplasmic), 3+ (>50% cytoplasmic)

  • Calculate cytoplasmic:nuclear ratio using digital image analysis for objective quantification

• Verification methodology:

  • Confirm antibody-based findings with molecular techniques (PCR, sequencing)

  • Correlate immunohistochemical patterns with specific mutation variants

  • Implement control slides with known NPM1 mutation status alongside test samples

• Sample preparation considerations:

  • Standardize fixation protocols (10% neutral buffered formalin, 24 hours) to maintain consistent subcellular localization

  • For bone marrow biopsies, limit decalcification time to preserve antigenicity

  • Process samples within 30 minutes of collection when analyzing fresh specimens

• Interpretation guidelines:

  • Strong cytoplasmic NPM1 staining with antibodies should prompt molecular confirmation

  • Absence of cytoplasmic staining has high negative predictive value for NPM1 mutations

  • Consider variant-specific staining patterns as different NPM1 mutation types may show subtle localization differences

While antibody-based detection provides valuable information, researchers should note that products are intended for research use only and not for diagnostic purposes without proper validation and regulatory approval .

What experimental approaches can address the functional relationship between NPM1 and MEIS1?

To investigate the functional relationship between NPM1 and MEIS1, researchers should consider these methodological approaches:

• Protein-protein interaction analysis:

  • Co-immunoprecipitation with NPM1 antibodies followed by MEIS1 detection

  • Proximity ligation assays to visualize potential interactions in situ

  • FRET or BiFC techniques to detect direct protein interactions in living cells

• Transcriptional regulation assessment:

  • ChIP-seq using both NPM1 and MEIS1 antibodies to identify co-regulated genomic regions

  • Sequential ChIP (ChIP-reChIP) to identify genomic sites co-bound by both factors

  • Correlate binding patterns with gene expression data from RNA-seq

• Functional dependency experiments:

  • Knockdown/knockout studies targeting NPM1 followed by assessment of MEIS1 binding patterns

  • Test whether NPM1 haploinsufficiency alters MEIS1 binding at specific loci such as SMC4

  • Assess therapeutic responses to Menin-MLL inhibitors in relation to NPM1 status and MEIS1 expression

• Animal model approaches:

  • Compare Npm1+/+ with Meis1 expression, Npm1+/- with Meis1 expression, and Npm1flox-cA/+ models

  • Assess leukemogenic potential and transcriptional profiles

  • Evaluate therapeutic vulnerabilities unique to each genetic combination

Research has established that NPM1 haploinsufficiency alters MEIS1-binding occupancies, particularly at the SMC4 promoter, and that the MEIS1-SMC4 axis represents a potential therapeutic target in NPM1-mutated AML .

How can NPM1 antibodies be utilized in investigating therapeutic resistance mechanisms?

For investigating therapeutic resistance mechanisms using NPM1 antibodies, researchers should implement these methodological approaches:

• Longitudinal sample analysis:

  • Compare NPM1 localization and expression patterns in matched diagnosis/relapse samples

  • Quantify changes in subcellular distribution using digital image analysis

  • Correlate shifts in NPM1 localization with treatment history and response

• Therapeutic response monitoring:

  • Assess NPM1 and MEIS1 expression changes during treatment with Menin-MLL inhibitors

  • Monitor the MEIS1-SMC4 axis as a resistance biomarker using NPM1 antibodies in combination with MEIS1 and SMC4 detection

  • Implement sequential sampling with standardized IHC protocols to ensure comparable results

• In vitro resistance modeling:

  • Develop resistant cell lines through drug exposure and analyze NPM1/MEIS1 patterns

  • Use NPM1 antibodies to track potential localization changes accompanying resistance

  • Combine with functional assays to correlate protein changes with phenotypic resistance

• Protein complex dynamics:

  • Employ NPM1 antibodies for immunoprecipitation followed by mass spectrometry

  • Compare protein interaction networks between sensitive and resistant states

  • Identify novel interaction partners potentially mediating resistance mechanisms

Research indicates that the MEIS1-SMC4 axis is a potential therapeutic target in NPM1c AML, and monitoring changes in this axis may provide insights into resistance mechanisms .

What approaches are recommended for studying NPM1 in combination with epigenetic modifications?

For studying NPM1 in relation to epigenetic modifications, researchers should implement these methodological approaches:

• Integrated ChIP-seq analysis:

  • Perform parallel ChIP-seq using NPM1 antibodies alongside antibodies targeting histone modifications (H3K4me3, H3K27me3, H3K27ac)

  • Implement sequential ChIP to identify genomic regions where NPM1 binding coincides with specific histone marks

  • Compare epigenetic landscapes between NPM1 wildtype, haploinsufficient, and mutant conditions

• Nucleolar organization assessment:

  • Combine NPM1 antibody staining with DNA methylation analysis (5-mC, 5-hmC immunostaining)

  • Correlate nucleolar NPM1 levels with rDNA methylation status

  • Implement 3D imaging to assess spatial relationships between NPM1 and epigenetic modifications

• Protein interaction studies:

  • Use NPM1 antibodies for co-immunoprecipitation followed by detection of epigenetic modifiers (DNMTs, HDACs, etc.)

  • Perform proximity ligation assays between NPM1 and epigenetic machinery components

  • Validate interactions with functional studies assessing epigenetic mark changes upon NPM1 manipulation

• Functional correlation experiments:

  • Manipulate NPM1 levels and assess global and locus-specific epigenetic changes

  • Combine with MEIS1 overexpression to determine collaborative effects on the epigenome

  • Correlate epigenetic patterns with transcriptional outputs using RNA-seq

This research area is particularly relevant given that NPM1 haploinsufficiency alters MEIS1-binding occupancies , which may involve epigenetic mechanisms that could be therapeutic targets.