NOL4L Antibody

What is NOL4L and why is it significant for research?

NOL4L (Nucleolar protein 4-like) is a nuclear protein encoded by a gene located on chromosome 20 (20q11.21) in humans. It plays critical roles in chromatin remodeling and transcriptional regulation, making it essential for maintaining genomic stability and gene expression. The protein is particularly significant in research because parts of the NOL4L gene have been found to fuse with RUNX1 and PAX5 in acute myeloid leukemia and acute lymphoblastic leukemia, respectively . Recent studies in zebrafish have shown that nol4l mRNA is expressed in multiple organs during embryogenesis, including parts of the brain, spinal cord, pronephros, hematopoietic cells, and gut . This diverse expression pattern suggests NOL4L may have tissue-specific functions worth investigating in various disease contexts.

How do I select the most appropriate NOL4L antibody for my specific research application?

When selecting a NOL4L antibody, consider these critical factors:

• Application compatibility: Different NOL4L antibodies are validated for specific applications. For instance, Abcam's ab237758 is suitable for WB, IHC-P, and ICC/IF techniques , while Assay Genie's PACO40186 is validated for ELISA, WB, IHC, and IF .

• Species reactivity: Most commercially available NOL4L antibodies are primarily validated for human samples , though some may cross-react with other species based on sequence homology.

• Immunogen specificity: Check the immunogen used. For example, ab237758 uses a recombinant fragment within human NOL4L amino acids 100-300 , while PACO40186 uses a similar region (101-300AA) . This is important for epitope-specific detection.

• Validation data: Review existing validation data in tissues relevant to your study. The antibodies have been tested in various cell lines including PC-3, A549, HepG2, and K562 .

• Published citations: Prioritize antibodies that have been successfully used in peer-reviewed publications related to your area of research.

What are the critical differences between NOL4 and NOL4L antibodies?

While NOL4 and NOL4L are related proteins, they are distinct targets requiring specific antibodies:

• Molecular weight differences: NOL4 antibodies typically detect proteins between 64-71 kDa , while NOL4L antibodies detect proteins around 47-48 kDa .

• Expression patterns: NOL4 is strongly expressed in brain tissue, testis tissue, and has been detected in rat brain tissue , whereas NOL4L shows a broader distribution pattern including thyroid, testis, adrenal gland, small intestine, and various cancer cell lines .

• Immunogen design: NOL4 antibodies like Proteintech's 14802-1-AP are generated using NOL4-specific fusion proteins , while NOL4L antibodies target regions specific to NOL4L protein, often focusing on the 100-300 amino acid region .

• Cross-reactivity profiles: Most NOL4L antibodies have been specifically validated for human samples , while some NOL4 antibodies have demonstrated cross-reactivity with mouse and rat samples .

• Application optimization: Dilution recommendations often differ, with NOL4 antibodies typically requiring 1:500-1:1000 dilutions for Western blot , while NOL4L antibodies may use varying dilutions depending on the application (e.g., 1:500 for WB, 1:100 for IHC) .

Researchers should carefully verify they are ordering the correct antibody (NOL4 vs. NOL4L) based on their target of interest to avoid experimental confusion and misinterpretation of results.

What are the optimal protocols for using NOL4L antibodies in Western blotting?

For optimal Western blot results with NOL4L antibodies, follow these methodological considerations:

• Sample preparation: Based on validation data, prepare whole cell lysates from human cell lines similar to those successfully tested (PC-3, A549, HepG2, K562) . Use standard lysis buffers containing protease inhibitors.

• Protein loading: Load 20-30 μg of total protein per lane on SDS-PAGE gels (10-12%).

• Antibody dilution: For Abcam's ab237758, use a 1:500 dilution . For Assay Genie's PACO40186, use between 1:500-1:5000 dilution, with 0.3μg/ml being an effective concentration based on published results .

• Detection system: Use HRP-conjugated secondary antibodies against rabbit IgG at 1:10,000 dilution, as this ratio has been validated in published protocols .

• Expected band size: Look for bands at approximately 47-48 kDa, which corresponds to the predicted molecular weight of NOL4L .

• Controls: Include positive control lysates from cells known to express NOL4L, such as PC-3 or HepG2 cells .

• Blocking: Use 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature to minimize background.

• Washing: Perform 3-5 washes with TBST for 5-10 minutes each after both primary and secondary antibody incubations.

The observed band at approximately 48 kDa in validated cell lines confirms specific detection of the NOL4L protein .

How can I optimize immunohistochemistry (IHC) procedures for NOL4L detection in tissue samples?

For successful IHC detection of NOL4L in tissues, implement these optimized procedures:

• Tissue preparation: Use 4% paraformaldehyde-fixed, paraffin-embedded tissue sections cut at 4-6 μm thickness. Validated tissues include thyroid, testis, adrenal gland, and small intestine .

• Antigen retrieval: Perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) for 15-20 minutes. This step is critical for unmasking NOL4L epitopes masked during fixation.

• Blocking endogenous activity: Block endogenous peroxidase activity with 3% H₂O₂ for 10 minutes, followed by protein blocking with 5% normal goat serum for 30 minutes.

• Antibody dilution: For Abcam's ab237758, use a 1:100 dilution . For Assay Genie's PACO40186, use dilutions between 1:20-1:200 . Incubate at 4°C overnight for optimal binding.

• Secondary detection: Apply appropriate HRP-conjugated secondary antibody against rabbit IgG for 30-60 minutes at room temperature.

• Visualization: Develop with DAB substrate and counterstain with hematoxylin.

• Controls: Include known positive tissues based on validation data (thyroid and testis are good positive controls) . Always include a negative control by omitting the primary antibody.

• Signal interpretation: NOL4L typically shows nuclear and nucleolar staining patterns, consistent with its role as a nucleolar protein.

For multiplex IHC studies, consider tyramide signal amplification (TSA) approaches which allow simultaneous detection of multiple markers while preserving tissue morphology.

What are the best approaches for immunofluorescence (IF/ICC) experiments with NOL4L antibodies?

For high-quality immunofluorescence detection of NOL4L in cultured cells:

• Cell culture preparation: Culture cells on glass coverslips to 70-80% confluence. HeLa cells have been validated for NOL4L detection , making them a reliable starting point.

• Fixation and permeabilization: Fix cells with 4% paraformaldehyde for 15 minutes at room temperature, then permeabilize with 0.2% Triton X-100 in PBS for 10 minutes.

• Blocking: Block non-specific binding with 5% normal goat serum in PBS containing 0.1% Triton X-100 for 1 hour at room temperature.

• Primary antibody incubation: Apply NOL4L antibody at optimized dilutions - 1:100 for ab237758 or 1:50-1:200 for PACO40186 . Incubate overnight at 4°C in a humidified chamber.

• Secondary antibody application: Use fluorophore-conjugated secondary antibodies such as Alexa Fluor 488®-conjugated goat anti-rabbit IgG at 1:500 dilution . Incubate for 1 hour at room temperature in the dark.

• Nuclear counterstaining: Counterstain nuclei with DAPI (1 μg/mL) for 5 minutes.

• Mounting and imaging: Mount slides using anti-fade mounting medium and image using confocal microscopy with appropriate filter sets.

• Expected localization: Look for primarily nuclear and nucleolar localization patterns, consistent with NOL4L's role as a nucleolar protein .

• Co-localization studies: Consider co-staining with nucleolar markers like fibrillarin to confirm nucleolar localization of NOL4L.

For quantitative analysis, use image analysis software to measure fluorescence intensity and co-localization coefficients with other proteins of interest.

How can I validate NOL4L antibody specificity in my experimental system?

Rigorous validation of NOL4L antibody specificity is essential for reliable research outcomes. Implement these comprehensive approaches:

• Genetic manipulation controls:

  • Generate CRISPR/Cas9 NOL4L knockouts in your cell line of interest

  • Use siRNA-mediated knockdown of NOL4L

  • Compare antibody signal between wild-type and NOL4L-depleted samples across all applications (WB, IHC, IF)

• Peptide competition assay: Pre-incubate the NOL4L antibody with excess immunizing peptide (the sequence used to generate the antibody, typically amino acids 100-300) . Signal disappearance confirms specificity.

• Multiple antibody validation: Test at least two NOL4L antibodies targeting different epitopes. Concordant results increase confidence in specificity.

• Cross-species reactivity assessment: While most NOL4L antibodies are validated for human samples , test reactivity in model organisms if relevant to your research, recognizing that sequence homology will determine cross-reactivity.

• Mass spectrometry verification: Perform immunoprecipitation with your NOL4L antibody followed by mass spectrometry to confirm that NOL4L is the primary precipitated protein.

• Recombinant protein detection: Test antibody against purified recombinant NOL4L protein alongside cell lysates to confirm expected molecular weight detection.

• Expression pattern correlation: Compare antibody staining patterns with known NOL4L mRNA expression patterns in tissues of interest .

This multi-faceted validation approach ensures that any observed signals genuinely represent NOL4L rather than non-specific or cross-reactive signals.

What are the key considerations when studying NOL4L in cancer models and oncology research?

When investigating NOL4L in cancer contexts, address these critical research considerations:

• Gene fusion analysis: NOL4L has been shown to fuse with RUNX1 and PAX5 in acute myeloid leukemia and acute lymphoblastic leukemia, respectively . Design experiments to detect these fusion proteins using antibodies that target regions preserved in the fusion proteins.

• Expression pattern characterization: Systematically analyze NOL4L expression across cancer types using tissue microarrays. Published data demonstrates NOL4L detection in prostate (PC-3), lung (A549), liver (HepG2), and leukemia (K562) cancer cell lines .

• Functional studies: Implement CRISPR/Cas9 or shRNA approaches to modulate NOL4L expression in cancer cells, then assess:

  • Proliferation and cell cycle progression

  • Migration and invasion capabilities

  • Response to therapeutics

  • Chromatin structure alterations

• Mechanistic investigations: As NOL4L is involved in chromatin remodeling and transcriptional regulation , perform:

  • ChIP-seq to identify NOL4L binding sites across the genome

  • RNA-seq after NOL4L modulation to identify regulated genes

  • Co-immunoprecipitation to identify protein interaction partners

• Clinical correlation analyses: Correlate NOL4L expression/localization with:

  • Patient survival outcomes

  • Treatment response

  • Disease progression markers

  • Histopathological features

• Subcellular localization dynamics: Monitor changes in NOL4L localization during cancer progression using fractionation techniques and immunofluorescence microscopy.

• Post-translational modification assessment: Investigate how phosphorylation, ubiquitination, or other modifications affect NOL4L function in cancer contexts.

These approaches will help establish whether NOL4L functions as an oncogene, tumor suppressor, or biomarker in specific cancer types.

How do expression patterns of NOL4L vary across different tissues and developmental stages?

NOL4L demonstrates complex expression patterns that vary by tissue type and developmental stage:

• Embryonic expression patterns: Studies in zebrafish embryos have revealed that nol4l mRNA is expressed in multiple developing organs, including:

  • Specific regions of the brain

  • Spinal cord

  • Pronephros

  • Hematopoietic cells

  • Gut

• Adult human tissue expression: Immunohistochemical studies using NOL4L antibodies have demonstrated expression in:

  • Thyroid tissue

  • Testis

  • Adrenal gland

  • Small intestine

• Cell line expression patterns: NOL4L protein has been detected in multiple human cell lines:

  • PC-3 (prostate adenocarcinoma)

  • A549 (lung carcinoma)

  • HepG2 (hepatocellular carcinoma)

  • K562 (chronic myelogenous leukemia)

  • HeLa (cervical adenocarcinoma)

• Subcellular localization: Predominantly nuclear and nucleolar localization has been observed in immunofluorescence studies , consistent with its predicted function in transcriptional regulation.

• Temporal regulation: While comprehensive temporal expression data across development is limited, the zebrafish studies suggest potential roles during organogenesis and tissue differentiation .

For researchers interested in NOL4L developmental biology, a systematic approach using antibodies validated for your species of interest combined with in situ hybridization techniques would provide complementary protein and mRNA expression data. Single-cell RNA-seq datasets could also be mined to identify cell type-specific expression patterns within complex tissues.

How can I overcome common challenges when using NOL4L antibodies in Western blotting?

When troubleshooting Western blot issues with NOL4L antibodies, implement these methodological solutions:

• No signal or weak signal:

  • Increase antibody concentration: Try 1:250 dilution instead of 1:500 for ab237758

  • Extend primary antibody incubation to overnight at 4°C

  • Increase protein loading to 40-50 μg per lane

  • Use enhanced sensitivity ECL substrate systems

  • Verify expression in your cell type; consider using validated positive controls like PC-3 or HepG2 cells

• Multiple bands or incorrect molecular weight:

  • Ensure complete protein denaturation by heating samples at 95°C for 5 minutes

  • Use freshly prepared samples with protease inhibitors to prevent degradation

  • Increase gel percentage (12-15%) for better resolution around 48 kDa

  • Validate bands using siRNA knockdown to identify specific NOL4L signal

  • Consider post-translational modifications or isoforms that may cause size shifts

• High background:

  • Increase blocking duration (2 hours) or concentration (5% to 7% milk/BSA)

  • Add 0.1% Tween-20 to antibody dilution buffers

  • Extend washing steps (5 washes, 10 minutes each)

  • Reduce secondary antibody concentration to 1:15,000 or 1:20,000

  • Prepare fresh buffers and filter solutions if necessary

• Membrane optimization:

  • For NOL4L detection, PVDF membranes may provide better results than nitrocellulose

  • Optimize transfer conditions: for 48 kDa proteins, 100V for 60-90 minutes is typically effective

  • Consider wet transfer systems for more consistent results

• Antibody-specific considerations:

  • Some batches may perform differently; check lot-specific validation data

  • Store antibody aliquots at -20°C to avoid freeze-thaw cycles

  • Centrifuge antibody vial before use to recover all material

What strategies can address poor signal-to-noise ratios in NOL4L immunohistochemistry?

To improve signal-to-noise ratios in NOL4L immunohistochemistry:

• Optimal antigen retrieval:

  • Compare citrate buffer (pH 6.0) versus EDTA buffer (pH 9.0) retrieval methods

  • Test different retrieval durations (10, 15, 20 minutes)

  • Allow slides to cool slowly in retrieval solution for 20 minutes after heating

  • Consider enzymatic retrieval with proteinase K as an alternative approach

• Fixation optimization:

  • Limit fixation time to 24 hours maximum

  • Use neutral-buffered formalin rather than other fixatives

  • Process tissues promptly after collection

  • For prospective studies, consider testing fixation times (6, 12, 24 hours)

• Blocking enhancements:

  • Implement dual blocking: first with hydrogen peroxide (3%, 10 minutes), then with protein block

  • Add 0.3% Triton X-100 to blocking solution to improve antibody penetration

  • Extend blocking time to 1-2 hours at room temperature

  • Use species-specific serum matching your secondary antibody

• Antibody dilution optimization:

  • Perform systematic dilution series from 1:20 to 1:200

  • Compare overnight 4°C versus 1-hour room temperature incubation

  • Add 0.05% Tween-20 to antibody diluent to reduce non-specific binding

  • Consider using antibody diluents containing background-reducing components

• Detection system enhancements:

  • Implement polymer-based detection systems for higher sensitivity

  • Consider tyramide signal amplification for low-expression targets

  • Optimize DAB development time with microscopic monitoring

  • Use automated staining platforms for consistent results

• Tissue-specific considerations:

  • Higher background is common in certain tissues (liver, kidney); titrate antibody accordingly

  • For thyroid tissue, which shows positive NOL4L staining , particular attention to blocking endogenous biotin may be necessary

  • Pre-absorb antibody with tissue powder from negative control tissues

How should I interpret contradictory results between different NOL4L antibodies or detection methods?

When facing contradictory results with NOL4L antibodies, apply this systematic analytical framework:

• Epitope-based analysis:

  • Map the specific epitopes recognized by each antibody

  • Antibodies targeting different regions of NOL4L (e.g., N-terminal vs. C-terminal) may detect different isoforms

  • Compare immunogen sequences: most NOL4L antibodies target amino acids 100-300 , but subtle differences may exist

• Method-specific considerations:

  • WB detects denatured proteins while IF/IHC detect native conformations

  • Some epitopes may be inaccessible in fixed tissues but accessible in denatured lysates

  • mRNA expression (RT-PCR/RNA-seq) may not correlate with protein levels due to post-transcriptional regulation

• Validation hierarchy implementation:

  • Establish a validation hierarchy: genetic knockdown/knockout > peptide competition > multiple antibody agreement

  • Perform side-by-side comparisons using identical samples and protocols

  • Quantify results using objective metrics rather than subjective assessments

• Technical variables analysis:

  • Document fixation methods, antigen retrieval, blocking conditions, and detection systems

  • Test whether contradictions persist across multiple cell lines/tissues

  • Consider lot-to-lot antibody variations by requesting lot-specific validation data

• Biological interpretation framework:

  • Consider context-dependent protein expression, localization, or modification

  • Evaluate whether contradictions reflect actual biological complexity rather than technical issues

  • Investigate potential splice variants or post-translational modifications using protein databases

• Resolution strategies:

  • Generate tagged NOL4L constructs (e.g., FLAG-tag) for antibody-independent detection

  • Implement mass spectrometry for unbiased protein identification

  • Use genetic approaches (CRISPR editing) to introduce epitope tags into endogenous NOL4L

  • Perform absolute quantification using recombinant protein standards

When publishing potentially contradictory results, transparently report all methodological details and discuss alternative interpretations of the data.

How can NOL4L antibodies be applied in chromatin immunoprecipitation (ChIP) studies?

While NOL4L antibodies haven't been extensively validated for ChIP applications in the provided resources, researchers can adapt them for chromatin studies following these methodological guidelines:

• Antibody selection criteria:

  • Choose antibodies validated for immunoprecipitation or those known to recognize native protein conformations

  • Select antibodies targeting DNA-binding domains if the exact NOL4L chromatin interaction mechanism is known

  • Consider generating ChIP-grade antibodies if commercial options yield suboptimal results

• Protocol optimization:

  • Begin with standard ChIP protocols for nuclear proteins

  • Test multiple crosslinking conditions: 1% formaldehyde for varying durations (5, 10, 15 minutes)

  • Optimize sonication conditions to generate 200-500 bp fragments

  • Test various antibody concentrations (2-10 μg per ChIP reaction)

  • Include sequential ChIP (re-ChIP) to identify genomic loci where NOL4L co-localizes with known interaction partners

• Controls and validation:

  • Include technical controls: IgG negative control, histone H3 positive control

  • Include biological controls: NOL4L-depleted cells

  • Validate enrichment at predicted binding sites using qPCR before proceeding to sequencing

  • Confirm antibody specificity in your experimental system using Western blotting

• Data analysis considerations:

  • Use appropriate peak calling algorithms suitable for transcription factors

  • Integrate with RNA-seq data to correlate binding with transcriptional outcomes

  • Perform motif analysis to identify potential DNA recognition sequences

  • Compare NOL4L binding sites with known chromatin features (histone modifications, chromatin accessibility)

• Advanced ChIP applications:

  • ChIP-seq: Genome-wide binding profile identification

  • CUT&RUN: Alternative to traditional ChIP with improved signal-to-noise ratio

  • ChIP-exo: Higher resolution binding site mapping

  • HiChIP: Combining chromatin conformation with protein binding

Given NOL4L's role in chromatin remodeling and transcriptional regulation , ChIP studies could provide valuable insights into its genomic targets and regulatory mechanisms.

What approaches can be used to study NOL4L protein-protein interactions?

To characterize NOL4L protein interaction networks, implement these complementary methods:

• Co-immunoprecipitation (Co-IP):

  • Use NOL4L antibodies to pull down NOL4L and associated proteins

  • Perform in both directions: NOL4L IP followed by partner detection and partner IP followed by NOL4L detection

  • Include appropriate controls (IgG, lysate from NOL4L-depleted cells)

  • Optimize lysis conditions to preserve nuclear and nucleolar protein interactions

  • Consider native versus crosslinked conditions to capture different interaction strengths

• Proximity labeling approaches:

  • Generate BioID-NOL4L or TurboID-NOL4L fusion proteins

  • Express in relevant cell types and induce proximity labeling

  • Purify biotinylated proteins and identify by mass spectrometry

  • This approach captures both stable and transient interactions in native cellular environments

• Mass spectrometry-based interaction profiling:

  • Perform immunoprecipitation with NOL4L antibodies

  • Analyze by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

  • Use quantitative approaches (SILAC, TMT) to distinguish specific from non-specific interactions

  • Implement crosslinking mass spectrometry (XL-MS) to map interaction interfaces

• Fluorescence-based interaction assays:

  • Fluorescence resonance energy transfer (FRET) between NOL4L and candidate partners

  • Fluorescence recovery after photobleaching (FRAP) to assess dynamics

  • Bimolecular fluorescence complementation (BiFC) to visualize interactions in living cells

  • Fluorescence correlation spectroscopy (FCS) for interaction kinetics

• Yeast two-hybrid screening:

  • Use NOL4L as bait to screen for novel interaction partners

  • Validate hits using orthogonal methods in mammalian systems

  • Consider membrane yeast two-hybrid for potential membrane-associated interactions

• Domain mapping:

  • Generate truncated versions of NOL4L to identify interaction domains

  • Perform mutagenesis of key residues to disrupt specific interactions

  • Use peptide arrays to map minimal binding motifs

Given NOL4L's roles in transcriptional regulation , focus particularly on interactions with chromatin modifiers, transcription factors, and nucleolar proteins to understand its functional networks.

How can I integrate NOL4L protein studies with transcriptomic and genomic data analysis?

To create a comprehensive multi-omics understanding of NOL4L function, implement these integrative approaches:

• Transcriptomic integration:

  • Perform RNA-seq after NOL4L modulation (knockdown/overexpression)

  • Compare differential gene expression patterns with:

    • NOL4L protein localization data from immunofluorescence studies

    • Known NOL4L-containing complex functions

    • Tissue-specific expression patterns observed in IHC studies

  • Analyze alternative splicing changes that may result from NOL4L's regulatory functions

• Chromatin landscape integration:

  • Combine NOL4L ChIP-seq data with:

    • Histone modification profiles (H3K4me3, H3K27ac, H3K9me3)

    • Chromatin accessibility data (ATAC-seq, DNase-seq)

    • RNA polymerase II occupancy

  • Generate genomic interaction maps using Hi-C or related methods

  • Overlay NOL4L binding sites with known regulatory elements

• Protein-centric multi-omics:

  • Correlate NOL4L protein levels (quantified by Western blot) with mRNA levels across tissues

  • Integrate NOL4L proteomics data with transcriptomics and genomics datasets

  • Use phosphoproteomics to identify signaling pathways that regulate NOL4L function

  • Implement ribosome profiling to assess translational impacts of NOL4L activity

• Disease-relevant integration:

  • Analyze genomic alterations (mutations, copy number variations) affecting NOL4L in disease databases

  • Examine differential expression of NOL4L in cancer datasets (TCGA, ICGC)

  • Correlate IHC-based NOL4L protein levels with patient outcomes

  • Integrate findings on NOL4L gene fusions in leukemia with functional data

• Computational approaches:

  • Apply network analysis to position NOL4L in regulatory networks

  • Use machine learning to identify patterns in multi-omics datasets related to NOL4L function

  • Implement predictive models for NOL4L-dependent phenotypes

  • Perform gene set enrichment analysis on NOL4L-regulated genes

• Visualization and analysis tools:

  • Use genome browsers (UCSC, IGV) to visualize multi-omics data at NOL4L locus

  • Implement Cytoscape for network visualization

  • Use R/Bioconductor packages for integrated analysis

  • Consider specialized multi-omics platforms (MultiOmics Factor Analysis)

This integrated approach provides a systems-level understanding of NOL4L function that transcends any single experimental methodology.