NFIX Antibody

What is NFIX and what cellular functions does it regulate?

NFIX (Nuclear Factor I/X) is a sequence-specific transcription factor belonging to the Nuclear Factor I family. It functions as a CCAAT-binding transcription factor and is also known by several aliases including CTF, NF1A, NF1-X, MRSHSS, NF-I/X, and SOTOS2 . The NFIX protein (Nuclear factor 1 X-type) plays crucial roles in regulating gene expression during development and in various cellular processes.

Research has shown that NFIX is particularly important in hematopoietic stem and progenitor cells (HSPCs), where it promotes ex vivo growth, cytokine hypersensitivity, and survival of primitive hematopoietic populations . NFIX has been identified as a transcriptional regulator of c-Mpl (the thrombopoietin receptor), revealing one molecular pathway through which NFIX influences HSPCs . This regulation appears to be direct, as chromatin immunoprecipitation studies have confirmed NFIX binding to the c-Mpl promoter region .

What types of NFIX antibodies are available for research applications?

Researchers can access several types of NFIX antibodies optimized for different experimental applications:

• Polyclonal antibodies: These are commonly generated in rabbits using synthetic peptides directed toward specific regions of human NFIX, such as the middle region . Polyclonal antibodies recognize multiple epitopes on the NFIX protein, potentially providing stronger signals in applications like Western blotting.

• Region-specific antibodies: These target defined regions of the NFIX protein:

  • Middle region antibodies (e.g., targeting AA 180-260)

  • Other region-specific antibodies targeting various domains (e.g., AA 291-390, AA 181-230, AA 201-440)

• Application-optimized antibodies: NFIX antibodies validated specifically for:

  • Western blotting (WB)

  • Immunohistochemistry (IHC)

  • Enzyme-linked immunosorbent assay (ELISA)

When selecting an NFIX antibody, researchers should consider the specific region of interest and ensure the antibody has been validated for their intended application.

How should NFIX antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of NFIX antibodies are critical for maintaining their activity and specificity. Based on manufacturer recommendations:

• Short-term storage: Store at 2-8°C for up to 1 week .

• Long-term storage: Store at -20°C in small aliquots to prevent freeze-thaw cycles . Repeated freeze-thaw cycles can degrade antibody quality and reduce binding efficiency.

• Working dilutions: Prepare fresh working dilutions on the day of the experiment whenever possible.

• Buffer considerations: Many NFIX antibodies are supplied in 1x PBS buffer with 0.09% (w/v) sodium azide and 2% sucrose . The sodium azide serves as a preservative, while sucrose helps maintain protein stability during freeze-thaw cycles.

• Handling precautions: Avoid contamination, and handle according to good laboratory practices, particularly since sodium azide is toxic.

Following these storage and handling recommendations will help ensure consistent and reliable results when using NFIX antibodies in research protocols.

How can I optimize Western blotting protocols using NFIX antibodies?

Optimizing Western blotting with NFIX antibodies requires careful attention to several parameters:

• Sample preparation:

  • Use fresh tissue/cell lysates whenever possible

  • Include protease inhibitors during lysis to prevent NFIX degradation

  • Normalize protein loading (30-50 μg total protein per lane is typically suitable)

• Antibody dilution and incubation:

  • Start with the manufacturer's recommended dilution (typically 1:500 to 1:2000)

  • Perform a dilution series to determine optimal concentration for your specific sample

  • Incubate primary antibody overnight at 4°C for optimal binding

• Blocking and washing:

  • Use 5% non-fat dry milk or BSA in TBST for blocking

  • Perform at least 3-5 washes (5-10 minutes each) with TBST after both primary and secondary antibody incubations

• Controls:

  • Include positive controls (tissues/cells known to express NFIX)

  • Consider using a blocking peptide control to confirm specificity

  • Use appropriate loading controls (β-actin, GAPDH, etc.)

• Detection:

  • Match your secondary antibody to the host species of your NFIX antibody (typically rabbit for polyclonal antibodies)

  • For weaker signals, consider using high-sensitivity ECL substrates or longer exposure times

The expected molecular weight of NFIX is approximately 55-60 kDa, though isoforms and post-translational modifications can result in multiple bands.

What are best practices for immunohistochemistry (IHC) with NFIX antibodies?

When using NFIX antibodies for immunohistochemistry, consider these methodological best practices:

• Tissue preparation:

  • Fixation: 10% neutral-buffered formalin is typically suitable

  • Section thickness: 4-6 μm is optimal for most applications

  • Deparaffinization and rehydration should be complete to ensure antibody access

• Antigen retrieval:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Pressure cooker or microwave methods may provide better results than water bath methods

• Blocking:

  • Block endogenous peroxidase activity with 0.3-3% hydrogen peroxide

  • Use normal serum (from the same species as the secondary antibody) to reduce background

• Antibody incubation:

  • Dilute according to manufacturer's recommendations (usually 1:100 to 1:500)

  • Incubate at 4°C overnight for optimal staining

  • Consider using a humidity chamber to prevent section drying

• Detection systems:

  • Polymer-based detection systems often provide better sensitivity with less background

  • Counterstain with hematoxylin to visualize tissue architecture

  • Use mounting media appropriate for your detection system (aqueous for fluorescent detection)

• Controls:

  • Include positive control tissues known to express NFIX

  • Include a negative control by omitting primary antibody

  • Consider using tissues from NFIX knockout models as specificity controls if available

Remember that optimal conditions may vary based on the specific NFIX antibody and tissue type being analyzed.

How can I validate the specificity of an NFIX antibody for my research?

Validating antibody specificity is crucial for generating reliable research data. For NFIX antibodies, consider these validation approaches:

• Molecular validation:

  • Western blot analysis showing a band of expected molecular weight (approximately 55-60 kDa)

  • Disappearance of signal when using a blocking peptide specific to the immunogen sequence

  • Signal reduction in samples with NFIX knockdown via siRNA or CRISPR-Cas9

• Immunogen sequence analysis:

  • Verify the immunogen sequence used to generate the antibody

  • For example, a middle region antibody might target: "YFVHTPESGQSDSSNQQGDADIKPLPNGHLSFQDCFVTSGVWNVTELVRV"

  • Check for potential cross-reactivity with other proteins using BLAST analysis

• Species cross-reactivity testing:

  • If you're working with multiple species, confirm reactivity in each species

  • Many NFIX antibodies have predicted homology across species: Human, Mouse, Rat, Cow, Dog, Guinea Pig, Horse, Rabbit, Zebrafish

  • Validate experimentally for your specific species of interest

• Orthogonal validation:

  • Compare results with a second NFIX antibody targeting a different epitope

  • Correlate protein detection with mRNA levels using RT-PCR or RNA-seq

  • Consider using multiple applications (WB, IHC, IF) to confirm consistent detection patterns

• Functional validation:

  • Confirm that observed phenotypes match known NFIX functions

  • For example, in hematopoietic research, changes in c-Mpl expression or cell survival would be consistent with NFIX function

Thorough validation ensures that your experimental observations truly reflect NFIX biology rather than antibody artifacts.

How can NFIX antibodies be used to study transcriptional regulation mechanisms?

NFIX antibodies can provide valuable insights into transcriptional regulation mechanisms through several advanced experimental approaches:

• Chromatin Immunoprecipitation (ChIP):

  • Use NFIX antibodies to immunoprecipitate chromatin fragments bound to NFIX

  • Research has demonstrated NFIX binding to the c-Mpl promoter region

  • Protocol considerations:

    • Cross-link cells with 1% formaldehyde

    • Sonicate chromatin to 200-500bp fragments

    • Immunoprecipitate with NFIX antibody (2-5 μg per reaction)

    • Analyze bound regions by qPCR or sequencing (ChIP-seq)

• Co-Immunoprecipitation (Co-IP):

  • Identify protein interaction partners of NFIX

  • Use NFIX antibodies to pull down NFIX and associated proteins

  • Analyze by mass spectrometry or Western blotting with antibodies against suspected interacting partners

• Electrophoretic Mobility Shift Assay (EMSA):

  • Study direct DNA binding of NFIX to target sequences

  • Use NFIX antibodies in supershift assays to confirm specificity

  • Include putative binding sites such as those identified in the c-Mpl promoter region

• Promoter-Reporter Assays:

  • Similar to studies with the c-Mpl promoter containing NFI binding sites subcloned into luciferase vectors

  • Test activity with and without NFIX overexpression

  • Use site-directed mutagenesis to evaluate specific binding site contributions

This multi-faceted approach can reveal not only which genes NFIX regulates but also the specific binding sites and mechanisms of regulation.

What role does NFIX play in hematopoietic stem cell regulation and how can this be studied?

NFIX has emerged as an important regulator of hematopoietic stem and progenitor cells (HSPCs). Research has shown that NFIX promotes ex vivo growth, cytokine hypersensitivity, and survival of primitive hematopoietic populations . Investigating this role requires specialized methodologies:

• Ex vivo culture systems:

  • Overexpression studies show NFIX+ HSPCs can withstand cytokine deprivation and display reduced apoptosis

  • Compare growth rates between control and NFIX-overexpressing cells under normal and reduced cytokine conditions

  • Measure selection for NFIX+ cells during extended culture periods

• Flow cytometry analysis:

  • Assess cell surface markers including:

    • c-Kit and Sca-1 for LSK (Lin-Sca-1+c-Kit+) cell identification

    • CD41 for megakaryocytic differentiation (increased in NFIX+ cells)

    • c-MPL (increased two-fold in NFIX+ cells)

  • Analyze cell cycle status and apoptosis (Annexin V staining)

• Functional assays:

  • Colony-forming unit (CFU) assays to assess progenitor function

  • Competitive repopulation assays to evaluate in vivo stem cell activity

  • Cytokine response studies, particularly with TPO/c-MPL signaling pathway

• Molecular mechanism investigations:

  • Gene expression analysis showing NFIX regulation of c-Mpl, Erg, and other HSPC genes

  • ChIP to identify direct transcriptional targets

  • Signaling pathway analysis (particularly TPO/c-MPL pathway)

These methodologies can help researchers elucidate the mechanisms through which NFIX regulates hematopoietic stem cell function and survival.

How can I investigate the relationship between NFIX and the thrombopoietin receptor (c-Mpl)?

The regulatory relationship between NFIX and c-Mpl (thrombopoietin receptor) represents an important molecular pathway in hematopoietic research. To investigate this relationship, consider these approaches:

• Gene expression analysis:

  • Quantify c-Mpl transcript levels in control vs. NFIX-overexpressing cells

  • Research has shown a two-fold increase in c-Mpl transcripts in NFIX+ cells (p = 0.028)

  • Use RT-qPCR to validate expression changes

• Protein expression analysis:

  • Measure c-MPL cell surface expression by flow cytometry

  • Western blotting to quantify total protein levels

  • Published data shows a two-fold increase in c-MPL cell surface antigen on NFIX+ cells (p = 0.042)

• Promoter analysis and direct binding:

  • Identify NFI binding sites in the c-Mpl promoter

  • Previous research identified four putative NFI binding sites in the c-Mpl promoter region

  • Use luciferase reporter assays with the c-Mpl promoter

  • Perform site-directed mutagenesis to evaluate the contribution of individual binding sites

• Functional relationship studies:

  • TPO removal or c-MPL neutralization experiments

  • Use of c-MPL neutralizing antibodies (e.g., AMM2)

  • Compare effects on cell growth, selection, and apoptosis between control and NFIX+ cells

  • Research shows NFIX+ cells are significantly more sensitive to loss of c-MPL stimulation (p = 0.0021)

• Downstream signaling analysis:

  • Evaluate activation of pathways downstream of c-MPL (JAK/STAT, MAPK, PI3K/AKT)

  • Compare phosphorylation status of key signaling molecules

  • Determine how NFIX overexpression affects response to TPO stimulation

These methodological approaches can provide a comprehensive understanding of how NFIX regulates c-Mpl expression and the functional consequences of this regulation in hematopoietic cells.

Why am I observing high background or non-specific binding with my NFIX antibody?

High background or non-specific binding can compromise experimental results. Consider these methodological solutions to common problems:

• Antibody-related factors:

  • Dilution issues: Try a more dilute antibody solution (1:1000 instead of 1:500)

  • Quality control: Check antibody purity (affinity-purified antibodies like those in search result typically give cleaner results)

  • Consider using a different NFIX antibody targeting another epitope region

  • Use freshly prepared working solutions of antibody

• Sample preparation issues:

  • Overfixation: Reduce fixation time or concentration

  • Incomplete blocking: Increase blocking time or concentration

  • Autofluorescence: Include a quenching step or use a different detection system

  • Cross-reactivity with endogenous biotin: Use a biotin-free detection system

• Protocol optimization:

  • More stringent washing: Increase wash duration, number, or detergent concentration

  • Better blocking: Try different blocking agents (BSA, normal serum, commercial blockers)

  • Reduce secondary antibody concentration

  • Include 0.1-0.3% Triton X-100 in antibody diluent to reduce non-specific membrane binding

• Validation steps:

  • Include a blocking peptide control (e.g., catalog # AAP32717 for anti-NFIX antibody)

  • Perform adsorption controls by pre-incubating antibody with recombinant NFIX

  • Include cell or tissue samples known to be negative for NFIX expression

• Detection system considerations:

  • High sensitivity detection systems may amplify background

  • Consider using polymer-based detection instead of avidin-biotin systems

  • Reduce substrate development time

Systematic troubleshooting of these factors can help achieve clean, specific NFIX detection.

How can I determine the optimal antibody concentration for detecting low levels of NFIX expression?

Detecting low NFIX expression levels requires careful optimization of antibody concentration and detection methods:

• Antibody titration:

  • Perform a dilution series (e.g., 1:100, 1:250, 1:500, 1:1000, 1:2000)

  • Use a positive control sample with known NFIX expression

  • Select the dilution that provides the best signal-to-noise ratio, not necessarily the strongest signal

  • Consider using concentrated antibody preparations (e.g., 0.5 mg/ml concentration)

• Sample enrichment techniques:

  • Immunoprecipitation before Western blotting

  • Cell sorting to enrich for populations with higher NFIX expression

  • Nuclear extraction (since NFIX is a nuclear protein)

  • Concentration of dilute samples

• Signal amplification methods:

  • For Western blotting:

    • Use high-sensitivity ECL substrates

    • Consider longer exposure times

    • Try fluorescent secondary antibodies with digital imaging

  • For IHC/ICC:

    • Use tyramide signal amplification (TSA)

    • Try polymer-based detection systems

    • Consider biotin-streptavidin amplification (if endogenous biotin is not an issue)

• Experimental controls:

  • Include a dilution series of recombinant NFIX protein to establish detection limits

  • Use samples with NFIX overexpression as positive controls

  • Compare results with a second NFIX antibody targeting a different epitope

• Optimization based on sample type:

  • Fresh vs. frozen tissue may require different antibody concentrations

  • Cell lines vs. primary cells may have different optimal conditions

  • Consider specialized fixation protocols for low-abundance nuclear proteins

Through methodical optimization, you can establish conditions that enable detection of even low levels of NFIX expression while maintaining specificity.

What controls should I include when studying NFIX in experimental models?

Proper controls are essential for generating reliable and interpretable data when studying NFIX. Include these controls in your experimental design:

• Antibody validation controls:

  • Blocking peptide control: Pre-incubate NFIX antibody with its specific blocking peptide

  • Isotype control: Use a non-specific IgG from the same host species and at the same concentration

  • Secondary-only control: Omit primary antibody to assess background from secondary detection

• Expression controls:

  • Positive control: Include samples known to express NFIX (based on literature)

  • Negative control: Use samples where NFIX expression is absent or knocked down

  • Overexpression control: Samples with forced NFIX expression serve as positive controls and help identify antibody specificity

• Functional controls:

  • For TPO/c-MPL signaling studies:

    • TPO removal or c-MPL neutralization (e.g., with AMM2 antibody)

    • Compare control vs. NFIX+ cells in different cytokine conditions

  • For transcriptional regulation studies:

    • Empty vector controls in promoter-reporter assays

    • Mutated binding site controls

• Technical controls:

  • Loading controls for Western blot (β-actin, GAPDH, histone H3 for nuclear extracts)

  • Housekeeping genes for RT-qPCR (GAPDH, β-actin, 18S rRNA)

  • Staining controls for flow cytometry (FMO - Fluorescence Minus One)

• Experimental design controls:

  • Time course experiments to capture dynamic changes

  • Dose-response relationships for treatments

  • Biological replicates (different donors/animals) and technical replicates

How can I optimize NFIX detection in specific cell types like hematopoietic stem cells?

Detecting NFIX in specific cell populations like hematopoietic stem cells (HSCs) requires specialized approaches:

• Cell isolation and enrichment:

  • For HSCs, use standard markers (LSK: Lin-Sca-1+c-Kit+) for flow cytometry sorting

  • Consider magnetic bead separation for pre-enrichment

  • Culture cells under conditions that maintain stemness (e.g., with appropriate cytokines)

  • Note that extended culture may affect immunophenotype, as NFIX+ LSK cells show accelerated loss of the LSK phenotype

• Detection method selection:

  • For rare populations like HSCs:

    • Flow cytometry provides single-cell resolution

    • Immunofluorescence allows visualization of subcellular localization

    • Western blotting may require larger cell numbers (pooled samples)

  • Consider multiparameter analysis combining NFIX with stem cell markers

• Protocol modifications:

  • Gentle fixation to preserve HSC markers and NFIX epitopes

  • Optimize permeabilization for nuclear antigen detection

  • Use low-background detection systems

  • For flow cytometry, include dead cell discrimination dyes

• Functional correlation:

  • Correlate NFIX expression with functional properties:

    • Apoptosis resistance (shown to be enhanced in NFIX+ HSPCs)

    • Cell cycle status

    • Colony-forming ability

    • Expression of target genes like c-Mpl

• Single-cell approaches:

  • Consider single-cell protein detection methods (CyTOF, CITE-seq)

  • Correlate protein expression with single-cell transcriptomics

  • Image-based single-cell analysis (imaging flow cytometry)

Research has shown that NFIX+ HSPCs display distinct properties, including resistance to cytokine deprivation and reduced apoptosis during ex vivo culture . These functional characteristics can help confirm successful detection of NFIX-expressing stem cell populations.

How is NFIX research contributing to our understanding of stem cell regulation and disease?

NFIX research is providing important insights into stem cell biology and disease mechanisms:

• Hematopoietic stem cell regulation:

  • NFIX promotes survival of immature hematopoietic cells via upregulation of the thrombopoietin receptor c-Mpl

  • This mechanism helps explain how NFIX contributes to HSPC maintenance and function

  • Understanding these pathways may improve ex vivo expansion protocols for clinical applications

• Transcriptional networks:

  • NFIX functions within broader transcriptional networks regulating stem cell fate

  • Research has identified connections to other important HSPC genes like Erg

  • These networks may provide therapeutic targets for hematological disorders

• Cell survival mechanisms:

  • NFIX+ cells show resistance to cytokine deprivation and reduced apoptosis

  • Understanding these survival mechanisms may inform approaches to enhance cell survival in therapeutic applications

  • May also provide insights into dysregulated survival in malignancies

• Differentiation pathways:

  • NFIX expression appears to influence megakaryocytic differentiation, as evidenced by increased CD41 expression

  • This suggests roles in lineage specification that warrant further investigation

• Disease implications:

  • NFIX is also known as SOTOS2, suggesting connections to Sotos syndrome

  • Further research may reveal roles in other developmental or hematological disorders

Continued research on NFIX will likely expand our understanding of normal stem cell regulation and provide insights into disease mechanisms and potential therapeutic approaches.

What emerging technologies might enhance NFIX research in the future?

Emerging technologies promise to advance NFIX research in several key areas:

• Advanced genomic approaches:

  • CUT&RUN or CUT&Tag for more precise mapping of NFIX binding sites

  • HiChIP or Micro-C to identify long-range chromatin interactions mediated by NFIX

  • CRISPR screening to identify functional partners or downstream effectors

  • Base editing or prime editing for precise genetic manipulation

• Single-cell technologies:

  • Single-cell multiomics (RNA-seq + ATAC-seq + protein detection)

  • Spatial transcriptomics to understand NFIX function in tissue context

  • Live cell imaging of NFIX dynamics using engineered reporter systems

  • Single-cell proteomics to detect low-abundance NFIX in specific cell populations

• Structural biology:

  • Cryo-EM of NFIX-DNA complexes

  • Hydrogen-deuterium exchange mass spectrometry to map binding interfaces

  • AlphaFold or other AI-based structural prediction tools for NFIX interactions

• Computational approaches:

  • Machine learning to predict NFIX binding sites and functional outcomes

  • Network analysis to position NFIX within broader regulatory networks

  • Integration of multi-omic datasets to build comprehensive models of NFIX function

• Advanced antibody technologies:

  • Recombinant antibodies with enhanced specificity

  • Nanobodies for improved access to nuclear antigens

  • Proximity labeling strategies using NFIX antibodies to identify interaction partners

These emerging technologies will provide deeper insights into NFIX biology and potentially reveal new therapeutic approaches targeting NFIX-regulated pathways.