HAX1 Antibody

What is HAX1 and why are HAX1 antibodies important for research?

HAX1 (HCLS1-associated protein X-1) is a 35 kDa protein consisting of 279 amino acids that is ubiquitously expressed across tissues. It functions as an anti-apoptotic protein with additional roles in cell motility, calcium homeostasis, and mitochondrial proteostasis . HAX1 antibodies are critical research tools because:

• HAX1 is implicated in severe congenital neutropenia (Kostmann disease)

• It regulates neutrophil adhesion and chemotaxis through RhoA

• It interacts with multiple cellular proteins including CLPB, cortactin, and Arp2/3

• Its overexpression has been reported in several tumors, including breast cancer

The diverse functions of HAX1 make specific and validated antibodies essential for investigating its role in normal physiology and disease states.

Western blotting for HAX1 requires careful optimization:

• Cell density: A minimum of 0.5 × 10^6 cells is recommended, with ≥1 × 10^6 cells being optimal for reliable detection

• Dilution ranges:

  • Mouse monoclonal antibodies: 1:1000-2000 (starting dilution 1:1000)

  • Rabbit polyclonal antibodies: 1:500-1000

  • Goat polyclonal antibodies: 1:1 μg/mL

• Protein visualization: The expected molecular weight of HAX1 is approximately 35 kDa

• Reducing conditions: Western blots should be conducted under reducing conditions using appropriate buffers (e.g., Immunoblot Buffer Group 2 for the R&D Systems antibody)

• Loading control: β-tubulin is an appropriate loading control as demonstrated in multiple studies

How should HAX1 antibodies be stored to maintain optimal activity?

Proper storage is critical for maintaining antibody performance:

• Short-term storage (up to 1 month): Store at 4°C

• Long-term storage: Store at -20°C

• Avoid freeze-thaw cycles: These degrade antibody quality and specificity

• Storage formulation: Typically 1mg/ml containing PBS, pH-7.4, with 0.1% Sodium Azide

• Shelf life: Approximately 12 months at -20°C and 1 month at 4°C when properly stored

How can researchers distinguish between HAX1 isoforms using specific antibodies?

HAX1 exists in different transcript variants that appear to have distinct functions. To distinguish between these isoforms:

• Targeting specific regions: Select antibodies that recognize regions unique to specific transcript variants

• Clinical relevance: Mutations affecting only transcript variant 1 are associated with congenital neutropenia, while mutations affecting both variants 1 and 2 cause both neutropenia and neurological symptoms including epilepsy and neurodevelopmental delay

• Tissue expression patterns: Transcript variant 2 is markedly expressed in human brain tissue compared to peripheral blood

• Antibody selection strategy:

  • For studying neurological manifestations: Choose antibodies recognizing both variants

  • For neutrophil-specific studies: Antibodies specific to variant 1 may be more appropriate

  • Verify the epitope region recognized by the antibody against known variant sequences

What methodological approaches can resolve conflicting results when using different HAX1 antibodies?

Discrepancies between results obtained with different HAX1 antibodies are not uncommon. To resolve such conflicts:

• Comparative antibody assessment: Test multiple antibodies side-by-side using identical samples and protocols

  • In one study, rabbit anti-HAX1 (Proteintech Group) provided more robust and consistent detection than mouse anti-HAX1 (BD Biosciences) when using the same cell extracts and imaging simultaneously

• Quantification methods:

  • Use band intensity ratios relative to loading controls

  • Example: The Odyssey imaging system (Li-Cor Biosciences) was used to calculate Hax1/tubulin intensity ratios across three independent blots for each antibody

• Antibody validation using knockdown/knockout systems:

  • Generate stably-expressing control shRNA and HAX1 shRNA cell lines

  • Both mouse anti-HAX1 and rabbit anti-HAX1 antibodies showed reduced detection in HAX1-deficient PLB-985 cells, confirming specificity

• Background assessment:

  • Test pre-immune serum and secondary antibodies alone

  • Some background in the rabbit 680nm channel was attributed to the goat anti-rabbit IgG secondary antibody rather than the primary HAX1 antibody

How do different fixation methods affect HAX1 localization studies using immunofluorescence?

HAX1 subcellular localization has been debated in the literature, with earlier studies suggesting primarily mitochondrial localization and recent findings indicating broader cytoplasmic distribution . When conducting immunofluorescence:

• Recommended fixation: Immersion fixation has been successfully used for detecting HAX1 in HepG2 cells

• Concentration for immunofluorescence: 10 µg/mL (for R&D Systems goat anti-human HAX-1)

• Counterstaining: DAPI is commonly used for nuclear visualization

• Expected localization: Primarily cytoplasmic, with specific staining localized to:

  • Cytoplasm

  • Cytoplasmic vesicles

  • Endoplasmic reticulum

  • Cell body (not exclusively mitochondrial as previously thought)

What strategies can be used to study HAX1-protein interactions using immunoprecipitation?

HAX1 interacts with numerous proteins, and immunoprecipitation (IP) is a key method for studying these interactions:

• TAG-based approaches:

  • Use FLAG-tagged HAX1 expressed in HEK293T cells followed by mass spectrometry to identify interacting partners

  • One study identified CLPB as a prominent binding partner using this approach

• Bidirectional co-immunoprecipitation:

  • Perform mutual interaction confirmation between HAX1 and target proteins

  • Example: Bidirectional co-IP confirmed the interaction between HAX1 and CLPB

• Endogenous antibody IP:

  • Use anti-HAX1 antibodies to pull down endogenous HAX1 and associated proteins

  • This approach has successfully confirmed HAX1-CLPB interaction in HL-60 human myeloid cells

• Controls:

  • Include appropriate IgG controls (as demonstrated in HAX1 RIP-seq experiments performed in five replicates)

  • Verify correct clustering by principal component analysis

How can HAX1 antibodies be used to investigate the role of HAX1 in neutrophil function and motility?

HAX1 plays a critical role in neutrophil adhesion and chemotaxis. To study these functions:

• Cell models:

  • PLB-985 cells differentiated into neutrophil-like cells are recommended

  • Primary neutrophils from patients with HAX1 mutations can provide valuable insights

• Functional assays with HAX1 knockdown/knockout models:

  • Chemotaxis assays using micropipette tips with fMLP gradients

  • Time-lapse imaging to quantify uropod length and detachment

  • Microfluidic gradient generators for live imaging

• Integrin-mediated adhesion assessment:

  • Quantify adhesion on different substrates (fibrinogen)

  • Use integrin function-blocking antibodies to determine specificity

• RhoA activity measurement:

  • HAX1-deficient cells display reduced RhoA activity

  • Activation of RhoA rescues adhesion defects in HAX1-deficient neutrophils

• Structure-function analysis:

  • The N-terminal 113 amino acids of HAX1 are sufficient to rescue neutrophil adhesion and chemotaxis defects

  • The C-terminal region (GFP-Hax1Δ113) cannot rescue these defects

How can HAX1 antibodies be used to investigate HAX1's role in RNA binding?

Recent research has identified HAX1 as an RNA-binding protein with potential involvement in ribosome biogenesis and translation:

• RNA immunoprecipitation sequencing (RIP-seq):

  • Use HAX1 antibodies to precipitate HAX1-RNA complexes

  • Perform in multiple replicates with appropriate IgG controls

  • Analyze enriched transcripts using adjusted p-value cutoffs (e.g., 0.05)

• Cross-linking and analysis of cDNA (CRAC):

  • More specific approach involving RNA crosslinking and two purification steps

  • Enables analysis of bound RNA regions and provides more specificity than RIP-seq

• Functional validation:

  • Compare HAX1 RNA targets with transcripts downregulated in HAX1 knockout cells

  • Assess ribosomal profiles, especially the ratio of small ribosomal subunit to monosome

• Enrichment analysis:

  • Use tools like DAVID (Database for Annotation, Visualization and Integrated Discovery)

  • Look for enrichment in RNA processing, translation, and ribosome biogenesis categories

Troubleshooting Common Issues

The choice of HAX1 antibody should be guided by the specific research question:

• For basic HAX1 detection in Western blotting:

  • Both mouse monoclonal (BD Biosciences) and rabbit polyclonal (Proteintech Group) antibodies reliably detect HAX1

  • Rabbit polyclonal antibodies generally provide more robust and consistent detection

• For studying neutrophil functions:

  • Mouse anti-Hax1 directed against amino acids 10-148

  • Rabbit anti-Hax1 directed against full-length Hax1

• For investigating RNA binding:

  • Antibodies suitable for immunoprecipitation with minimal background

• For localization studies:

  • Goat Anti-Human HAX-1 Antigen Affinity-purified Polyclonal Antibody has been validated for immunofluorescence in HepG2 cells

• For investigating HAX1 transcript variants:

  • Select antibodies targeting specific regions present or absent in different variants

What does the most recent research reveal about HAX1 function that might influence antibody application strategies?

Recent discoveries about HAX1 function that impact antibody selection and application include:

• RNA binding capacity:

  • HAX1 binds transcripts involved in translation, ribosome biogenesis, and rRNA processing

  • This suggests new applications for HAX1 antibodies in RNA immunoprecipitation studies

• Mitochondrial proteostasis regulation:

  • HAX1 interacts with CLPB, affecting mitochondrial function

  • This interaction may influence antibody epitope accessibility in certain experimental conditions

• Collective cell migration regulation:

  • HAX1 impacts collective (but not single-cell) migration

  • Suggests the importance of cell-cell contacts in HAX1 function

  • May require different fixation and staining approaches for visualizing HAX1 at cell-cell junctions

• HAX1 transcript variants and disease correlation:

  • Mutations affecting only transcript variant 1 associate with neutropenia

  • Mutations affecting both variants 1 and 2 cause neutropenia plus neurological symptoms

  • Requires careful antibody selection to distinguish variant-specific functions