HOXA4 Antibody, HRP conjugated

What is the biological function of HOXA4?

HOXA4 is a sequence-specific transcription factor that functions as part of a developmental regulatory system providing cells with specific positional identities on the anterior-posterior axis. It binds to sites in the 5'-flanking sequence of its coding region with various affinities. The consensus sequences of the high and low affinity binding sites are 5'-TAATGA[CG]-3' and 5'-CTAATTTT-3', respectively. As a homeobox protein, HOXA4 plays critical roles in regulating gene expression, morphogenesis, and differentiation during embryonic development .

What is the correct molecular weight of HOXA4 protein?

Based on extensive validation studies, the true molecular weight of HOXA4 protein is approximately 37-39 kDa. This has been confirmed through multiple methodologies including siRNA knockdown experiments, forced expression of full-length HOXA4, and validation with multiple commercially available antibodies. Researchers should be aware that a prominent non-specific band at approximately 30-33 kDa is often mistakenly identified as HOXA4 in Western blot analyses .

What are the recognized synonyms for HOXA4?

HOXA4 is also known by several synonyms including: Homeobox protein Hox-A4, Homeobox protein Hox-1.4, Homeobox protein Hox-1D, HOX1D, and in database annotations as HXA4_HUMAN. Understanding these alternative designations is important when conducting literature searches or cross-referencing different antibody products .

How can I validate the specificity of a HOXA4 antibody for my experiments?

A rigorous validation approach for HOXA4 antibodies should include:

• Testing in cell lines with known HOXA4 expression profiles (both positive and negative controls)

• siRNA knockdown experiments targeting HOXA4 to confirm reduction of the specific band

• Overexpression studies with full-length HOXA4 to verify band augmentation

• Cross-validation with multiple antibodies targeting different HOXA4 epitopes

• Western blot analysis focusing on the ~37-39 kDa band rather than the non-specific ~30-33 kDa band

These validation steps are essential as published studies have demonstrated that certain commercial antibodies detect strong non-specific bands that do not correspond to HOXA4 expression levels .

What are the documented specificity issues with commercial HOXA4 antibodies?

Several specificity concerns have been documented with commercial HOXA4 antibodies:

• A prominent non-specific band at ~30-33 kDa is often detected with commercial antibodies

• This non-specific band is present in HOXA4-negative cell lines (including SKOV-3 and A2780)

• The non-specific band is insensitive to HOXA4 siRNA treatment

• The non-specific band does not increase with forced HOXA4 expression

• Additional non-specific bands at ~46-48 kDa may also appear

In contrast, the authentic HOXA4 band at ~37-39 kDa correlates with mRNA expression levels, diminishes with siRNA treatment, and increases with exogenous expression .

How can I distinguish between specific and non-specific bands in Western blots?

To differentiate between specific and non-specific HOXA4 signals:

Some researchers recommend cutting the membrane just below the 37 kDa marker prior to immunoblotting to prevent the strong signal from the ~30-33 kDa non-specific band from interfering with quantitation of the authentic HOXA4 band .

What are the recommended applications for HOXA4 antibody, HRP conjugated?

The HOXA4 antibody, HRP conjugated is primarily recommended for:

• ELISA (Enzyme-Linked Immunosorbent Assay): Direct detection without secondary antibody

• Western blotting: With careful attention to identifying the correct ~37-39 kDa band

• Immunohistochemistry: With appropriate controls to distinguish specific from non-specific staining

For all applications, validation of specificity is crucial given the documented issues with non-specific binding .

What are the optimal conditions for Western blotting with HOXA4 antibodies?

For optimal Western blot detection of HOXA4:

• Focus on the ~37-39 kDa region for true HOXA4 detection

• Use longer exposure times to detect the often fainter true HOXA4 band

• Consider physical separation of the membrane below 37 kDa to eliminate interference from the intense non-specific band

• Include positive controls (cells with known HOXA4 expression) and negative controls (HOXA4-negative cell lines like SKOV-3)

• Normalize quantification to appropriate loading controls

• Validate critical findings with siRNA knockdown or overexpression experiments

These optimizations are particularly important given the tendency of some commercial antibodies to produce strong non-specific bands .

What are the recommended storage and handling conditions for HOXA4 antibody, HRP conjugated?

For optimal preservation of antibody activity:

• Store at -20°C in the manufacturer-provided buffer (typically containing 50% glycerol)

• The standard preservation buffer contains: 0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4

• Avoid repeated freeze-thaw cycles by preparing small working aliquots

• Allow antibody to equilibrate to room temperature before opening to prevent condensation

• For long-term storage, -80°C may provide better preservation of the HRP conjugate activity

• Verify activity periodically with positive control samples

How can I address inconsistencies between HOXA4 mRNA and protein detection?

When HOXA4 mRNA and protein data appear contradictory:

• Verify you are analyzing the correct HOXA4 band (~37-39 kDa) rather than non-specific bands

• Compare results using multiple antibodies targeting different HOXA4 epitopes

• Validate protein detection with siRNA knockdown experiments

• Consider post-transcriptional regulation mechanisms that might affect protein expression

• Examine protein stability and degradation pathways

• Verify primer specificity for mRNA detection

• Use absolute quantification methods for both mRNA and protein when possible

What might cause false positive signals in immunofluorescence studies with HOXA4 antibodies?

False positive signals in immunofluorescence may result from:

• Non-specific binding to cellular components, particularly in perinuclear regions

• Cross-reactivity with structurally similar proteins

• Inadequate blocking or washing steps

• Secondary antibody cross-reactivity

• Autofluorescence from fixatives or cellular components

Research has shown that even HOXA4-negative cell lines (SKOV-3) can exhibit strong perinuclear staining with certain HOXA4 antibodies, highlighting the importance of proper controls and validation strategies .

What control experiments are essential when studying HOXA4 protein expression?

Essential control experiments include:

• Positive controls: Cell lines with confirmed HOXA4 expression

• Negative controls: HOXA4-negative cell lines (e.g., SKOV-3, A2780)

• siRNA knockdown controls: To confirm specificity of detected bands

• Overexpression controls: Transfection with HOXA4 expression constructs

• Secondary antibody-only controls: To identify non-specific binding

• Multiple antibody validation: Using different antibodies targeting distinct HOXA4 epitopes

• Subcellular fractionation controls: To verify localization patterns

These controls are particularly important given the documented specificity issues with certain commercial HOXA4 antibodies .

How can I optimize immunoprecipitation protocols for HOXA4?

For successful HOXA4 immunoprecipitation:

• Select antibodies validated for immunoprecipitation applications

• Use native conditions when possible to preserve protein-protein interactions

• Pre-clear lysates thoroughly to reduce non-specific binding

• Include appropriate negative controls (IgG, HOXA4-negative cell lines)

• Validate pulled-down protein by Western blot using a different HOXA4 antibody

• Consider crosslinking approaches for transient or weak interactions

• Optimize antibody concentrations and incubation conditions empirically

Given the specificity issues with some HOXA4 antibodies, validation of immunoprecipitation results with multiple approaches is strongly recommended .

What methodological approaches are recommended for studying HOXA4 binding to target DNA sequences?

For investigating HOXA4-DNA interactions:

• Chromatin Immunoprecipitation (ChIP) with validated HOXA4 antibodies

• Electrophoretic Mobility Shift Assays (EMSA) with recombinant HOXA4

• DNA affinity precipitation using oligonucleotides containing HOXA4 consensus binding sequences (5'-TAATGA[CG]-3' and 5'-CTAATTTT-3')

• Reporter gene assays with HOXA4 binding site mutations

• In vitro binding assays with purified components

• Next-generation sequencing approaches (ChIP-seq) for genome-wide binding patterns

When designing oligonucleotides for these studies, incorporate the established consensus sequences for high and low affinity binding .

How can I investigate potential cross-reactivity between HOXA4 antibodies and other HOX family proteins?

To assess potential cross-reactivity:

• Test the antibody against recombinant proteins from multiple HOX family members

• Compare detection patterns in cell lines with known expression profiles of various HOX proteins

• Perform competitive binding experiments with recombinant HOX proteins

• Use specific knockdown of individual HOX genes to determine specificity

• Compare epitope sequences across HOX family members for potential shared recognition sites

• Consider using epitope-tagged HOXA4 constructs and tag-specific antibodies as an alternative approach

What factors should be considered when using HOXA4 antibody, HRP conjugated for ELISA?

For optimal ELISA performance:

• Determine appropriate antibody dilutions through titration experiments

• Create standard curves using recombinant HOXA4 protein for quantification

• Optimize blocking buffers to minimize background signal

• Consider sandwich ELISA format using capture and detection antibodies targeting different epitopes

• Validate specificity through competition with soluble HOXA4 protein

• Optimize substrate selection and development time for the HRP conjugate

• Include controls for non-specific binding assessment

The direct HRP conjugation eliminates the need for secondary antibody incubation, potentially reducing background and improving specificity .

How can HOXA4 antibodies be applied to study developmental expression patterns?

For developmental expression studies:

• Select antibodies validated for the specific tissue and fixation methods

• Include appropriate developmental stage controls based on known HOXA4 expression patterns

• Use multiple detection methods (immunohistochemistry, Western blot, in situ hybridization)

• Correlate protein detection with mRNA expression data

• Consider laser capture microdissection for region-specific analysis

• Use confocal microscopy for precise subcellular localization

• Include knockout or knockdown models as negative controls where available

Given HOXA4's role in anterior-posterior patterning, spatial resolution in detection is particularly important for developmental studies .