HOXA11 Antibody, HRP conjugated

What is HOXA11 and what biological functions does it serve?

HOXA11 (Homeobox protein Hox-A11) is a sequence-specific transcription factor that belongs to the AbdB homeobox family. It plays crucial roles in developmental regulatory systems by providing cells with specific positional identities on the anterior-posterior axis . HOXA11 governs multiple biological processes including:

• Embryonic development of the vertebrate central nervous system, heart, axial skeleton, limbs, gut, and urogenital tract

• Female fertility through regulation of endometrial development and uterine formation during embryogenesis

• Cell apoptosis in acute myeloid leukemia (AML), with its expression predicting improved response to cytarabine (Ara-C) treatment

• Regulation of gene expression during the mid-luteal phase of the menstrual cycle to facilitate blastocyst implantation

The HOXA11 protein is primarily localized in the nuclei of cells and functions by binding to specific DNA sequences to activate transcription of target genes.

What detection methods are compatible with HOXA11 Antibody, HRP conjugated?

HOXA11 Antibody, HRP conjugated is primarily designed for the following detection methods:

• ELISA (Enzyme-Linked Immunosorbent Assay): The most common application, with a typical detection range of 15.625-1000 pg/ml and sensitivity around 9.375 pg/ml

• Western Blotting: Though unconjugated antibodies are often preferred, HRP-conjugated versions eliminate the need for secondary antibody incubation

• Immunohistochemistry (IHC): For tissue sections when properly diluted

• Immunocytochemistry (ICC)/Immunofluorescence (IF): For cellular localization studies

The direct HRP conjugation offers advantages in reducing non-specific binding and background noise that can occur with two-step detection systems. The recommended working dilutions vary by application but typically range from 1:200 to 1:1000 for optimal results .

How should researchers prepare biological samples for HOXA11 detection using HRP-conjugated antibodies?

Proper sample preparation is crucial for successful detection of HOXA11. Based on the ELISA kit protocols and antibody documentation, researchers should consider:

• Cell/Tissue Lysate Preparation:

  • Use appropriate lysis buffers containing protease inhibitors to prevent protein degradation

  • For nuclear proteins like HOXA11, nuclear extraction protocols are recommended

  • Typical sample volumes needed: Serum (50 μL), Plasma (50 μL), Cell Culture Supernatant (100 μL), Cell/Tissue lysate (100 μL)

• Sample Clearing:

  • Centrifuge samples at 1000×g for 10 minutes to remove cellular debris

  • Filter through 0.22 μm filters if necessary to remove particulates

• Quantification:

  • Determine protein concentration using Bradford or BCA assays

  • Normalize sample loading to ensure consistent protein amounts

• Denaturation for Western Blotting:

  • Heat samples at 95°C for 5 minutes in reducing sample buffer containing SDS and DTT or β-mercaptoethanol

  • For HOXA11 detection, 20-50 μg of total protein is typically loaded per lane

• Fixation for ICC/IF:

  • 4% paraformaldehyde fixation for 15-20 minutes at room temperature

  • Permeabilization with 0.1-0.5% Triton X-100 is crucial as HOXA11 is a nuclear protein

How can HOXA11 Antibody, HRP conjugated be utilized to study the relationship between HOXA11 expression and cancer progression?

HOXA11 has been implicated in various cancer types, including acute myeloid leukemia (AML) and oral squamous cell carcinoma (OSCC). Researchers can leverage HRP-conjugated HOXA11 antibodies to investigate these connections through several methodological approaches:

• Expression Level Analysis in Clinical Samples:

  • Use ELISA to quantify HOXA11 protein levels in patient samples

  • Compare expression between normal and tumor tissues to establish correlation with disease progression

  • Research has shown that HOXA11 expression can predict AML response to Ara-C treatment, making it a potential biomarker for therapeutic response

• Functional Studies Using Cell Models:

  • After gene knockdown or overexpression of HOXA11, use the antibody in Western blots to confirm changes in protein levels

  • Correlate HOXA11 expression with cellular phenotypes such as proliferation, apoptosis, and invasion

  • Studies have demonstrated that HOXA11 regulates apoptosis-related genes, including NF-κB inhibitor α, transcription factor p65, and transformation-related protein p53

• Cancer Subtype Classification:

  • Develop ELISA-based tissue microarrays to screen multiple patient samples

  • Correlate HOXA11 expression profiles with clinical parameters and outcomes

  • In OSCC, HOXA11 expression has been linked to differentiation status, T stage, N stage, and clinical stage

• Mechanistic Studies:

  • Investigate HOXA11's interaction with miRNAs and long non-coding RNAs

  • For instance, HOXA11-AS has been shown to promote OSCC cell proliferation and invasion by regulating miR-518a-3p and PDK1

What methodological considerations are important when using HOXA11 Antibody, HRP conjugated in ELISA-based transcription factor activity assays?

When implementing HOXA11 transcription factor activity assays using HRP-conjugated antibodies, researchers should address several critical methodological aspects:

• Nuclear Extract Preparation:

  • Since HOXA11 functions as a transcription factor, proper nuclear extraction is essential

  • Use specialized nuclear extraction buffers containing protease inhibitors

  • Verify extraction efficiency using nuclear markers (e.g., Lamin A/C) as controls

• DNA Binding Specificity:

  • HOXA11 binds to specific DNA sequences, so oligonucleotide design for capturing active HOXA11 is crucial

  • Include both positive controls (known HOXA11 binding sequences) and negative controls (mutated sequences)

  • Consider competitive binding assays to confirm specificity

• Transcription Factor Activity Measurement Protocol:

  • Use appropriate plate coating with consensus DNA sequence for HOXA11 binding

  • Nuclear extracts containing active HOXA11 will bind to these sequences

  • HRP-conjugated HOXA11 antibody then detects bound transcription factor

  • Total assay time is typically around 4.5 hours

• Signal Detection Optimization:

  • Optimize substrate incubation time (typically 15-30 minutes) to achieve optimal signal-to-noise ratio

  • Control TMB (3,3',5,5'-Tetramethylbenzidine) coloration carefully to avoid oversaturation

  • Read absorbance at 450 nm with reference at 620 nm for optimal results

• Data Interpretation:

  • Include a standard curve using recombinant HOXA11 protein for quantitative analysis

  • Normalize to total protein concentration or housekeeping transcription factors

  • Compare transcription factor activity between experimental conditions rather than relying solely on absolute values

What are the critical controls that should be included when using HOXA11 Antibody, HRP conjugated in research experiments?

Proper experimental controls are essential for ensuring the validity and reliability of results when using HOXA11 Antibody, HRP conjugated. Researchers should incorporate the following controls:

• Positive Controls:

  • Cell/tissue samples known to express HOXA11 (e.g., rat brain tissue, human placenta tissue, HT1080 cells)

  • Recombinant HOXA11 protein as a standard for quantification

  • In AML studies, use cell lines with confirmed HOXA11 expression like those harboring MLL translocations

• Negative Controls:

  • Cell lines with confirmed absence of HOXA11 expression

  • Immunizing peptide competition assay to confirm specificity

  • When examining tissues, include those known not to express significant HOXA11 levels

• Technical Controls:

  • Background control (without primary antibody) to assess non-specific binding

  • Isotype control (irrelevant IgG of the same isotype and conjugate) to evaluate Fc receptor binding

  • Loading control (housekeeping protein) for Western blotting to normalize protein loading

• Validation Controls:

  • Antibody specificity verification using HOXA11 knockdown or knockout samples

  • Protein array testing against 364 human recombinant protein fragments to check for cross-reactivity

  • Multiple antibody approach using different HOXA11 antibodies targeting different epitopes

• Procedural Controls:

  • Inclusion of standard curves in ELISA applications

  • Step-by-step protocol validation with time course and dilution series

  • Inter-assay and intra-assay variation assessment

How can researchers troubleshoot weak or non-specific signals when using HOXA11 Antibody, HRP conjugated?

When facing challenges with weak signals or non-specific binding, consider these methodological approaches:

• For Weak Signals:

  • Increase antibody concentration (within manufacturer's recommended range)

  • Extend incubation time (e.g., overnight at 4°C instead of 1-2 hours at room temperature)

  • Enhance signal development by optimizing substrate incubation time

  • Ensure proper antigen retrieval for fixed samples

  • Verify sample preparation preserves HOXA11 epitope integrity

• For High Background/Non-specific Signals:

  • Increase washing frequency and duration between steps

  • Optimize blocking conditions (try different blockers like 5% BSA or 5% non-fat milk)

  • Dilute antibody further (within recommended range)

  • Pre-adsorb antibody with non-specific proteins

  • Use more stringent washing buffers (higher salt concentration)

• For Inconsistent Results:

  • Standardize sample collection and processing

  • Avoid freeze-thaw cycles of antibody (aliquot upon first use)

  • Maintain consistent incubation times and temperatures

  • Use freshly prepared reagents and buffers

  • Calibrate detection instruments regularly

• For Unexpected Band Sizes (Western Blot):

  • Consider post-translational modifications affecting mobility

  • Check for proteolytic degradation by adding more protease inhibitors

  • Verify sample denaturation conditions

  • Test alternative gel percentages for better resolution

  • HOXA11 should appear around 30-34 kDa

• For Cross-Reactivity Issues:

  • Perform peptide competition assays with the immunizing peptide

  • Test the antibody on known negative samples

  • Consider using more specific monoclonal antibodies

  • Validate results with orthogonal methods (e.g., mass spectrometry)

What are the advantages of recombinant production of HOXA11-HRP conjugates compared to conventional chemical conjugation methods?

Recombinant production of antibody-HRP conjugates represents an advanced approach over traditional chemical conjugation. While not specific to HOXA11 in the search results, the principles apply to all antibody-HRP conjugates:

• Improved Homogeneity and Stoichiometry:

  • Recombinant conjugates have a strictly determined 1:1 stoichiometry between antibody and HRP

  • Chemical conjugation results in heterogeneous mixtures with variable numbers of HRP molecules per antibody

• Preserved Functional Activity:

  • Recombinant production maintains both enzymatic activity of HRP and antigen-binding properties of the antibody

  • Chemical conjugation often results in partial inactivation of the enzyme or reduced antibody specificity

• Reproducibility and Consistency:

  • Batch-to-batch variation is minimized with recombinant production

  • Expression systems like Pichia pastoris produce consistent conjugates with defined characteristics

• Simplified Production Process:

  • Gene expression systems allow for scalable production

  • Secreted forms simplify purification procedures

• Design Flexibility:

  • Genetic constructions permit precise positioning of HRP relative to antibody fragments

  • Linker sequences can be optimized for spatial arrangement and activity

  • Documented examples show functional differences between N-terminal and C-terminal placement of HRP

The technical approach involves creating expression vectors that enable the fusion of HRP with antibody fragments (e.g., Fab) via linker sequences like (Gly₄Ser)₃, followed by expression in suitable systems like P. pastoris .

How can researchers utilize HOXA11 antibodies to investigate HOXA11-related long non-coding RNAs in disease pathogenesis?

Long non-coding RNAs (lncRNAs) related to HOXA11, particularly HOXA11-AS (antisense), have emerged as important regulators in cancer development. Researchers can employ HOXA11 antibodies in conjunction with molecular techniques to investigate their roles:

• Correlation Studies:

  • Use HOXA11 antibodies to quantify protein levels while simultaneously measuring HOXA11-AS expression through RT-qPCR

  • Analyze the relationship between HOXA11 protein and HOXA11-AS in clinical samples

  • Studies have demonstrated that HOXA11-AS is upregulated in oral squamous cell carcinoma and correlates with advanced clinical features

• Mechanistic Investigations:

  • After HOXA11-AS knockdown or overexpression, measure changes in HOXA11 protein expression using HRP-conjugated antibodies

  • Determine whether HOXA11-AS regulates HOXA11 expression or functions independently

  • HOXA11-AS has been shown to target miR-518a-3p in OSCC, creating a complex regulatory network

• Cellular Localization Studies:

  • Use immunofluorescence with HOXA11 antibodies alongside FISH (Fluorescent In Situ Hybridization) for HOXA11-AS

  • Determine co-localization patterns that might suggest functional interactions

  • This approach can reveal whether HOXA11-AS regulates HOXA11 in specific cellular compartments

• Extracellular Vesicle (EV) Analysis:

  • HOXA11-AS has been detected in extracellular vesicles from prostate cancer cells

  • Use HOXA11 antibodies to determine if the protein is also packaged into EVs

  • Differential ultracentrifugation can be used to isolate EVs following established protocols

• Translation Regulation Studies:

  • HOXA11 mRNA contains translation inhibitory elements (TIEs) with upstream open reading frames (uORFs)

  • Investigate whether HOXA11-AS affects these regulatory elements using reporter assays

  • Complement these studies with protein level measurements using HOXA11 antibodies

What emerging technologies might enhance the utility of HOXA11 Antibody, HRP conjugated in research?

Several technological advancements hold promise for expanding the applications of HOXA11 Antibody, HRP conjugated:

• Multiplexed Detection Systems:

  • Development of multiplexed ELISA platforms that allow simultaneous detection of HOXA11 alongside other HOX proteins

  • Integration with microfluidic systems for high-throughput screening

  • Incorporation into antibody arrays for comprehensive HOX family profiling

• Single-Cell Analysis Technologies:

  • Adaptation of HRP-conjugated antibodies for mass cytometry (CyTOF) applications

  • Integration with single-cell Western blotting platforms for heterogeneity studies

  • Coupling with spatial transcriptomics to correlate protein expression with gene expression patterns

• In Vivo Imaging Applications:

  • Development of HRP-conjugated antibody fragments for intravital microscopy

  • Creation of activatable probes that generate signal only upon binding to HOXA11

  • Incorporation into clearing-compatible protocols for whole-organ imaging

• Nanobody and Alternative Scaffold Technologies:

  • Engineering smaller HOXA11-binding fragments with improved tissue penetration

  • Development of non-antibody binding proteins (e.g., affibodies, DARPins) conjugated to HRP

  • These smaller formats may offer advantages in certain applications requiring better tissue penetration

• Artificial Intelligence Integration:

  • Development of machine learning algorithms to analyze complex patterns in HOXA11 expression data

  • Automated image analysis tools for quantifying immunohistochemistry results

  • Predictive models correlating HOXA11 levels with disease progression or treatment response

How might HOXA11 Antibody, HRP conjugated contribute to the development of precision medicine approaches in cancer treatment?

HOXA11 Antibody, HRP conjugated could play a significant role in advancing precision medicine approaches for cancer:

• Biomarker Development:

  • HOXA11 expression predicts response to Ara-C in AML patients

  • Standardized ELISA-based tests using HRP-conjugated antibodies could help stratify patients for specific therapeutic regimens

  • Correlation of HOXA11 expression with genetic mutations (e.g., MLL translocations, RAS signaling mutations) could guide targeted therapy selection

• Companion Diagnostics:

  • Development of diagnostic tests that use HOXA11 antibodies to identify patients likely to respond to specific treatments

  • Integration into multiplexed assay panels that assess multiple predictive biomarkers simultaneously

  • Potential incorporation into point-of-care testing platforms for rapid assessment

• Therapeutic Monitoring:

  • Serial measurement of HOXA11 levels during treatment to assess therapeutic efficacy

  • Early detection of resistance development through changes in HOXA11 expression patterns

  • Adjustment of treatment regimens based on dynamic changes in biomarker profiles

• Novel Therapeutic Target Identification:

  • Identification of downstream pathways regulated by HOXA11 using antibody-based approaches

  • Discovery of small molecule modulators of HOXA11 activity through screening assays

  • Development of therapeutic approaches targeting HOXA11-AS and related non-coding RNAs

• Liquid Biopsy Applications:

  • Detection of circulating HOXA11 protein or HOXA11-expressing extracellular vesicles

  • Monitoring treatment response through minimally invasive sampling

  • Early detection of recurrence based on biomarker reappearance in circulation

The combination of sensitive detection methods using HRP-conjugated antibodies with comprehensive patient profiling could significantly advance personalized treatment approaches for HOXA11-related malignancies.