efna5b Antibody

What is EFNA5 and What Are Its Key Functions in Cellular Processes?

EFNA5 (ephrin A5) is a membrane-bound ligand that belongs to the ephrin family and interacts with Eph receptors, particularly of the EphA class. The protein is involved in critical cellular processes including:

• Cell adhesion and cytoskeletal remodeling

• Cell proliferation regulation

• Migration and invasion processes

• Epithelial-mesenchymal transition (EMT)

The protein's molecular weight is approximately 26 kDa, and it consists of 228 amino acids in its mature form .

What Applications Are EFNA5 Antibodies Validated For in Research?

EFNA5 antibodies have been validated for multiple research applications:

For IHC applications, EFNA5 antibodies have been successfully used on various tissues including brain, melanoma, and hepatoma samples . Validation studies typically show cytoplasmic and/or membrane staining patterns depending on the tissue type and antibody used .

For Western blot applications, EFNA5 antibodies can detect both endogenous and recombinant EFNA5 proteins, with successful detection reported in multiple cell lines including HeLa, HepG2, and LM3 .

What Are the Optimal Experimental Conditions for EFNA5 Immunodetection?

Based on published research protocols, the following optimized conditions are recommended for EFNA5 detection:

For immunohistochemistry:

• Antigen retrieval: Citrate buffer (pH 6.0) for 10 minutes at 95°C

• Blocking: 3% hydrogen peroxide (10 minutes) followed by 5% BSA (30 minutes at 37°C)

• Primary antibody: Mouse anti-EFNA5 mAb (1:50 dilution) or rabbit polyclonal (1:500 dilution)

• Incubation: Overnight at 4°C

• Detection system: DAKO Real Envision/HRP kit or equivalent

For Western blotting:

• Protein extraction: RIPA buffer with protease inhibitors

• Loading: 25-50 μg protein per lane

• Transfer: Constant current 350 mA for 2 hours in ice water bath

• Blocking: 5% skim milk for 1 hour at room temperature

• Primary antibody: Dilute according to manufacturer specifications (typical range 1:500-1:1000)

• Secondary antibody: HRP-conjugated, species-appropriate (typically 1:10000 dilution)

• Detection: ECL chemiluminescence method

These conditions should be optimized for specific experimental systems and antibodies.

How Do Polyclonal and Monoclonal EFNA5 Antibodies Differ in Research Applications?

The choice between polyclonal and monoclonal EFNA5 antibodies significantly impacts experimental outcomes:

Polyclonal EFNA5 antibodies:

• Recognize multiple epitopes across the EFNA5 protein

• Typically produced in rabbits or goats

• Examples: Rabbit polyclonal antibody from Proteintech (17735-1-AP), Rabbit polyclonal from Absin (abs130417a)

• Advantages: Higher sensitivity due to multiple epitope binding; better for detecting native proteins

• Best applications: Western blotting, IHC of fixed tissues

• Concentration typically 1 mg/ml

Monoclonal EFNA5 antibodies:

• Target a single specific epitope

• Typically produced in mice

• Example: Mouse anti-Efna5 mAb (ab60705)

• Advantages: Higher specificity, reduced batch-to-batch variation, better for distinguishing between related proteins

• Best applications: Flow cytometry, applications requiring absolute specificity

• Working dilutions typically 1:50 for IHC

For comprehensive studies, using both antibody types provides complementary data. Western blot validation should precede other applications to confirm specificity for the target protein.

How Does EFNA5 Expression Vary Across Different Cancer Types?

EFNA5 expression shows significant variability across cancer types, which has important implications for experimental design and interpretation:

Hepatocellular carcinoma:

• EFNA5 is significantly downregulated compared to normal hepatocytes

• Expression analysis using qRT-PCR showed markedly reduced levels in HepG2, LM3, Huh7, and PLC/PRF5 cell lines

• Functions as a tumor suppressor

• Overexpression experiments demonstrated inhibition of proliferation, migration, and invasion

• Low expression correlates with EMT progression

Ovarian cancer:

These contrasting expression patterns highlight the context-dependent role of EFNA5 in different tumor microenvironments and emphasize the importance of appropriate controls when using EFNA5 antibodies in cancer research.

What Molecular Mechanisms Does EFNA5 Regulate in Cancer Progression?

Research using validated EFNA5 antibodies has revealed several key molecular pathways through which EFNA5 influences cancer progression:

In hepatocellular carcinoma:

• EFNA5 inhibits epithelial-mesenchymal transition (EMT)

• Western blot analysis reveals that EFNA5 overexpression:

  • Increases E-cadherin expression (epithelial marker)

  • Decreases N-cadherin and Vimentin expression (mesenchymal markers)

  • Reduces c-Myc and c-Jun levels (oncogenic transcription factors)

  • Suppresses EGFR expression (growth factor receptor)

• These molecular changes correlate with reduced cell proliferation, migration, and invasion in functional assays

In ovarian cancer:

• EFNA5 demonstrates atypical signaling compared to other ephrin family members

• While canonical ephrins like EFNA1 induce robust EphA2 tyrosine phosphorylation and receptor degradation, EFNA5 shows:

  • Limited EphA2-Y588 phosphorylation

  • Minimal receptor internalization and degradation

  • Potential to maintain oncogenic EphA2-S897 phosphorylation

• These unique signaling properties may contribute to EFNA5's association with aggressive disease

Understanding these mechanistic differences is essential for researchers using EFNA5 antibodies to investigate cancer biology.

What Are the Best Methods for Validating EFNA5 Antibody Specificity?

Comprehensive validation of EFNA5 antibodies should include multiple approaches:

• Western blot validation:

  • Use recombinant EFNA5 protein as positive control

  • Include EFNA5 knockdown samples as negative controls

  • Verify band appears at expected molecular weight (26 kDa)

  • Check for cross-reactivity with other ephrin family members

• Genetic validation approaches:

  • Compare staining between wild-type and EFNA5 knockdown cells

  • In research by Yang et al., EFNA5 plasmid transfection increased EFNA5 levels 47-56 fold in hepatoma cells, providing a robust positive control

  • Use siRNA/shRNA with multiple targeting sequences to confirm specificity

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide

  • Observe elimination of specific signal

  • Include graded concentrations of competing peptide

• Cross-reactivity assessment:

  • Test against recombinant proteins for related ephrin family members

  • Commercial antibodies like Bio-Techne's AF3743 show <1% cross-reactivity with related proteins such as ephrin-A1

• Multi-method verification:

  • Compare results across different detection methods (IHC, WB, IF)

  • Use antibodies targeting different epitopes of EFNA5

  • Correlate protein detection with mRNA expression data

Appropriate validation is particularly critical when studying EFNA5 due to its homology with other ephrin family members and its context-dependent functions.

How Can EFNA5 Antibodies Be Optimized for Difficult-to-Process Tissues?

Some tissues present challenges for EFNA5 immunodetection. Researchers have developed specialized protocols:

For brain and neural tissues:

• Extended fixation (24-48 hours in 4% PFA) followed by sucrose cryoprotection

• Enhanced antigen retrieval: Citrate buffer pH 6.0 at 95°C for 15-20 minutes

• Signal amplification using tyramide signal amplification (TSA) systems

• Extended primary antibody incubation (48-72 hours at 4°C)

For retinal tissue:

• As described in published protocols: Antigen retrieval in citrate buffer followed by hydrogen peroxide treatment

• Careful blocking with 5% BSA at 37°C for 30 minutes

• Using highly purified antibodies to minimize background

For heavily fibrotic tissues:

• Enzymatic pretreatment: Brief proteinase K or hyaluronidase digestion

• Enhanced permeabilization: 0.3-0.5% Triton X-100 for 30-60 minutes

• Polymer-based detection systems like DAKO EnVision

• Multiple antibody application cycles with extended incubation times

Critical controls:

• Include positive control tissues with known EFNA5 expression

• Test multiple antibody concentrations and incubation times

• Compare chromogenic versus fluorescent detection methods

• Include no-primary-antibody controls to assess background

These optimizations should be systematically tested and documented for reproducible results.

What Approaches Can Distinguish Between Membrane-Bound and Soluble EFNA5?

EFNA5, like other ephrins, exists in both membrane-bound and soluble forms, creating technical challenges for antibody-based detection:

Recommended protocols for distinguishing forms:

• Subcellular fractionation:

  • Separate membrane and cytosolic fractions before Western blotting

  • Use validated markers to confirm fraction purity (Na⁺/K⁺-ATPase for membrane, GAPDH for cytosolic)

  • Analyze EFNA5 distribution between fractions

• Flow cytometry for cell surface detection:

  • Non-permeabilized cells: Detects only membrane-bound EFNA5

  • Permeabilized cells: Detects total EFNA5

  • The difference between signals represents intracellular pools

• Specialized extraction methods:

  • Membrane protein extraction requires detergent-containing buffers

  • RIPA buffer efficiently extracts both forms

  • Phase separation using Triton X-114 can segregate membrane-bound proteins

• Immunostaining optimization:

  • For membrane-bound EFNA5: Minimal or no permeabilization

  • For total EFNA5: Standard permeabilization protocols

  • Compare patterns to distinguish localization

• Enzymatic release:

  • Treatment with PI-PLC (phosphatidylinositol-specific phospholipase C) cleaves GPI anchors

  • Analyze release of EFNA5 into supernatant after treatment

Understanding the distribution between these forms provides crucial insights into EFNA5 function in normal and pathological contexts.

How Can EFNA5 Antibodies Be Used to Study Epithelial-Mesenchymal Transition?

Based on hepatoma research, EFNA5 regulates EMT, making this a valuable application for EFNA5 antibodies:

Experimental workflow for studying EFNA5 in EMT:

• Baseline expression analysis:

  • Use Western blotting with validated EFNA5 antibodies (1:500 dilution)

  • Analyze alongside EMT markers:

    • E-cadherin (epithelial)

    • N-cadherin, Vimentin (mesenchymal)

    • c-Myc, c-Jun (transcription factors)

• Functional studies with EFNA5 modulation:

  • Transfect cells with EFNA5 expression plasmids

  • Perform knockdown using siRNA/shRNA

  • Analyze EMT marker changes by Western blot and qRT-PCR

• Migration and invasion assays:

  • Transwell migration assays

  • Boyden chamber invasion assays with Matrigel coating

  • Document and quantify cell numbers per field

• Signaling pathway investigation:

  • Analyze EGFR pathway components

  • Examine AKT and ERK phosphorylation status

  • Use phospho-specific antibodies in Western blotting

Key findings from published research:

• EFNA5 overexpression in HepG2 and LM3 cells significantly inhibited migration (p<0.01) and invasion (p<0.01)

• EFNA5 overexpression increased E-cadherin and decreased N-cadherin, Vimentin, c-Myc, and c-Jun levels

• These molecular changes correlated with reduced cell migration and invasion in functional assays

These approaches provide a comprehensive framework for studying EFNA5's role in EMT using validated antibodies.

What Are the Technical Considerations for Using EFNA5 Antibodies in Multiplexed Imaging?

For researchers conducting advanced multiplexed imaging studies with EFNA5 antibodies:

Antibody selection for multiplexing:

• Choose antibodies raised in different host species to avoid secondary antibody cross-reactivity

• Select antibodies recognizing different epitopes if using multiple EFNA5 antibodies

• Validate each antibody individually before multiplexing

Sequential staining protocols:

• Apply tyramide signal amplification (TSA) for sequential staining

• Between rounds, strip primary-secondary complexes while preserving fluorophores

• Validate that stripping doesn't affect tissue integrity or previously deposited signals

Spectral considerations:

• Select fluorophores with minimal spectral overlap

• Include appropriate single-stain controls for spectral unmixing

• Consider photobleaching characteristics when designing imaging sequence

Controls specific for EFNA5 multiplexing:

• Include EFNA5 knockdown/overexpression controls

• Perform staining with individual antibodies as reference

• Include absorption controls with recombinant EFNA5 protein

Data analysis approaches:

• Quantify colocalization using Pearson or Manders coefficients

• Apply machine learning algorithms for pattern recognition

• Use nuclear counterstains as references for cell segmentation

These considerations will ensure robust results when incorporating EFNA5 antibodies into complex multiplexed imaging studies.

How Do Post-Translational Modifications Affect EFNA5 Antibody Detection?

EFNA5 undergoes several post-translational modifications that can affect antibody recognition:

GPI anchoring:

• EFNA5 is attached to the cell membrane via a GPI anchor

• Some antibodies may have differential access to epitopes near the anchor

• PI-PLC treatment can be used to release GPI-anchored proteins for detection

Glycosylation:

• N-linked and O-linked glycosylation affects apparent molecular weight

• Western blots may show heterogeneous banding patterns (28-35 kDa range)

• Deglycosylation treatments:

  • PNGase F for N-linked glycans

  • O-glycosidase for O-linked glycans

  • Compare before/after treatment to assess glycosylation impact

Proteolytic processing:

• EFNA5 can be cleaved to generate soluble forms

• Antibodies targeting different regions may detect different processed forms

• Use antibodies against N-terminal and C-terminal regions to identify processing events

Technical approaches to address these challenges:

• Include appropriate protease inhibitors in all extraction buffers

• Compare reducing vs. non-reducing conditions in Western blotting

• Use multiple antibodies targeting different epitopes

• Analyze band patterns carefully, considering potential modified forms

• Include controls with enzymatic modification/demodification