AFAP1L2 Antibody

What molecular and structural characteristics define AFAP1L2 protein and how do these impact antibody selection?

AFAP1L2 (actin filament associated protein 1-like 2), also known as XB130, is an 818 amino acid adaptor protein containing several key structural domains including two pleckstrin homology (PH) domains, multiple SH2 and SH3 binding motifs, and a coiled-coil region . While its calculated molecular weight is approximately 91 kDa, it typically appears at around 130 kDa in Western blots due to extensive post-translational modifications .

For antibody selection, researchers should consider:

• Epitope location relative to functional domains

• Whether phosphorylation-specific detection is required

• The need to distinguish between the four known isoforms

• Cross-reactivity with related proteins (AFAP1, AFAP1L1)

Appropriate controls should include AFAP1L2-expressing tissues (thyroid, spleen) and cell lines (A549), with thymus tissue serving as an excellent positive control for both human and mouse studies .

What is the tissue-specific expression pattern of AFAP1L2 and how does this inform experimental design?

AFAP1L2 demonstrates variable expression across tissues, with highest levels in:

• Spleen and thyroid (strong expression)

• Kidney, brain, lung, and pancreas (lower expression)

This tissue-specific expression pattern should inform experimental design in several ways:

• Selection of appropriate positive control tissues (thymus tissue is validated for WB, IHC, and IP applications)

• Choice of model systems (cell lines like A549 express detectable AFAP1L2 levels)

• Interpretation of expression data in disease contexts (e.g., altered expression in hepatocellular carcinoma)

When designing experiments to study AFAP1L2 function in specific tissues, researchers should validate antibody performance in their particular tissue of interest rather than assuming uniform detection efficiency across all tissue types.

What key signaling pathways involve AFAP1L2 and how can antibody-based approaches elucidate these mechanisms?

AFAP1L2 participates in several critical signaling pathways that can be investigated using appropriately validated antibodies:

• Src tyrosine kinase pathway: AFAP1L2 both enhances Src kinase activity and serves as a substrate

• AFAP1L2-SRC-FUNDC1 axis: Regulates mitophagy, with implications for cancer drug resistance

• EGF receptor signaling: Contributes to phosphorylation of Akt and GSK3β

• RET/PTC kinase pathway: Particularly relevant in thyroid cancer

To effectively study these pathways, researchers should:

• Use phospho-specific antibodies to monitor activation states

• Employ co-immunoprecipitation approaches to detect protein-protein interactions

• Combine AFAP1L2 antibodies with antibodies against pathway components in multiplexed assays

• Validate findings with both gain-of-function and loss-of-function approaches

What criteria should guide AFAP1L2 antibody selection for specific experimental applications?

Selection of an appropriate AFAP1L2 antibody requires consideration of multiple technical parameters:

Additionally, consider:

• Species reactivity (human and mouse are most commonly validated)

• Monoclonal vs. polyclonal (monoclonals offer higher specificity; polyclonals may provide stronger signals)

• Host species (rabbit polyclonal and mouse monoclonal options are available)

• Immunogen used (peptide vs. recombinant protein)

What validation protocols are necessary to confirm AFAP1L2 antibody specificity and performance?

Comprehensive validation of AFAP1L2 antibodies should include:

• Western blot analysis:

  • Confirm single band at ~130 kDa (despite calculated 91 kDa weight)

  • Test multiple concentrations (e.g., 1 μg/mL and 2 μg/mL)

  • Include positive controls (A549 cells, thymus tissue)

• Knockdown/knockout validation:

  • Compare signal between wild-type and AFAP1L2-depleted samples

  • Document complete or significant reduction in signal intensity

• Cross-reactivity assessment:

  • Test across species if cross-reactivity is claimed

  • Evaluate potential cross-reactivity with other AFAP family members

• Application-specific validation:

  • For IHC: Demonstrate specific staining in known positive tissues with appropriate controls

  • For IP: Confirm pulled-down protein identity by Western blot

  • For IF: Verify expected subcellular localization patterns

How can researchers reconcile discrepancies between calculated and observed molecular weights of AFAP1L2?

The notable difference between AFAP1L2's calculated molecular weight (91 kDa) and its observed migration (~130 kDa) on SDS-PAGE requires careful consideration:

• Post-translational modifications:

  • AFAP1L2 contains numerous phosphorylation sites

  • Phosphorylation by Src and other kinases significantly affects migration

• Experimental approaches to address this discrepancy:

  • Phosphatase treatment of lysates to assess contribution of phosphorylation

  • Deglycosylation experiments if glycosylation is suspected

  • Mass spectrometry analysis to identify specific modifications

  • Comparison with recombinant unmodified protein

• Interpretation guidelines:

  • Always include positive controls showing the established 130 kDa band

  • Be cautious of signals at exactly 91 kDa without validation

  • Consider the possibility of proteolytic processing or alternative isoforms

This discrepancy highlights the importance of thorough validation when working with AFAP1L2 antibodies.

What protocol optimizations improve AFAP1L2 detection in Western blot applications?

For optimal AFAP1L2 detection by Western blot:

Sample preparation:

• Use fresh tissue/cell lysates with RIPA or similar buffer

• Include both protease AND phosphatase inhibitors (critical for maintaining phosphorylation state)

• Denature samples at 95°C for 5 minutes in loading buffer containing SDS and reducing agent

Gel electrophoresis and transfer:

• Use 8-10% polyacrylamide gels for optimal resolution of the 130 kDa protein

• Transfer to PVDF membranes (preferred over nitrocellulose for high molecular weight proteins)

• Employ wet transfer systems for more efficient transfer of large proteins

Antibody incubation:

• Blocking: 3% non-fat dry milk in TBST

• Primary antibody dilution: 1:500-1:2000

• Incubation: Overnight at 4°C for optimal signal-to-noise ratio

• Secondary antibody: HRP-conjugated anti-rabbit or anti-mouse IgG (depending on primary)

Detection:

• ECL-based detection systems are suitable for most applications

• Exposure time optimization is critical (start with 1 second exposure)

• For weak signals, consider enhanced chemiluminescence substrates

These optimizations will help ensure consistent and specific detection of AFAP1L2 in Western blot applications.

What are the critical parameters for successful immunohistochemical detection of AFAP1L2?

Successful immunohistochemical detection of AFAP1L2 requires attention to several critical parameters:

Tissue preparation:

• Fixation: 10% neutral buffered formalin (24-48 hours)

• Processing: Standard paraffin embedding

• Sectioning: 4-5 μm thickness

Antigen retrieval:

• Primary recommendation: TE buffer pH 9.0

• Alternative method: Citrate buffer pH 6.0

• Heat-induced epitope retrieval (pressure cooker or microwave)

Antibody application:

• Blocking: Serum-based blocking appropriate to secondary antibody

• Primary antibody dilution: 1:50-1:500

• Incubation time: Overnight at 4°C (preferred for maximal sensitivity)

• Detection system: Compatible with primary antibody host species

Controls:

• Positive tissue control: Human thymus tissue shows specific staining

• Negative controls: Primary antibody omission and isotype controls

• Scoring system: Establish clear criteria for intensity and distribution assessment

These parameters should be systematically optimized for each specific AFAP1L2 antibody to achieve consistent and specific staining.

How should immunoprecipitation protocols be adapted for AFAP1L2 studies, particularly for interaction analyses?

For effective immunoprecipitation of AFAP1L2 and associated proteins:

Lysis conditions:

• Use non-denaturing lysis buffers (e.g., NP-40 or Triton X-100 based)

• Include both protease and phosphatase inhibitors

• Maintain cold temperature throughout to preserve protein-protein interactions

Immunoprecipitation procedure:

• Antibody amount: 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

• Pre-clearing: Incubate lysate with protein A/G beads before adding antibody

• Binding: Rotate overnight at 4°C to maximize precipitation efficiency

• Washing: Multiple gentle washes to remove non-specific binding

Co-immunoprecipitation considerations:

• For SRC interaction studies, include sodium orthovanadate to preserve phosphorylation

• For FUNDC1 interaction studies, consider membrane fraction enrichment

• Cross-linking may be necessary for weaker or transient interactions

Verification:

• Confirm successful immunoprecipitation by Western blot for AFAP1L2

• Probe for interaction partners (SRC, FUNDC1) on the same blot

• Include IgG control to identify non-specific binding

Mouse thymus tissue serves as an excellent positive control for AFAP1L2 immunoprecipitation studies .

How can researchers address specificity issues when employing AFAP1L2 antibodies?

To resolve specificity issues with AFAP1L2 antibodies:

Common specificity problems:

• Multiple bands in Western blot

• Unexpected cellular localization in IF

• Non-specific tissue staining in IHC

• False positive co-immunoprecipitation results

Troubleshooting approaches:

• Antibody titration:

  • Test multiple dilutions (typically 1:500-1:2000 for WB; 1:50-1:500 for IHC)

  • Optimize primary and secondary antibody concentrations independently

• Blocking optimization:

  • Compare milk vs. BSA blocking (5% BSA may reduce background for phospho-epitopes)

  • Extend blocking time (1-2 hours at room temperature)

• Sample preparation adjustments:

  • Ensure complete protein denaturation for Western blot

  • Optimize fixation and antigen retrieval for IHC and IF

• Alternative validation:

  • Use multiple antibodies targeting different epitopes

  • Compare with genetic knockdown/knockout models

  • Peptide competition assay to confirm specificity

• Consider cross-reactivity:

  • Evaluate potential cross-reactivity with AFAP1 and AFAP1L1

  • Sequence alignment analysis to identify regions of homology

Systematic application of these approaches will help establish the specificity of signals obtained with AFAP1L2 antibodies.

How should researchers interpret varying results from different AFAP1L2 antibodies across experimental methods?

When facing discrepancies between different AFAP1L2 antibodies or techniques:

Analysis framework:

• Epitope considerations:

  • Map epitopes of different antibodies relative to functional domains

  • Consider accessibility of epitopes in different experimental contexts

  • Evaluate whether post-translational modifications might mask epitopes

• Isoform specificity:

  • AFAP1L2 has at least four known isoforms

  • Determine which isoforms each antibody detects

  • Correlate expression patterns with known isoform distribution

• Technical variables:

  • Native vs. denatured conditions affect epitope accessibility

  • Fixation methods can alter antigen detection in IHC/IF

  • Sample preparation may affect protein modification status

• Validation approach:

  • Use orthogonal methods (mRNA analysis, mass spectrometry)

  • Genetic manipulation (overexpression, knockdown) to verify specificity

  • Consider tissue/cell type-specific factors affecting detection

• Consensus building:

  • Prioritize results validated by multiple antibodies

  • Weight evidence from antibodies with more extensive validation

  • Consider the biological context when interpreting results

This structured approach helps reconcile seemingly contradictory results and build a more accurate understanding of AFAP1L2 biology.

What quality control measures ensure reliable results in longitudinal AFAP1L2 studies?

For consistent results in longitudinal AFAP1L2 studies:

Antibody management:

• Aliquot antibodies upon receipt to avoid freeze-thaw cycles

• Store according to manufacturer recommendations (typically -20°C)

• Track lot numbers and test new lots against previous ones

Sample preparation standardization:

• Standardize lysis/extraction protocols

• Process all comparative samples simultaneously

• Use consistent protein quantification methods

Experimental controls:

• Include unchanged reference/housekeeping proteins

• Run inter-assay calibrators across experiments

• Maintain consistent positive controls (A549 cells, thymus tissue)

Documentation practices:

• Record complete antibody information (catalog number, lot, RRID)

• Document all protocol modifications

• Maintain detailed records of imaging/acquisition parameters

Validation frequency:

• Re-validate antibodies after prolonged storage

• Periodically confirm specificity with knockdown/overexpression controls

• Review published literature for updates on antibody performance

These quality control measures help ensure that observed changes reflect true biological variation rather than technical artifacts.

How can researchers design experiments to investigate AFAP1L2's role in mitophagy and cancer drug resistance?

Recent research has identified AFAP1L2 as a key regulator of mitophagy with implications for cancer drug resistance . To investigate this function:

Experimental design strategy:

• Cell model establishment:

  • Generate AFAP1L2 knockdown and overexpression in relevant cancer cell lines

  • Create sorafenib-resistant hepatocellular carcinoma models

  • Compare AFAP1L2 expression between sensitive and resistant cells

• Molecular pathway analysis:

  • Assess AFAP1L2-SRC-FUNDC1 axis activation

  • Monitor SRC and FUNDC1 phosphorylation status using phospho-specific antibodies

  • Evaluate downstream effects on mitochondrial function

• Mitophagy assessment:

  • Measure expression of mitochondrial membrane proteins (TOMM20)

  • Monitor LC3B lipidation (LC3B-II:LC3B-I ratio)

  • Perform confocal microscopy for colocalization of TOMM20 with LC3B and LAMP1

  • Assess mitochondrial membrane potential and ROS production

• Therapeutic targeting:

  • Test artesunate as an AFAP1L2-targeting agent

  • Evaluate combination strategies to overcome resistance

  • Monitor changes in IC50 values for sorafenib with AFAP1L2 modulation

Expected outcomes:

• AFAP1L2 knockdown should increase drug sensitivity

• AFAP1L2 overexpression should confer resistance

• Changes in resistance should correlate with alterations in mitophagy markers

• Therapeutic targeting of AFAP1L2 should restore drug sensitivity

This experimental framework provides a comprehensive approach to understanding AFAP1L2's role in drug resistance via mitophagy regulation.

What approaches can effectively characterize AFAP1L2 interactions with binding partners in different cellular contexts?

To characterize AFAP1L2 protein interactions:

Interaction detection strategies:

• Affinity-based approaches:

  • Co-immunoprecipitation with AFAP1L2 antibodies followed by Western blot for binding partners

  • Pull-down assays with recombinant AFAP1L2 domains

  • Protein microarrays to identify novel interaction partners

• Proximity-based methods:

  • Proximity ligation assay (PLA) to visualize protein interactions in situ

  • FRET/BRET analysis for dynamic interaction studies

  • BioID or APEX2 proximity labeling to identify interaction networks

• Domain mapping:

  • Create deletion constructs of AFAP1L2 to identify critical interaction regions

  • Generate point mutations in SH2/SH3 binding motifs

  • Perform peptide competition assays with synthetic peptides

• Context-dependent analysis:

  • Compare interactions under different cellular stresses

  • Evaluate how phosphorylation status affects binding partner selection

  • Assess tissue-specific interaction networks

Application to known interactions:

• SRC: Focus on phosphorylation-dependent interactions and functional consequences

• FUNDC1: Investigate links to mitophagy regulation and mitochondrial function

• RET/PTC: Explore relevance to thyroid cancer biology

These approaches will provide a comprehensive understanding of how AFAP1L2 functions as an adaptor protein in different cellular contexts.

What methodological approaches can determine the functional significance of AFAP1L2 post-translational modifications?

AFAP1L2 undergoes extensive post-translational modifications that affect its function. To investigate these:

Modification mapping strategies:

• Phosphorylation analysis:

  • Phospho-specific antibodies for known sites

  • Phospho-enrichment followed by mass spectrometry

  • Phosphatase treatment to confirm phosphorylation contribution to MW shift

  • In vitro kinase assays to identify responsible kinases

• Functional mutant generation:

  • Phosphomimetic mutations (S/T→D/E)

  • Phospho-null mutations (S/T→A)

  • Domain deletion/mutation to prevent specific modifications

• Dynamic modification assessment:

  • Time-course experiments following stimulation

  • Inhibitor treatments to block specific modification pathways

  • Correlation of modification status with functional outcomes

• Structural consequences:

  • Analyze how modifications affect protein-protein interactions

  • Assess impact on subcellular localization

  • Determine effects on protein stability and turnover

Application to AFAP1L2 biology:

• Focus on SRC-mediated phosphorylation sites

• Investigate how phosphorylation affects interaction with FUNDC1

• Determine how modifications regulate mitophagy induction

These approaches will help establish the causal relationships between AFAP1L2 modifications and their functional consequences in normal and disease states.