ACVRL1 Antibody

What is ACVRL1 and why is it an important research target?

ACVRL1, commonly known as Activin Receptor-Like Kinase 1 (ALK1), is a type I receptor in the Transforming Growth Factor-beta (TGF-β) superfamily. This receptor is primarily expressed in endothelial cells and vascular tissues, where it maintains vascular integrity. It forms heteromeric complexes with type II receptors to transduce signals via SMAD transcription factors . ACVRL1 specifically interacts with Bone Morphogenetic Proteins (BMPs), particularly BMP9 and BMP10, to activate SMAD1/5/8 phosphorylation, regulating endothelial cell proliferation, maturation, and migration .

Research interest in ACVRL1 is heightened by its implication in diseases such as hereditary hemorrhagic telangiectasia (HHT) and its recently discovered role in cancer progression mechanisms, including resistance to multitarget tyrosine kinase inhibitors in colorectal cancer .

Selecting the appropriate ACVRL1 antibody requires consideration of several factors:

• Target species: Confirm reactivity with your experimental species. Most ACVRL1 antibodies are validated for human samples, with many also recognizing mouse and rat ACVRL1 .

• Target domain/epitope: Different antibodies target specific regions of ACVRL1:

  • N-terminal antibodies (e.g., targeting AA 38-68) for detecting full-length protein

  • Extracellular domain antibodies for cell surface detection

  • C-terminal antibodies for specific downstream signaling studies

• Antibody format: Consider whether monoclonal or polyclonal antibodies are more suitable for your application:

  • Monoclonal antibodies offer higher specificity but recognize a single epitope

  • Polyclonal antibodies provide stronger signals by recognizing multiple epitopes

• Validation evidence: Review published literature using the antibody. For example, search results indicate that Abcam's antibodies have been used in peer-reviewed publications .

• Application-specific validation: Select antibodies with validation data for your specific application (WB, IHC, FC, etc.) .

What controls should be implemented when working with ACVRL1 antibodies?

Proper controls are essential for interpreting results with ACVRL1 antibodies:

• Positive controls: Use tissues or cell lines known to express ACVRL1:

  • Endothelial cell lines (MS1, RAEC)

  • Macrophage cell lines (J774)

  • Vascular tissues (heart, aorta)

• Negative controls: Include samples with minimal ACVRL1 expression or employ:

  • Primary antibody omission

  • Isotype controls

  • Secondary antibody-only controls

• Blocking peptide controls: Several manufacturers provide blocking peptides that can be used to confirm antibody specificity . Western blot analysis showing signal elimination when antibodies are preincubated with blocking peptides provides strong evidence of specificity .

• Knockout/knockdown validation: If available, use ACVRL1 knockout or knockdown samples to validate antibody specificity .

• Cross-reactivity assessment: Test for potential cross-reactivity with other ALK family members, particularly in applications like Western blotting .

How can researchers optimize ACVRL1 antibodies for studying receptor activation and signaling?

Studying ACVRL1 activation and signaling requires careful experimental design:

• Phosphorylation detection: To study ACVRL1 activation:

  • Stimulate cells with BMP9 (100 pg/mL) for approximately 20 minutes after serum starvation

  • Use phospho-SMAD1/5/9 antibodies to assess downstream activation

  • Include appropriate time points (5-30 minutes) to capture signaling dynamics

• Co-immunoprecipitation (Co-IP) optimization:

  • Use epitope-tagged ACVRL1 constructs (HA-tag, FLAG-tag) for Co-IP experiments

  • When studying interactions with partner proteins like endoglin or BMPR2, consider using crosslinking agents to stabilize transient interactions

  • For endogenous protein interactions, use detergent conditions that preserve membrane protein interactions (0.5-1% NP-40 or Triton X-100)

• Receptor complex formation studies:

  • Implement proximity ligation assays to visualize ACVRL1 interactions with type II receptors

  • Consider FRET or BRET approaches for live-cell analysis of receptor dynamics

  • Use flow cytometry with extracellular domain-specific antibodies to analyze cell surface expression levels

What methodological approaches are recommended for investigating ACVRL1's role in disease mechanisms?

Recent research has implicated ACVRL1 in multiple disease processes, requiring specific methodological approaches:

• Cancer research applications:

  • RNA sequencing can identify ACVRL1 expression changes in response to treatments (e.g., mTKIs in colorectal cancer)

  • Mass spectrometry (LC-MS) after immunoprecipitation can identify novel ACVRL1 binding partners

  • Western blotting to compare ACVRL1 expression between tumor and normal tissues

  • IHC for assessing ACVRL1 expression patterns in different tumor types

• Vascular disease models:

  • Use site-directed mutagenesis to create disease-relevant ACVRL1 variants for functional studies

  • Implement reporter assays to assess how mutations affect SMAD-dependent transcriptional activation

  • Co-expression studies with endoglin to analyze receptor complex formation in HHT models

• Therapeutic target validation:

  • Gain/loss-of-function experiments to assess the biological function of ACVRL1 in drug resistance

  • Ubiquitination assays to study post-translational regulation

  • Chromatin immunoprecipitation to identify transcriptional targets

How do different experimental conditions affect ACVRL1 antibody performance?

Experimental conditions significantly impact ACVRL1 antibody performance:

• Antigen retrieval for IHC/ICC:

  • Heat-mediated antigen retrieval in EDTA buffer (pH 8.0) is recommended for optimal ACVRL1 detection in paraffin-embedded tissues

  • This is particularly important for detecting membrane proteins like ACVRL1

• Western blot optimization:

  • SDS-PAGE conditions: 5-20% gradient gels provide better resolution for the ~55 kDa ACVRL1 protein

  • Transfer conditions: 150mA for 50-90 minutes to nitrocellulose membranes

  • Blocking: 5% non-fat milk in TBS is effective for reducing background

  • Antibody incubation: Overnight at 4°C with concentrations around 0.5-2 μg/mL

• Flow cytometry considerations:

  • For cell surface detection, use antibodies targeting extracellular domains

  • Avoid permeabilization when studying surface expression

  • Use live intact cells with gentle fixation protocols

• Sample preparation impact:

  • Lysate preparation method affects detection: Membrane fractions may show different ACVRL1 patterns than whole cell lysates

  • Phosphatase inhibitors are essential when studying receptor activation

  • Detergent selection influences membrane protein extraction efficiency

What are the latest methodological advances in studying ACVRL1 protein-protein interactions?

Recent research has employed sophisticated methods to study ACVRL1 interactions:

• Mass spectrometry-based interactome analysis:

  • Transfection with ACVRL1-Flag followed by immunoprecipitation and LC-MS has identified novel interacting partners

  • Comparison with vector controls helps identify specific interactors

  • Peptide ratios >2 between experimental and control samples suggest potential interactions

• Functional validation approaches:

  • Co-immunoprecipitation confirms direct interactions

  • Proximity-dependent biotin identification (BioID) can identify proximal proteins in living cells

  • Domain mapping using truncation mutants helps identify specific interaction regions

• Studying ACVRL1-USP15-GPX2 interactions:

  • Recent research identified that ACVRL1 stabilizes GPX2 via interaction with deubiquitination enzyme USP15

  • This discovery used ubiquitination assays to demonstrate functional consequences of these interactions

  • Similar approaches can be applied to study other ACVRL1 binding partners

How should researchers interpret contradictory results from different ACVRL1 antibodies?

When facing contradictory results from different ACVRL1 antibodies:

• Epitope considerations:

  • Different antibodies target distinct regions (N-terminal, extracellular, C-terminal), which may be differentially accessible in certain applications

  • Some epitopes might be masked by protein-protein interactions or post-translational modifications

  • Compare epitope locations across antibodies showing discrepancies

• Validation approach:

  • Use multiple antibodies targeting different epitopes to confirm results

  • Implement blocking peptide controls to verify specificity

  • Consider genetic approaches (siRNA knockdown, CRISPR knockout) to validate antibody specificity

• Experimental conditions assessment:

  • Fixation methods can affect epitope accessibility, particularly for membrane proteins

  • Denaturation conditions in Western blotting may impact epitope recognition

  • Buffer compositions can influence antibody-antigen interactions

• Publication track record:

  • Consult literature using specific antibodies to determine reliability in similar applications

  • Contact manufacturers for additional validation data not present in datasheets

What are common pitfalls when working with ACVRL1 antibodies and how can they be avoided?

Common pitfalls and their solutions include:

• Non-specific binding:

  • Problem: Background signals in Western blots or IHC

  • Solution: Optimize blocking conditions (5% milk or BSA) , increase washing steps, and titrate antibody concentrations

• False positive signals:

  • Problem: Signals in negative control samples

  • Solution: Validate with blocking peptides , use multiple antibodies targeting different epitopes, and include proper controls

• Inconsistent results across different lots:

  • Problem: Variability between antibody batches

  • Solution: Document lot numbers, request validation data for specific lots, and consider monoclonal antibodies for greater consistency

• Low signal strength:

  • Problem: Weak detection despite known expression

  • Solution: Optimize protein loading, consider more sensitive detection methods, and use signal enhancement systems

• Lack of signal in fixed tissues:

  • Problem: Poor detection in fixed samples despite protein presence

  • Solution: Optimize antigen retrieval (EDTA buffer, pH 8.0) , test different fixation protocols, and consider fresh-frozen samples

How do research findings about ACVRL1 in different disease contexts influence antibody selection and experimental design?

Research on ACVRL1 in disease contexts informs experimental approaches:

• Hereditary Hemorrhagic Telangiectasia (HHT) research:

  • Select antibodies validated in vascular tissues

  • Consider antibodies that can detect specific mutations associated with HHT

  • Design experiments to assess how mutations affect receptor localization and signaling

• Cancer research applications:

  • Recent findings show ACVRL1 drives resistance to multitarget tyrosine kinase inhibitors in colorectal cancer

  • Select antibodies validated in relevant cancer cell lines and tissues

  • Design experiments to study ACVRL1 interactions with USP15 and GPX2

• Angiogenesis research:

  • Use antibodies validated in endothelial cells

  • Design experiments to study ACVRL1 interactions with BMP9/BMP10

  • Consider the role of endoglin as a co-receptor

• Drug development research:

  • Select antibodies that can distinguish between activated and non-activated forms

  • Design experiments to test how inhibitors affect ACVRL1 expression and activity

  • Consider how genetic variability might influence drug responses