ADAM9 Antibody

What is ADAM9 and why is it a significant target for antibody development?

ADAM9 (also known as MDC9 or Meltrin-γ) is a member of the ADAM family, which belongs to the zinc metalloprotease superfamily. ADAM9 contains multiple functional domains including a signal peptide, prodomain, metalloprotease domain, disintegrin domain, cysteine-rich domain, EGF-like domain, transmembrane domain, and cytoplasmic tail . It plays crucial roles in cell migration, proliferation, and invasion, making it a significant target in cancer research . ADAM9 is widely expressed in normal tissues but becomes overexpressed in multiple cancer types, including prostate cancer, pancreatic cancer, gastric cancer, and small cell lung cancer, where it has been linked to invasion and metastasis . This differential expression pattern makes ADAM9 particularly valuable as both a biomarker and therapeutic target.

What are the key structural domains of ADAM9 that different antibodies may target?

ADAM9 antibodies can be designed to target different domains of the protein, each offering unique research applications:

• Cytoplasmic domain antibodies: These recognize the intracellular portion of ADAM9 and are useful for studying signaling interactions. The cytoplasmic tail contains proline-rich sequences that can bind to SH3 domains of proteins like Src, functioning as SH3 ligand domains .

• Ectodomain antibodies: These target the extracellular portion (Ala206-Asp697) and are valuable for studying ADAM9's interactions with extracellular substrates .

• Metalloprotease domain antibodies: These focus on the catalytic region containing the zinc-binding motif HEXGHXXGXXHD, critical for ADAM9's proteolytic function .

• Disintegrin and cysteine-rich domain antibodies: These target regions involved in cell adhesion through interactions with integrins .

How can I distinguish between different isoforms of ADAM9 in my experiments?

To distinguish between ADAM9 isoforms:

• Western blotting with domain-specific antibodies: ADAM9 exists in multiple forms, including a long membrane-bound form (ADAM9-L, ~100 kDa pro-form and ~80 kDa mature form) and an alternatively spliced shorter secreted form (~50 kDa) . Using appropriate antibodies that recognize specific domains allows differentiation between these isoforms.

• Sample preparation considerations: Under reducing conditions in Western blots, you can detect bands at approximately 110 kDa (pro-ADAM9-L), 80 kDa (mature ADAM9-L), and 50 kDa (secreted ADAM9-S) depending on the cell type and antibody used .

• Cell type selection: Different cell lines express varying levels of ADAM9 isoforms. For example, in Western blot analyses, HeLa cells show bands at 78 kDa and 110 kDa, while WI-38 human lung fibroblasts and U-87 MG glioblastoma cells show similar patterns but with potentially different intensities .

What criteria should researchers consider when selecting an ADAM9 antibody for their specific application?

When selecting an ADAM9 antibody, consider:

• Target domain specificity: Determine which domain of ADAM9 is relevant to your research question. For studying proteolytic activity, select antibodies targeting the metalloprotease domain. For cell signaling studies, cytoplasmic domain antibodies are more appropriate .

• Species reactivity: Ensure the antibody recognizes ADAM9 from your experimental species. Some antibodies show cross-reactivity between human, mouse, rat, and monkey ADAM9, while others are species-specific .

• Application compatibility: Verify the antibody has been validated for your specific application (Western blotting, immunoprecipitation, immunohistochemistry). For example, some antibodies work well at 1:1000 dilution for Western blots but may require different optimization for other techniques .

• Isoform recognition: Determine if the antibody detects specific isoforms or all forms. Some antibodies detect both unprocessed pro-forms and active forms of ADAM9, while others are more selective .

• Validation data: Look for antibodies with knockout validation data, as these provide the strongest evidence of specificity .

How can researchers validate the specificity of their ADAM9 antibody?

To validate ADAM9 antibody specificity:

• Knockout cell line controls: Compare antibody reactivity between parental cells and ADAM9 knockout cells. As demonstrated with HeLa cells, specific ADAM9 bands should be detectable in parental lines but absent in knockout lines .

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide before application to samples. Specific signals should be blocked or significantly reduced.

• siRNA knockdown: Transfect cells with ADAM9-specific siRNA and confirm reduced signal intensity compared to non-targeting controls.

• Multiple antibody approach: Use different antibodies targeting distinct epitopes of ADAM9 to confirm consistent results.

• Recombinant protein controls: Include purified ADAM9 protein (full-length or domain-specific) as a positive control to verify antibody recognition.

What are the key differences between polyclonal and monoclonal ADAM9 antibodies in terms of research applications?

Polyclonal ADAM9 antibodies:

• Recognize multiple epitopes, providing stronger signals through cumulative binding

• Offer greater tolerance to protein denaturation, making them suitable for various applications

• Example: Rabbit polyclonal antibodies raised against GST-ADAM9-cytoplasmic tail

• Particularly useful for detecting low-abundance ADAM9 in samples

• May show batch-to-batch variation

Monoclonal ADAM9 antibodies:

• Target single epitopes, offering higher specificity

• Provide consistent results across experiments with minimal batch variation

• More suitable for quantitative analyses where epitope accessibility is consistent

• Better for distinguishing closely related family members (e.g., other ADAM proteins)

• May have limited application range if the single epitope is compromised in certain techniques

Selection depends on your experimental needs: use polyclonals for maximum sensitivity or monoclonals for maximum specificity.

What are the optimal conditions for using ADAM9 antibodies in Western blotting experiments?

For optimal Western blotting with ADAM9 antibodies:

• Sample preparation:

  • Use RIPA or NP-40 based lysis buffers with protease inhibitors

  • Include metalloprotease inhibitors (e.g., EDTA) to prevent autodegradation

  • Perform under reducing conditions using Immunoblot Buffer Group 1 for optimal results

• Antibody dilutions and incubations:

  • Primary antibody: Typically 1:1000 dilution (e.g., for Cell Signaling Technology #2099)

  • Secondary antibody: HRP-conjugated anti-species IgG (e.g., anti-goat IgG for R&D Systems AF939)

  • Incubate primary antibody overnight at 4°C for best results

• Expected bands:

  • Pro-ADAM9-L: ~100-110 kDa

  • Mature ADAM9-L: ~78-80 kDa

  • Secreted ADAM9-S: ~50 kDa

• Controls:

  • Positive control: Cell lines known to express ADAM9 (e.g., WI-38, U-87 MG, HeLa)

  • Negative control: ADAM9 knockout cell lines

  • Loading control: GAPDH or β-actin to normalize expression levels

How can ADAM9 antibodies be effectively used in immunohistochemistry to study tissue expression patterns?

For effective immunohistochemical detection of ADAM9:

• Tissue preparation:

  • Use 3-5 μm sections from formalin-fixed paraffin-embedded (FFPE) tissues

  • Perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Antibody protocol:

  • Block endogenous peroxidase activity with hydrogen peroxide

  • Use protein blocking solution to reduce background

  • Apply ADAM9 antibody at optimized dilution (e.g., 1:800 for FFPE tissues)

  • Visualize using appropriate detection system like poly-link secondary antibody and peroxidase kit

  • Counterstain with hematoxylin for nuclear visualization

• Interpretation:

  • ADAM9 shows positive staining as brown signals in target tissues

  • Co-staining with cell-type specific markers (e.g., S100 for Schwann cells) helps identify ADAM9-expressing cell populations

  • Compare expression levels between pathological tissues and normal controls quantitatively

• Controls:

  • Include positive controls (tissues known to express ADAM9)

  • Include negative controls (primary antibody omission or isotype control)

  • Consider including comparison to mRNA expression data for validation

What approaches can be used to quantify ADAM9 expression levels across different experimental samples?

To quantify ADAM9 expression:

• Western blot densitometry:

  • Capture digital images of immunoblots

  • Use image analysis software (ImageJ, Image Lab, etc.) to quantify band intensities

  • Normalize ADAM9 signals to loading controls

  • Compare fold changes relative to control samples

• qRT-PCR for mRNA expression:

  • Design primers specific to ADAM9 mRNA

  • Use reference genes for normalization

  • Calculate relative expression using ΔΔCt method

  • Can detect significant overexpression (e.g., 8.8-fold higher in vestibular schwannoma compared to controls)

• Flow cytometry:

  • Use fluorescently labeled ADAM9 antibodies for cell surface expression

  • Analyze population shifts to determine expression levels

  • Particularly useful for heterogeneous populations

• ELISA/Sandwich immunoassay:

  • Employ capture and detection antibodies targeting different ADAM9 epitopes

  • Generate standard curves with recombinant ADAM9 protein

  • Quantify ADAM9 levels in cell lysates or biological fluids

How can ADAM9 antibodies be used to investigate the relationship between ADAM9 expression and cancer progression?

ADAM9 antibodies enable several approaches for investigating cancer progression:

• Tissue microarray analysis:

  • Screen ADAM9 expression across multiple patient samples simultaneously

  • Correlate expression with clinical parameters and patient outcomes

  • Assess differential expression between tumor and adjacent normal tissues

  • Example: In vestibular schwannoma, ADAM9 expression strongly correlates with functional impairment (r~1, p=0.01)

• Functional studies with blocking antibodies:

  • Use domain-specific antibodies to inhibit ADAM9 functions

  • Assess effects on cancer cell migration, invasion, and proliferation

  • Compare with genetic knockout/knockdown approaches to validate findings

• Isoform-specific investigations:

  • Differentiate between membrane-bound and secreted ADAM9 forms

  • Study their distinct roles in cancer progression

  • The secreted isoform of ADAM9 has been associated with higher hearing impairment in vestibular schwannoma patients

• Signaling pathway analysis:

  • Investigate how ADAM9 interacts with other signaling molecules

  • Study phosphorylation status of the cytoplasmic tail

  • Examine how ADAM9 contributes to cancer cell adaptations under stress conditions, as ADAM9 overexpression is observed under oxidative stress with increased levels of reactive oxygen species

What are the current approaches for using ADAM9 antibodies in developing targeted cancer therapies?

ADAM9 antibodies are central to developing targeted cancer therapies through:

• Antibody-drug conjugates (ADCs):

  • ADCs targeting ADAM9 are being developed for clinical applications

  • Example: IMGC936 was an ADC targeting ADAM9 developed by ImmunoGen and MacroGenics

  • Although AbbVie terminated IMGC936 development in March 2024 due to unsatisfactory phase I results, MacroGenics continues advancing MGC028, another ADAM9-targeted ADC

• Target validation strategies:

  • Antibodies help validate ADAM9 as a therapeutic target in various cancers

  • Confirm ADAM9 overexpression in target tissues versus normal tissues

  • Demonstrate functional relevance through antibody-mediated blocking studies

• Companion diagnostics development:

  • ADAM9 antibodies can identify patients likely to respond to ADAM9-targeted therapies

  • Stratify patients based on ADAM9 expression levels or specific isoform patterns

  • Help monitor treatment response through sequential tissue or liquid biopsies

• Combination therapy approaches:

  • Study how ADAM9 inhibition might synergize with other treatment modalities

  • Investigate resistance mechanisms to ADAM9-targeted therapies

  • Develop rational combination strategies based on pathway interactions

How can researchers investigate cross-talk between ADAM9 and other signaling pathways using specialized antibody techniques?

To investigate ADAM9 signaling cross-talk:

• Co-immunoprecipitation (Co-IP):

  • Use ADAM9 antibodies to pull down protein complexes

  • Identify interacting partners through mass spectrometry or Western blotting

  • Examine interactions with specific signaling molecules (e.g., Src through the proline-rich sequences in ADAM9's cytoplasmic tail)

• Proximity ligation assay (PLA):

  • Visualize protein-protein interactions in situ

  • Combine ADAM9 antibodies with antibodies against potential interacting partners

  • Quantify interaction signals at subcellular resolution

• Phospho-specific antibody approaches:

  • Use phospho-specific antibodies to detect activated forms of ADAM9

  • Study how phosphorylation of the cytoplasmic tail influences signaling

  • Track signaling cascade activation downstream of ADAM9

• Chromatin immunoprecipitation (ChIP) sequencing:

  • Study transcriptional changes following ADAM9 modulation

  • Identify gene networks influenced by ADAM9 signaling

  • Connect ADAM9 to broader cellular programs in cancer cells

What are common issues encountered with ADAM9 antibodies in Western blotting and how can they be resolved?

Common issues and solutions:

• Multiple unexpected bands:

  • Cause: Cross-reactivity with other ADAM family members

  • Solution: Validate antibody specificity using ADAM9 knockout controls

  • Alternative: Try domain-specific antibodies that have less cross-reactivity

• Weak or no signal:

  • Cause: Insufficient protein loading or antibody concentration

  • Solution: Increase protein amount (25-50 μg) or antibody concentration

  • Alternative: Try a more sensitive detection system (ECL Plus vs. standard ECL)

• Inconsistent detection of isoforms:

  • Cause: Sample preparation affecting protein extraction or degradation

  • Solution: Include metalloprotease inhibitors in lysis buffer

  • Alternative: Compare results using antibodies targeting different domains

• High background:

  • Cause: Insufficient blocking or washing

  • Solution: Optimize blocking (5% BSA often works better than milk for phospho-epitopes)

  • Alternative: Increase washing duration and detergent concentration

• Size discrepancies:

  • Cause: Post-translational modifications affecting migration

  • Solution: Compare with recombinant protein controls

  • Alternative: Use glycosidase treatment to remove glycosylation and assess true protein size

How can researchers address non-specific binding when using ADAM9 antibodies in immunohistochemistry?

To reduce non-specific binding in immunohistochemistry:

• Optimization of antibody concentration:

  • Perform titration experiments to determine optimal dilution

  • Start with manufacturer recommendations (e.g., 1:800 dilution) and adjust as needed

• Blocking procedures:

  • Use adequate blocking (3-5% normal serum from the same species as secondary antibody)

  • Consider dual blocking with both serum and BSA for challenging tissues

  • Add 0.1-0.3% Triton X-100 for better antibody penetration in thicker sections

• Antigen retrieval optimization:

  • Compare different retrieval methods (heat-induced vs. enzymatic)

  • Test different pH conditions (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

  • Adjust retrieval time to balance epitope exposure and tissue preservation

• Controls and countervalidation:

  • Use ADAM9-negative tissues as negative controls

  • Perform peptide competition assays to confirm specificity

  • Compare staining patterns with in situ hybridization for ADAM9 mRNA

• Signal amplification system selection:

  • Choose appropriate detection systems based on expression levels

  • For low expression, consider tyramide signal amplification

  • For co-localization studies, use fluorescent secondary antibodies with minimal spectral overlap

What strategies can researchers employ to optimize ADAM9 antibody performance in co-immunoprecipitation experiments?

For optimal co-immunoprecipitation:

• Lysis buffer optimization:

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

  • Include appropriate protease and phosphatase inhibitors

  • Adjust salt concentration (150-300mM NaCl) to balance specificity and interaction strength

• Antibody selection and conjugation:

  • Choose antibodies validated for immunoprecipitation (e.g., Cell Signaling #2099 at 1:100 dilution)

  • Consider directly conjugated antibodies to avoid heavy chain interference

  • For difficult targets, epitope-tagged ADAM9 constructs might provide cleaner results

• Bead selection and pre-clearing:

  • Magnetic beads often provide better recovery than agarose

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Use control IgG from the same species as your primary antibody

• Cross-linking strategies:

  • Consider reversible cross-linking to stabilize transient interactions

  • DSP (dithiobis[succinimidyl propionate]) preserves complexes while allowing release during elution

• Elution and detection optimization:

  • Use gentle elution conditions to preserve interactions

  • For Western blot detection, use HRP-conjugated protein A/G or light chain-specific secondary antibodies

  • For mass spectrometry, consider on-bead digestion to minimize contamination

How might emerging antibody technologies enhance the study of ADAM9 in disease mechanisms?

Emerging technologies for ADAM9 research:

• Single-domain antibodies (nanobodies):

  • Smaller size allows access to cryptic epitopes in ADAM9

  • Better tissue penetration for in vivo imaging

  • Potential for intracellular delivery to block ADAM9 function

• BiTE (Bispecific T-cell Engager) antibodies:

  • Combine ADAM9 targeting with T-cell recruitment

  • Potential for enhancing immune response against ADAM9-overexpressing tumors

  • May overcome limitations of current ADAM9 ADC approaches

• Antibody fragments for super-resolution microscopy:

  • Smaller probes improve resolution in imaging studies

  • Allow detailed subcellular localization of ADAM9

  • Enable real-time tracking of ADAM9 dynamics in live cells

• Activatable antibody conjugates:

  • Design antibody-drug conjugates activated by tumor microenvironment conditions

  • Improve therapeutic window by reducing off-target effects

  • Particularly relevant given ADAM9's wide expression pattern

What are the current challenges in developing highly specific antibodies against ADAM9 versus other ADAM family members?

Major challenges include:

How can researchers design experiments to investigate the therapeutic potential of ADAM9 antibodies in preclinical models?

Designing preclinical experiments:

• Target validation studies:

  • Compare ADAM9 expression in patient-derived xenografts versus normal tissues

  • Correlate expression with tumor aggressiveness and treatment response

  • Identify optimal patient populations for ADAM9-targeted therapy

• Antibody functional characterization:

  • Test antibodies for their ability to block ADAM9 proteolytic activity

  • Assess effects on cancer cell phenotypes (migration, invasion, etc.)

  • Determine mechanism of action (blocking protein interactions, inducing internalization, etc.)

• In vivo efficacy studies:

  • Evaluate tumor growth inhibition in xenograft models

  • Assess metastasis prevention in appropriate models

  • Study pharmacokinetics and biodistribution using labeled antibodies

• Combination therapy approaches:

  • Test ADAM9 antibodies with standard chemotherapies

  • Evaluate synergy with targeted therapies (e.g., tyrosine kinase inhibitors)

  • Investigate combinations with immune checkpoint inhibitors

• Toxicity and safety assessment:

  • Evaluate on-target/off-tumor effects given ADAM9's wide expression

  • Assess immune-related adverse events with therapeutic antibodies

  • Determine appropriate dosing schedules to balance efficacy and safety

By systematically addressing these aspects, researchers can advance ADAM9 antibodies from bench to bedside, potentially developing new therapeutic options for cancers with ADAM9 overexpression.