TIMP3 Antibody

What is TIMP3 and why is it significant in biological research?

TIMP3 (Tissue Inhibitor of Metalloproteinase 3) is a critical regulatory protein that inhibits matrix metalloproteinases (MMPs), a group of enzymes involved in extracellular matrix (ECM) degradation. Unlike other TIMP family members, TIMP3 uniquely inhibits ADAM17 and binds irreversibly to the ECM, making it an essential regulator of tissue remodeling and integrity. TIMP3 functions by binding to the catalytic zinc cofactor of MMPs, preventing their proteolytic activity . Its significance extends to nervous system development, tissue regeneration, and visual perception, with abnormal expression implicated in multiple pathological conditions including cardiovascular diseases and Sorby's fundus dystrophy .

How do TIMP3 antibodies differ from other TIMP family antibodies in terms of specificity and applications?

TIMP3 antibodies detect a protein with unique properties compared to other TIMP family members. While all TIMPs inhibit MMPs, TIMP3 is distinguished by:

• Its exclusive ability to effectively inhibit ADAM17/TACE

• High affinity for binding to the extracellular matrix components

• Irreversible binding to zinc-dependent MMPs

These characteristics make TIMP3 antibodies particularly valuable for studying ECM regulation, ADAM17-mediated signaling pathways, and vascular inflammation that aren't detectable with other TIMP antibodies .

What are the recommended dilutions and applications for TIMP3 antibodies in different experimental contexts?

Optimal dilutions for TIMP3 antibodies vary by application and specific antibody product:

For optimal results, researchers should titrate the antibody in each testing system and perform preliminary experiments with positive control tissues such as human placenta, mouse brain, or rat brain tissues for Western blotting applications . Different antibody clones may have varying sensitivities and specificities, so validation with both positive and negative controls is essential.

How should researchers optimize antigen retrieval for TIMP3 antibody staining in immunohistochemistry?

For optimal TIMP3 detection in immunohistochemistry:

• Primary recommendation: Use TE buffer at pH 9.0 for antigen retrieval

• Alternative method: Citrate buffer at pH 6.0 may also be effective

The choice between these methods should be determined empirically for each tissue type. Human stomach tissue has been validated as a positive control for TIMP3 immunostaining. When optimizing:

• Test both buffer systems on serial sections

• Evaluate signal-to-noise ratio

• Consider tissue-specific factors that may influence epitope accessibility

• Adjust incubation times (typically 15-30 minutes) based on preliminary results

Different fixation methods may require additional optimization steps for successful TIMP3 immunodetection .

What controls should be included when validating TIMP3 antibodies in experimental systems?

Comprehensive validation of TIMP3 antibodies requires several controls:

• Positive tissue controls: Use tissues known to express TIMP3:

  • Human placenta tissue

  • Mouse and rat brain tissues

  • Human stomach tissue (for IHC)

• Negative controls:

  • Primary antibody omission

  • Isotype control (rabbit IgG at equivalent concentration)

  • TIMP3 knockout/knockdown samples where available

• Specificity controls:

  • Pre-absorption with immunizing peptide

  • Comparison of staining patterns across multiple TIMP3 antibodies targeting different epitopes

  • Western blot validation showing expected molecular weight (20-30 kDa)

• Cross-reactivity assessment:

  • Testing on tissues from different species (human, mouse, rat)

  • Evaluation in tissues expressing other TIMP family members

How are TIMP3 expression and function altered in atherosclerotic plaques, particularly in subjects with type 2 diabetes?

TIMP3 expression is significantly reduced in atherosclerotic plaques from subjects with type 2 diabetes compared to those with normal glucose tolerance (NGT). This reduction has functional consequences:

• Molecular consequences:

  • Increased ADAM17 activity

  • Elevated MMP9 activity

  • Enhanced vascular inflammation

• Cellular localization:

  • Immunohistochemistry shows that both macrophages and smooth muscle cells express TIMP3 in NGT subjects

  • This expression pattern is diminished in diabetic subjects

• Clinical correlations:

  • Negative correlation between TIMP3 expression and LDL cholesterol (r = -0.29; P < 0.03)

  • Negative correlation between TIMP3 expression and A1C levels (r = -0.31; P < 0.02)

These findings suggest that TIMP3 downregulation may be a key mechanism linking diabetes to accelerated atherosclerosis, offering potential therapeutic targets for intervention .

What is the relationship between TIMP3 and Sorsby's fundus dystrophy, and how can antibodies help study this condition?

Sorsby's fundus dystrophy (SFD) is an inherited macular degeneration caused by mutations in the TIMP3 gene. Key aspects of this relationship include:

• Mutation characteristics:

  • Point mutations in the C-terminal domain of TIMP3 are causative

  • The Ser181Cys mutation is commonly associated with SFD

  • These mutations do not affect TIMP3's MMP-inhibitory function

• Antibody applications in SFD research:

  • Detection of mutant TIMP3 accumulation in Bruch's membrane

  • Evaluation of normal vs. mutant TIMP3 localization

  • Investigation of downstream effects on VEGF signaling

  • Analysis of protein dimerization or aggregation

• Methodological considerations:

  • Antibodies recognizing both wild-type and mutant TIMP3 are needed for comparative studies

  • N-glycosylation at Asn184 may affect antibody binding but not inhibitory function

  • Techniques to distinguish between matrix-bound and soluble TIMP3 are critical for understanding pathogenesis

Research using TIMP3 antibodies helps elucidate how these mutations lead to protein accumulation, ECM disruption, and ultimately visual impairment .

How does TIMP3 regulate cerebral blood flow, and what experimental approaches can assess this function?

TIMP3 plays a crucial role in regulating cerebral arterial tone and blood flow responses through inhibition of ADAM17 and subsequent HB-EGF signaling. Key methodological approaches include:

• Functional assessment techniques:

  • Whisker stimulation to evaluate cerebrovascular reactivity

  • Measurement of cerebral blood flow responses to acetylcholine or adenosine

  • Pharmacological studies using ADAM17 inhibitors (GW413333X) versus ADAM10 inhibitors (GI254023X)

• Molecular mechanisms:

  • TIMP3 inhibits ADAM17, which processes HB-EGF

  • This pathway affects voltage-dependent potassium channel (Kv) number in cerebral arterial myocytes

  • The balance between TIMP3 and ADAM17 activity modulates cerebral blood flow

• Experimental models:

  • CADASIL mouse model (a genetic small vessel disease)

  • Hypomorphic ADAM17 mice to confirm specificity

  • Exogenous ADAM17 or HB-EGF administration to rescue cerebral arterial tone

This research reveals TIMP3 as a significant regulator of cerebrovascular function with implications for small vessel diseases of the brain .

How does TIMP3 interact with VEGF signaling, and what methodologies can assess this interaction?

TIMP3 modulates angiogenesis through its interaction with VEGF signaling pathways in a concentration-dependent manner:

• Binding characteristics:

  • TIMP3 binds to VEGFR2 with an IC50 of 3.3-4.5 μg/ml

  • This binding affinity is significantly lower than VEGF's affinity for VEGFR2 (IC50 of 8.5-15 ng/ml)

  • The C-terminal domain of TIMP3 mediates this interaction

• Concentration-dependent effects:

  • Higher concentrations inhibit angiogenesis

  • Lower concentrations may paradoxically promote angiogenesis, though the mechanism remains unclear

• Methodological approaches:

  • Competitive binding assays with labeled VEGF

  • VEGFR2 phosphorylation analysis

  • Endothelial tube formation assays

  • In vivo angiogenesis models (e.g., matrigel plug assay, retinal vascularization)

  • Proximity ligation assays to detect TIMP3-VEGFR2 interactions

• Research considerations:

  • Distinguish between direct VEGFR2 binding and indirect effects via MMP inhibition

  • Account for concentration-dependent biphasic effects

  • Consider cross-talk with other signaling pathways

What is the mechanism of TIMP3 interaction with AT2R, and how can this be experimentally investigated?

TIMP3 interacts with the Angiotensin II type 2 receptor (AT2R) but not type 1 receptor (AT1R), presenting a unique signaling pathway:

• Interaction domains:

  • The N-terminal domain of TIMP3 binds to the C-terminal sequence (aa 235-363) of AT2R

  • This is distinct from VEGFR2 binding, which involves TIMP3's C-terminal domain

• Functional consequences:

  • Potential additive effects on apoptosis in cancer cells

  • Inhibition of VEGF-induced angiogenesis

  • The anti-angiogenic effect may be independent of this interaction

• Experimental approaches:

  • Co-immunoprecipitation to confirm protein-protein interaction

  • Domain mapping using truncated protein constructs

  • siRNA knockdown of AT2R to assess TIMP3-dependent functions

  • Dual overexpression systems to evaluate additive effects

  • Apoptosis assays in relevant cell types (normal vs. cancer cells)

• Research implications:

  • TIMP3 may simultaneously bind VEGFR2 (via C-terminus) and AT2R (via N-terminus)

  • The functional significance in cardiovascular disease remains to be fully elucidated

  • This interaction represents a potential new therapeutic target

How does the N-terminal domain of TIMP3 contribute to its inhibitory function against MMPs and ADAMs?

The N-terminal domain of TIMP3 is critical for its inhibitory activity against both MMPs and ADAMs:

• Structural elements:

  • Ridge formed by N-TIMP3 residues Cys1-Thr2-Cys3-Ser4-Pro5

  • sC-sD loop (Ser64-Glu-Ser-Leu-Cys68)

  • Cys1-Cys68 disulfide bond

• Inhibitory mechanism:

  • These structural elements insert into the MMP/ADAM active site

  • The Cys1 residue lies above the catalytic zinc ion

  • Mutation of Cys1 to Ser results in complete loss of MMP-inhibitory activity

• Experimental approaches:

  • Site-directed mutagenesis of key residues

  • Enzyme activity assays with purified MMPs/ADAMs

  • Structural studies (X-ray crystallography, NMR)

  • Fluorimetric assays to measure inhibition efficacy

  • Comparison with other TIMP family members to identify TIMP3-specific features

• Research considerations:

  • The N-terminal domain is sufficient for inhibitory activity (N-TIMP3)

  • The C-terminal domain assists inhibition by interacting with hemopexin domains of certain MMPs

  • TIMP3 can form trimolecular complexes with proMMP2 and MT3-MMP

What are common technical challenges when using TIMP3 antibodies, and how can they be overcome?

Researchers frequently encounter several challenges when working with TIMP3 antibodies:

• Specificity issues:

  • Cross-reactivity with other TIMP family members

  • Solution: Validate with multiple antibodies targeting different epitopes or use TIMP3 knockout samples

• Detection sensitivity:

  • Low endogenous expression levels in some tissues

  • Solution: Consider signal amplification methods (TSA), longer exposure times, or Superclonal™ antibodies that combine multiple recombinant monoclonal antibodies for enhanced sensitivity

• Matrix binding interference:

  • TIMP3's high affinity for ECM can complicate extraction

  • Solution: Use specialized extraction buffers containing detergents or chaotropic agents to release matrix-bound TIMP3

• Post-translational modifications:

  • Glycosylation may affect antibody binding

  • Solution: Test multiple antibodies recognizing different epitopes; compare results with enzymatic deglycosylation

• Nonspecific background in IHC:

  • Particularly in tissues with abundant ECM

  • Solution: Optimize blocking (use bovine serum albumin at 0.1% concentration for 20μl sizes), increase antibody dilution, and use appropriate antigen retrieval methods (TE buffer pH 9.0 or alternative citrate buffer pH 6.0)

How can researchers distinguish between free and ECM-bound TIMP3 in experimental systems?

Distinguishing between soluble and ECM-bound TIMP3 requires specialized experimental approaches:

• Sequential extraction method:

  • Initial extraction with physiological buffers (PBS) to isolate free TIMP3

  • Followed by extraction with detergent-containing buffers to release loosely bound TIMP3

  • Final extraction with chaotropic agents (urea, guanidine HCl) to release tightly matrix-bound TIMP3

• In situ analysis techniques:

  • Immunofluorescence with minimal permeabilization to detect surface-bound TIMP3

  • Dual staining with ECM component antibodies (collagen, proteoglycans) to assess colocalization

  • Proximity ligation assays to detect TIMP3-ECM protein interactions

• Functional assays:

  • Comparison of MMP inhibitory activity in soluble versus ECM fractions

  • Assessment of relative resistance to washout in cell culture systems

  • ADAM17 activity assays in different cellular compartments

• Analytical considerations:

  • Matrix-bound TIMP3 may have altered epitope accessibility

  • Different antibodies may preferentially detect free versus bound forms

  • Cross-validation with techniques that don't rely on antibody recognition (e.g., mass spectrometry)

What approaches can be used to study the role of TIMP3 in regulating ADAM17 activity in various disease models?

Investigating TIMP3-mediated regulation of ADAM17 in disease contexts requires multiple complementary approaches:

• Activity assays:

  • Fluorimetric assays using ADAM17-specific substrates

  • Analysis of ADAM17 substrate shedding (TNF-α, HB-EGF, etc.)

  • Comparison between normal and disease tissues/models

• Pharmacological interventions:

  • Use of selective ADAM inhibitors (GW413333X for ADAM17)

  • Control experiments with ADAM10 inhibitors (GI254023X) to confirm specificity

  • Dose-response studies to determine IC50 values

• Genetic approaches:

  • TIMP3 overexpression or knockdown/knockout

  • ADAM17 hypomorphic mouse models

  • Patient samples with TIMP3 mutations or polymorphisms

• In vivo functional assays:

  • Measuring cerebral blood flow responses following whisker stimulation

  • Assessment of vascular responses to acetylcholine or adenosine

  • Rescue experiments with exogenous ADAM17 or HB-EGF

• Tissue-specific analyses:

  • Comparative studies in atherosclerotic plaques from diabetic versus non-diabetic subjects

  • Investigation of TIMP3/ADAM17 balance in various vascular beds

  • Analysis of downstream signaling pathways (e.g., EGFR activation)

These approaches collectively allow for comprehensive assessment of how TIMP3 regulates ADAM17 in pathological conditions, providing insights into potential therapeutic interventions .

How is TIMP3 expression regulated by SirT1, and what experimental design can investigate this relationship?

The relationship between SirT1 (Sirtuin 1) and TIMP3 expression has significant implications for vascular health:

• Regulatory mechanism:

  • SirT1 levels positively correlate with TIMP3 expression in vivo

  • Inhibition of SirT1 activity reduces TIMP3 expression in smooth muscle cells

  • SirT1 overexpression increases TIMP3 promoter activity

• Experimental design considerations:

  • Chromatin immunoprecipitation (ChIP) to detect SirT1 binding to TIMP3 promoter

  • Luciferase reporter assays with TIMP3 promoter constructs

  • Modulation of SirT1 activity using activators (resveratrol) or inhibitors (EX-527)

  • Western blot and qPCR for TIMP3 expression following SirT1 manipulation

  • Analysis in disease models, particularly diabetes-associated atherosclerosis

• Clinical relevance:

  • Both TIMP3 and SirT1 are reduced in atherosclerotic plaques from diabetic subjects

  • This relationship may explain the accelerated atherosclerosis observed in diabetes

  • Therapeutic strategies targeting this pathway could potentially modify disease progression

• Methodological challenges:

  • Distinguishing direct vs. indirect effects of SirT1 on TIMP3 expression

  • Accounting for cell type-specific differences in regulation

  • Translating in vitro findings to in vivo significance

What emerging technologies are advancing TIMP3 antibody development and application in research?

Several innovative approaches are enhancing the utility of TIMP3 antibodies in research:

• Recombinant Superclonal™ antibody technology:

  • Combines multiple recombinant monoclonal antibodies

  • Provides polyclonal-like sensitivity with monoclonal-like specificity

  • Ensures reproducibility by eliminating biological variability

  • Optimal for detecting low-abundance TIMP3 in complex samples

• Enhanced validation methodologies:

  • Use of CRISPR-Cas9 knockout validation

  • Orthogonal method validation (mass spectrometry)

  • Physiological context validation in multiple cell types

  • Application-specific testing (IHC, ICC-IF, WB)

• Advanced imaging applications:

  • Super-resolution microscopy for subcellular localization

  • Multiplexed immunofluorescence for pathway analysis

  • Intravital imaging for in vivo TIMP3 dynamics

  • FRET-based sensors for detecting TIMP3-target interactions

• Therapeutic development applications:

  • Use of antibodies to modulate TIMP3 function

  • Development of bispecific antibodies targeting TIMP3 and its interaction partners

  • Antibody-drug conjugates for targeted delivery

  • Tools for pharmacodynamic biomarker assessment in clinical trials

These technological advances provide researchers with more sensitive, specific, and reproducible tools for investigating TIMP3 biology and its role in disease processes .

How can TIMP3 antibodies be applied to identify novel therapeutic targets in cardiovascular and neurodegenerative diseases?

TIMP3 antibodies serve as valuable tools for uncovering new therapeutic opportunities:

• Cardiovascular disease applications:

  • Identification of TIMP3 reduction in atherosclerotic plaques from diabetic patients

  • Assessment of ADAM17 and MMP9 overactivity as downstream consequences

  • Correlation with clinical parameters (LDL cholesterol, A1C levels)

  • Stratification of patient samples to identify responders to TIMP3-based therapies

• Cerebrovascular applications:

  • Investigation of TIMP3-sensitive pathways in cerebral small vessel disease

  • Analysis of ADAM17/HB-EGF axis in regulating cerebral blood flow

  • Identification of voltage-dependent potassium channels as downstream effectors

  • Development of pharmacological interventions targeting specific pathway components

• Neurodegenerative disease approaches:

  • Evaluation of TIMP3 expression patterns in affected brain regions

  • Analysis of ECM integrity in relation to TIMP3 levels

  • Investigation of TIMP3's role in blood-brain barrier function

  • Assessment of interactions with disease-specific proteins

• Multi-omics integration:

  • Combination of TIMP3 antibody-based proteomics with transcriptomics

  • Correlation with metabolomic profiles to identify affected pathways

  • Network analysis to discover novel TIMP3-dependent mechanisms

  • Validation of therapeutic targets using genetic and pharmacological approaches

This multifaceted approach enables identification of novel therapeutic targets within TIMP3-regulated pathways that could be exploited for disease intervention .