TIMP1 Antibody

What is TIMP1 and what considerations are important when selecting TIMP1 antibodies?

TIMP1 (Tissue Inhibitor of Metalloproteinase-1) is a secreted protein of approximately 23,171 daltons that inhibits matrix metalloproteinases (MMPs) . When selecting TIMP1 antibodies, researchers should consider:

• Target epitope location: Some antibodies target the C-terminal domain (e.g., Anti-TIMP1 antibody [C2C3])

• Applications needed: Different clones perform optimally in specific applications:

  • For IHC on FFPE tissues, clone VT7 demonstrates superior sensitivity when used with heat-induced epitope retrieval (HIER)

  • For Western blot, multiple validated antibodies detect bands at ~23-28 kDa

• Species reactivity: Confirm cross-reactivity with your experimental model (human, mouse, rat)

• Immunoglobulin subtype: Most TIMP1 antibodies are IgG1 subtype

How should TIMP1 antibodies be validated prior to experimental use?

Proper validation requires multiple complementary approaches:

• Western blotting: Confirm antibody detects the expected molecular weight band (~23-28 kDa)

  • Example: Human TIMP-1 antibody (AF970) detected specific bands at ~25-26 kDa in human prostate, lung tissue, and cancer cell lines

  • Boster PA1385 antibody detected bands at ~28 kDa in U-87MG, U2OS, and PC-3 cell lysates

• Specificity controls:

  • Use isotype-matched control antibodies (e.g., Aspergillus niger glucose oxidase)

  • Pre-absorption with recombinant TIMP1 protein should eliminate signal

  • BLAST analysis of epitope sequences against human proteome

• Positive control tissues:

  • Islets of Langerhans in normal pancreas

  • Epithelial cells of hyperplastic prostate

  • Tumor cells of medullary thyroid carcinoma

What are the optimal conditions for immunohistochemical detection of TIMP1?

Based on comparative studies of anti-TIMP1 antibodies, researchers should consider:

• Epitope retrieval method: Heat-induced epitope retrieval (HIER) in target retrieval solution (pH 6.1) provides superior results compared to no retrieval or proteolytic treatment

• Antibody concentration: Optimal concentration varies by clone:

  • VT7 shows high sensitivity at 1-10 μg/mL with HIER

  • Other clones may require higher concentrations (see table below)

Data adapted from comparative study of TIMP-1 antibody clones

How can TIMP1 antibodies be used to investigate immune cell interactions in cancer research?

Recent research reveals TIMP1's emerging role in immune regulation, particularly in cancer contexts:

• Dendritic cell (DC) studies:

  • Use anti-TIMP1 antibodies to detect TIMP1 secretion by myeloid DCs and T cells via immunoblotting of conditioned media

  • Investigate TIMP1's effect on MHC-I expression in DCs using flow cytometry after exposure to recombinant TIMP1

• Methodological approach:

  • Stimulate primary human DCs with IFN-γ/LPS and T cells with anti-CD3/CD28 antibodies

  • Collect conditioned media and quantify TIMP1 secretion via Western blot

  • In parallel experiments, treat FLT3L-differentiated myeloid BMDCs with recombinant TIMP1 (100 ng/mL) alone or combined with tumor cell-derived extracellular vesicles

  • Assess MHC-I expression changes via flow cytometry

Research finding: TIMP1 significantly upregulates MHC-I expression in classical type 1 dendritic cells (cDC1), especially when combined with tumor antigen exposure, suggesting its potential to enhance antigen presentation .

What approaches can be used to study TIMP1's role in epithelial-mesenchymal transition (EMT)?

TIMP1 has been implicated in promoting EMT in breast epithelial cells:

• Experimental design:

  • Generate cell lines stably overexpressing TIMP1 using retroviral transduction

  • Analyze EMT marker expression using:

    • Semi-quantitative RT-PCR for mRNA changes

    • Immunoblotting for protein level changes

    • Immunostaining in 3D matrigel culture

• Key markers to assess:

  • Epithelial markers: E-cadherin, β-catenin, γ-catenin (expected to decrease)

  • Mesenchymal markers: vimentin, N-cadherin, fibronectin (expected to increase)

Research finding: TIMP1 overexpression resulted in loss of E-cadherin and gain of vimentin at both mRNA and protein levels, consistent with EMT induction .

How can researchers investigate TIMP1's role in neurological disorders?

Recent studies have identified TIMP1 as a potential biomarker in temporal lobe epilepsy (TLE):

• Tissue expression analysis:

  • Use immunofluorescence staining to determine cellular localization of TIMP1

  • Quantify expression changes using qRT-PCR and Western blot

• Experimental approach:

  • For human studies: Compare hippocampal tissue from TLE patients versus controls

  • For animal models: Use kainic acid-induced epileptic mice

  • Primer sequences for qRT-PCR:

    • Human TIMP1: Forward: TTCCAGTCCCGTCACCTT; Reverse: CAGGCTTCAGCTTCCACTC

    • Mouse TIMP1: Forward: TCACTGTTTGTGGACGGA; Reverse: AGGCTTCAGGTCATCGG

Research finding: TIMP1 was found to be mainly located in cortical neurons with scant expression in cortical gliocytes, and showed decreased expression in TLE patients and kainic acid-induced epileptic mice .

What is the evidence for TIMP1's interaction with Amyloid Precursor Protein (APP) and how can this be studied?

Recent discovery identifies TIMP1 as a novel ligand for APP:

• Detection methods:

  • Unbiased ligand-receptor-capture-screening

  • Pull-down assays

  • Confocal microscopy

  • In silico docking studies

• Experimental approach:

  • Test recombinant TIMP1 variants to map interaction domains

  • Employ interference strategies to block binding

  • Measure functional outcomes such as glucose uptake and cytokine expression in monocytes

Research finding: TIMP1 interacts with APP family members (APP and APLP2) through its C-terminal domain, triggering proinflammatory cytokine expression in monocytes. In pancreatic cancer patients, TIMP1 plasma levels correlate with monocyte activation marker sCD163 .

How can TIMP1 antibodies contribute to investigating its potential role in immunotherapy response?

With TIMP1's newly discovered role in dendritic cell function, researchers can:

• Assess correlation with immunotherapy outcomes:

  • Use TIMP1 antibodies for IHC in tumor biopsies to correlate expression with response to immune checkpoint therapies

  • Perform spatial transcriptomic analysis of immune compartments in tumor microenvironment

• Mechanistic studies:

  • Investigate TIMP1's impact on immunoproteasome/TAP complex components:

    • Measure PSMB8 and TAP-1 expression changes in DCs upon TIMP1 stimulation using Western blot

    • Assess functional consequences for MHC-I peptide loading and presentation

Research finding: High TIMP1 levels in metastatic melanoma correlate with increased CD8+ T cell infiltration and survival, and TIMP1 expression in immune compartments associates with an HLA-A/MHC-I peptide loading signature in lymph nodes .

What are the optimal conditions for studying TIMP1 secretion by immune cells?

To accurately measure TIMP1 secretion and its regulation:

• Cell culture conditions:

  • For BMDCs: Differentiate using FLT3L method which enhances tumor antigen presentation capacity

  • For T cells: Use primary human T cells isolated from PBMCs

• Stimulation protocols:

  • BMDCs: Stimulate with 100 ng/mL LPS

  • Human DCs: Use 10 ng/mL LPS and 1000 IU/mL recombinant human IFN-γ

  • T cells: Activate with anti-CD3/CD28 Dynabeads for 3 days

• Detection methods:

  • Collect conditioned media after 24h stimulation

  • Use immunoblotting with validated anti-TIMP1 antibodies

  • Consider cross-species differences (human vs. mouse) when selecting antibodies

Research finding: Both unstimulated DCs and T cells secrete detectable TIMP1, but stimulated BMDCs show significant increase in TIMP1 secretion, suggesting activation-dependent regulation .

How can researchers address inconsistent results when using TIMP1 antibodies across different applications?

Inconsistencies may arise due to several factors:

• Epitope accessibility issues:

  • Different epitopes may be masked in various applications

  • Solution: Try antibodies targeting different epitopes or use multiple antibodies

• Fixation and preparation effects:

  • In IHC, different antibody clones show varying sensitivity to fixation methods

  • VT7 performs optimally with HIER, while VT2 works better with proteolytic treatment

• Protein modification detection:

  • TIMP1 may undergo post-translational modifications affecting antibody binding

  • Expected molecular weight ranges from 23-28 kDa depending on modification state

• Recommended approach:

  • Validate each antibody specifically for your application

  • Include appropriate controls (recombinant TIMP1, isotype controls)

  • Consider using multiple antibody clones to confirm findings

What factors influence the detection of TIMP1 in tissue sections from different origins?

Based on comprehensive antibody characterization studies:

• Tissue-specific considerations:

  • Endocrine tissues: VT7 antibody at 1 μg/mL with HIER shows consistent strong staining across normal and neoplastic endocrine tissues

  • Neuroendocrine cells: Detection in colon varies by antibody clone (VT1 shows no reactivity)

  • Cancer tissues: Fibroblast-like cells in melanoma show variable staining with different clones and protocols

• Non-specific binding concerns:

  • Some antibody clones (e.g., VT7 at 10 μg/mL) may show non-specific staining

  • Solution: Titrate antibody concentration and include absorption controls

Data adapted from tissue-specific TIMP1 immunoreactivity study

How might TIMP1 antibodies be used to develop new biomarkers for cancer immunotherapy?

Based on recent findings linking TIMP1 to immune regulation:

• Potential biomarker applications:

  • Combined measurement of plasma TIMP1 with sCD163 as prognostic indicators in pancreatic cancer

  • Assessment of TIMP1 expression in immune compartments to predict immunotherapy response

• Methodological approaches:

  • Develop multiplexed IHC panels combining TIMP1 with immune cell markers

  • Analyze TIMP1 in liquid biopsies from patients receiving immune checkpoint inhibitors

  • Correlate TIMP1 levels with CD8+ T cell infiltration in tumor microenvironment

Research finding: TIMP1 presence in liquid biopsies may serve as a biomarker to select patients for combined immunotherapy targeting both immune checkpoints and astrocyte-mediated local immunosuppression in brain metastases .

What experimental designs would best elucidate the mechanistic details of TIMP1's roles beyond MMP inhibition?

To investigate TIMP1's non-canonical functions:

• Structure-function studies:

  • Use domain-specific antibodies to distinguish between N-terminal (MMP inhibition) and C-terminal (cytokine activity) functions

  • Create domain deletion mutants to identify regions responsible for specific functions

• Receptor-binding investigations:

  • Employ proximity ligation assays to visualize TIMP1 interactions with receptors like APP and CD63

  • Use FRET or BiFC to monitor TIMP1-receptor interactions in live cells

• Signaling pathway analysis:

  • Combine TIMP1 treatment with pathway inhibitors to delineate downstream effects

  • Use phospho-specific antibodies to track activation of signaling cascades following TIMP1 binding

Research finding: TIMP1 triggers monocyte activation through its C-terminal domain via APP, suggesting potential therapeutic implications for inflammatory diseases .