MMP19 Antibody

What epitope regions of MMP19 are targeted by commercially available antibodies?

Commercial MMP19 antibodies target different epitope regions of the protein, with most focusing on either the N-terminal or C-terminal domains. C-terminal antibodies typically target regions between amino acids 344-373 or 250-350, while N-terminal antibodies often target the first 100 amino acids . The choice of epitope region can significantly impact experimental outcomes due to:

• Differential accessibility in native proteins

• Varied conservation across species

• Distinct functional domains that may be exposed differently during processing

For studying full-length MMP19, antibodies targeting the C-terminal region (such as those binding amino acids 344-373) have demonstrated reliable detection in Western blot applications with observed molecular weights of 57-60 kDa .

What applications are MMP19 antibodies validated for?

MMP19 antibodies have been validated for multiple research applications with varying performance characteristics:

Most published research has utilized Western blot and IHC applications, with K562 cells and human placenta tissue serving as positive controls for antibody validation .

How should I validate the specificity of a new MMP19 antibody?

Proper validation of MMP19 antibodies requires a multi-step approach:

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide before application. This should abolish specific signals, as demonstrated with antibody ab53146 in immunohistochemical analysis of human breast carcinoma tissue .

• Knockout/knockdown controls: Compare staining patterns between wild-type samples and those with reduced MMP19 expression. MMP19-deficient mice models are available for such validations .

• Multiple antibody comparison: Use antibodies targeting different epitopes of MMP19 to confirm consistent detection patterns.

• Predicted reactivity assessment: Consider cross-reactivity based on epitope conservation. For example, antibody ABIN2779560 shows predicted reactivity of 100% with human MMP19, while showing 86% with mouse, 93% with rat, and varying levels with other species .

• Positive control selection: For human samples, K562 cells, HeLa cells, and placental tissue have shown reliable MMP19 expression .

How can I use MMP19 antibodies to investigate its role in cancer progression?

MMP19 expression has been associated with increased mortality in non-small cell lung cancer (NSCLC) . To investigate MMP19's role in cancer progression:

• Expression profiling: Utilize immunohistochemistry with MMP19 antibodies to evaluate expression patterns in tumor tissues versus adjacent normal tissues. Published data shows MMP19 protein expression is increased in lung cancer tumors compared to histologically normal tissues .

• Prognostic correlation: Quantify MMP19 expression levels and correlate with patient outcomes. Gene expression data from three independent datasets demonstrated that increased MMP19 expression conferred poorer prognosis in NSCLC .

• Functional studies: Combine antibody detection with in vitro assays to correlate MMP19 expression with:

  • Epithelial-mesenchymal transition (EMT) markers

  • Migration and invasion capacities

  • Expression of prometastasis genes

• Mechanistic investigation: Use co-immunoprecipitation with MMP19 antibodies to identify interaction partners in cancer cells.

• Therapeutic potential: Evaluate MMP19 as a potential biomarker for disease severity and outcome in human cancer samples using validated antibodies for IHC applications .

An example workflow could include immunostaining of melanoma tissues using anti-MMP19 antibodies at 10 μg/mL after heat-induced epitope retrieval with HRP-DAB detection systems, which has successfully demonstrated MMP19 localization to cytoplasm and plasma membranes in cancer cells .

What approaches can be used to study MMP19's role in T cell development and immune responses?

MMP19 plays a pivotal role in T cell development and T cell-mediated cutaneous immune responses . Research approaches to investigate this function include:

• Comparative immunophenotyping: Compare wild-type and MMP19-deficient mice for:

  • T cell populations in thymus, lymph nodes, and peripheral blood

  • CD4+/CD8+ T cell ratios

  • Thymocyte maturation stages

• Functional immune assays: Utilize contact hypersensitivity (CHS) models to assess:

  • Inflammatory cell influx

  • Keratinocyte proliferation

  • CD8+ T cell activation in draining lymph nodes

• Cytokine profiling: Measure production of:

  • Lymphotactin

  • Interferon-inducible T cell α chemoattractant (I-TAC)

  • Other proinflammatory cytokines

• Thymocyte development analysis: Assess thymocyte proliferation rates and CD4+CD8+ double-positive cell populations, which were found to be augmented in MMP19-deficient mice .

• Tissue-specific expression: Use immunohistochemistry with anti-MMP19 antibodies to detect expression patterns in lymphoid tissues and skin during immune responses.

In published models, MMP19-deficient mice showed impaired T cell-mediated immune reactions characterized by limited inflammatory cell influx, reduced keratinocyte proliferation, and fewer activated CD8+ T cells in draining lymph nodes during contact hypersensitivity responses .

What are the optimal conditions for Western blot detection of MMP19?

For optimal Western blot detection of MMP19, consider the following technical parameters:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors for cell/tissue lysis

  • Process samples quickly to prevent degradation

  • Validated positive controls include K562 cells, HeLa cells, and human placenta tissue

• Gel electrophoresis:

  • 8-10% SDS-PAGE gels provide optimal resolution for MMP19 (57-60 kDa)

  • Include reducing conditions (β-mercaptoethanol)

• Transfer conditions:

  • PVDF membranes are preferred over nitrocellulose for MMP19 detection

  • Semi-dry or wet transfer at 100V for 1-2 hours, keeping the buffer cold

• Antibody incubation:

  • Primary antibody dilutions: 1:500-1:2000 depending on the specific antibody

  • Overnight incubation at 4°C typically yields better results than short incubations

  • For secondary detection, HRP-conjugated anti-rabbit IgG has been successfully used

• Detection system:

  • Enhanced chemiluminescence (ECL) systems provide good sensitivity

  • Expected band sizes: 57-60 kDa for full-length MMP19, with additional bands at 50 kDa reported in some tissues

• Validation controls:

  • Peptide competition should eliminate specific bands

  • Multiple antibodies targeting different epitopes can confirm specificity

Western blot analysis of human small intestine tissue lysates showed specific bands for MMP19 at approximately 57 kDa and 50 kDa, likely representing different processing forms of the enzyme .

What are the recommended protocols for immunohistochemical detection of MMP19?

For optimal immunohistochemical detection of MMP19 in tissue sections:

• Tissue preparation:

  • Formalin-fixed paraffin-embedded (FFPE) tissues require deparaffinization and rehydration

  • Fresh frozen sections should be fixed with 4% paraformaldehyde in PBS

• Antigen retrieval:

  • Heat-induced epitope retrieval with TE buffer pH 9.0 is recommended

  • Alternative: citrate buffer pH 6.0 with pressure cooker or microwave heating

  • Specific antibodies may have different optimal retrieval conditions

• Blocking:

  • 1% BSA/0.1% Triton X-100 for 15 minutes has been effective

  • Alternative: 5% normal serum from the species of the secondary antibody

• Primary antibody incubation:

  • Dilutions range from 1:20-1:200 depending on the antibody

  • Overnight incubation at 4°C typically provides optimal results

  • For human melanoma tissues, 10 μg/mL of goat anti-human MMP19 antibody has been effective

• Detection systems:

  • For brightfield microscopy: HRP-DAB systems work well

  • For fluorescence: Alexa Fluor-conjugated secondary antibodies

• Co-labeling options:

  • MMP19 antibodies have been successfully co-labeled with GFAP for studies in human donor eyes

  • Sequential staining protocols may be necessary for certain combinations

• Controls:

  • Positive controls: human breast carcinoma, melanoma, and liver cancer tissues have shown reliable MMP19 expression

  • Negative controls: omission of primary antibody or peptide competition

The antibody concentration, incubation time, and detection system should be optimized for each tissue type and fixation method.

How can MMP19 antibodies be used in cancer research?

MMP19 has demonstrated significant associations with cancer progression, making it a valuable target for oncology research:

• Prognostic biomarker evaluation:

  • Studies have shown increased MMP19 gene and protein expression in lung cancer tumors compared to normal tissues

  • In three independent datasets, increased MMP19 expression correlated with poorer prognosis in NSCLC

  • Antibody-based detection methods can quantify MMP19 expression levels in patient samples

• Metastasis mechanism investigations:

  • MMP19 overexpression promotes epithelial-mesenchymal transition, migration, and invasiveness in NSCLC cell lines

  • Antibodies can track MMP19 expression changes during these processes

  • Co-localization studies using MMP19 antibodies with EMT markers can reveal mechanistic insights

• Regulatory pathways:

  • miR-30 isoforms have been identified as regulators of MMP19 expression in lung cancer

  • Antibody-based detection can verify miRNA effects on protein expression

• Functional domains analysis:

  • MMP19 with mutations at the catalytic site showed impaired ability to promote EMT

  • Domain-specific antibodies can help characterize functional regions

• Therapeutic target validation:

  • MMP inhibitors have undergone clinical development with improved selectivity profiles

  • Antibodies can assess target engagement in pre-clinical models

Practical applications include immunohistochemical analysis of human breast carcinoma and melanoma samples, where MMP19 antibodies have demonstrated specific cytoplasmic and membrane staining patterns .

How can I investigate MMP19's role in extracellular matrix remodeling?

MMP19 degrades multiple extracellular matrix (ECM) components, making it an important target for studying matrix remodeling:

• Substrate specificity profiling:

  • MMP19 hydrolyzes collagen type IV, laminin, nidogen, nascin-C isoform, fibronectin, and type I gelatin

  • Co-immunoprecipitation with MMP19 antibodies can identify novel substrates

  • Western blotting can detect cleavage fragments of known substrates

• Tissue-specific ECM degradation:

  • MMP19 degrades aggrecan and cartilage oligomeric matrix protein (COMP) in arthritic disease models

  • Immunohistochemistry with MMP19 antibodies can map expression to areas of active matrix remodeling

• Angiogenesis mechanisms:

  • MMP19 has demonstrated both pro- and anti-angiogenic functions

  • It processes plasminogen to generate angiostatin-like molecules

  • It creates an environment promoting ECM retention of soluble VEGF

  • Co-staining with MMP19 and endothelial markers can reveal spatial relationships

• In vitro degradation assays:

  • Zymography complemented with immunoblotting can correlate MMP19 expression with degradative activity

  • Inhibition studies with specific MMP19 antibodies can confirm enzymatic function

• Genetic models:

  • MMP19-deficient mice do not show overt developmental abnormalities but display altered immune responses

  • Comparative analysis of ECM composition between wild-type and knockout models using MMP19 antibodies can reveal physiological substrates

With its wide expression in stratum basale keratinocytes, smooth muscle cells, epiphysial cartilage chondrocytes, and monocytes/macrophages, MMP19 represents an important target for understanding tissue-specific matrix remodeling processes.

How can I differentiate between active and latent forms of MMP19 using antibodies?

Distinguishing between pro-MMP19 and active MMP19 requires specific methodological approaches:

• Domain-specific antibodies:

  • Pro-domain specific antibodies can selectively recognize latent MMP19

  • Catalytic domain antibodies may recognize both forms

  • C-terminal antibodies typically detect both forms

• Western blot analysis:

  • Pro-MMP19 appears at approximately 57-59 kDa

  • Active MMP19 shows reduced molecular weight (~50 kDa) after pro-domain removal

  • Multiple bands observed in Western blots may represent different activation states

• Activation-state specific assays:

  • Activity-based probes combined with immunoprecipitation using MMP19 antibodies

  • Zymography followed by Western blotting with MMP19 antibodies

  • FRET-based activity assays correlated with antibody detection

• Structural information:

  • Human proMMP-19 is 490 amino acids in length

  • The catalytic domain contains the zinc-binding region targeted by some antibodies

  • Mature MMP19 forms after cleavage between Leu97 and Leu98

• Recombinant protein controls:

  • Compare antibody reactivity with recombinant pro-MMP19 versus activated MMP19

  • Use site-directed mutagenesis to create catalytically inactive mutants as controls

Research indicates that human MMP19 is processed to generate forms of 57 kDa and 50 kDa, with the latter likely representing an activated form of the enzyme .

What approaches can be used to study MMP19 interactions with other proteins?

To investigate MMP19's interactions with other proteins:

• Co-immunoprecipitation (Co-IP):

  • Use MMP19 antibodies (0.5-4.0 μg per 1-3 mg lysate) to pull down protein complexes

  • Analyze precipitated proteins by mass spectrometry to identify novel binding partners

  • Confirm interactions by reverse Co-IP using antibodies against candidate interactors

  • Human placenta tissue has been validated for MMP19 immunoprecipitation

• Proximity ligation assay (PLA):

  • Detect in situ protein-protein interactions using MMP19 antibodies paired with antibodies against suspected interactors

  • Provides spatial information about interaction locations within cells/tissues

  • Requires careful antibody validation to prevent false positives

• Bimolecular fluorescence complementation (BiFC):

  • Generate fusion proteins of MMP19 and potential interactors with split fluorescent protein fragments

  • Validate interactions using antibodies against MMP19 and partner proteins

• Pull-down assays with substrate candidates:

  • Immobilize recombinant MMP19 or use MMP19 antibodies for immunoprecipitation

  • Incubate with potential substrate proteins

  • Analyze binding and/or cleavage using Western blotting

• TIMP interaction studies:

  • MMP19 activity is regulated by tissue inhibitors of metalloproteinases (TIMPs)

  • TIMP-2 C-terminal domain interactions with MMP19 have been studied

  • Co-immunoprecipitation with MMP19 antibodies can confirm TIMP binding

Research has shown that a TIMP-1 chimera with the TIMP-2 C-terminal domain (T1:T2) was a much more effective inhibitor of MMP19 than wild-type TIMP-1, highlighting the importance of protein-protein interactions in regulating MMP19 activity .