ADAMTSL4 Antibody

What is ADAMTSL4 and what are its key structural features?

ADAMTSL4 (ADAMTS-like protein 4) is a secreted glycoprotein belonging to the ADAMTS family of proteins, with a predicted molecular mass of approximately 116.5 kDa. Unlike typical ADAMTS proteins, ADAMTSL4 lacks the catalytic domain characteristic of other family members but retains features important for protein interactions that influence tissue stability and extracellular matrix organization . The protein contains multiple thrombospondin type I repeats, which are critical for its interactions with extracellular matrix components. ADAMTSL4 is involved in positive regulation of apoptosis and may facilitate fibrillin-1 (FBN1) microfibril biogenesis, contributing to the structural integrity of tissues . Mutations in ADAMTSL4 result in recessively inherited isolated ectopia lentis, a dysgenesis of the fibrillin-1-rich zonule of Zinn, highlighting its essential role in ocular development and function .

What are the common applications for ADAMTSL4 antibodies in research?

ADAMTSL4 antibodies are employed in multiple research applications with specific protocols optimized for each method:

For Western blot applications, researchers should anticipate detecting ADAMTSL4 at various molecular weights due to extensive post-translational modifications, particularly glycosylation. When performing immunohistochemistry, optimal staining is typically achieved in tissues including skeletal muscle, liver, and colon cancer samples following appropriate antigen retrieval . For co-localization studies of ADAMTSL4 with other extracellular matrix components such as fibrillin-1, immunofluorescence with methanol fixation has proven effective in preserving protein interactions .

Which tissues show the highest expression of ADAMTSL4?

ADAMTSL4 exhibits a distinctive tissue expression pattern that researchers should consider when selecting positive controls for antibody validation:

ADAMTSL4 is strongly expressed in colon, heart, leukocytes, liver, lung, skeletal muscle, spleen, testis, and placenta . Weaker expression is observed in bone marrow, brain tissue, kidney, and pancreas . Studies in fetal tissues reveal robust expression in heart, kidney, liver, lung, and skeletal muscle, with lower levels in fetal brain and skin . Within the eye, ADAMTSL4 shows particularly high expression in the equatorial lens epithelium, which serves as one insertion site for the ciliary zonule . Additional ocular expression is detected in the corneal stroma, corneal epithelium, iris (pupillary and ciliary zones), and retinal pigment epithelium (RPE) .

For researchers working with ocular tissues, the lens epithelium represents an ideal positive control for ADAMTSL4 antibody validation, as in situ hybridization studies have confirmed strong and consistent mRNA expression in this region throughout development and in adult tissues . When working with non-ocular tissues, skeletal muscle, liver, and colon samples have demonstrated reliable ADAMTSL4 immunoreactivity in validated immunohistochemistry protocols .

What fixation and sample preparation methods are recommended for ADAMTSL4 detection?

Optimal detection of ADAMTSL4 requires careful consideration of fixation and sample preparation methods, which vary by application:

For immunohistochemistry on paraffin-embedded tissues (IHC-P), formalin fixation followed by paraffin embedding is the standard approach. After sectioning, antigen retrieval is crucial - TE buffer at pH 9.0 is most commonly recommended, though citrate buffer at pH 6.0 can serve as an alternative . This step is essential for unmasking epitopes that may be cross-linked during fixation.

For immunofluorescence in cultured cells, ice-cold methanol fixation (7 minutes) followed by air drying has been successfully employed in studies examining ADAMTSL4 and fibrillin-1 co-localization . After fixation, blocking with 5% normal goat serum in PBS before antibody incubation minimizes non-specific binding .

For Western blot applications, sample preparation typically involves cell or tissue lysis in the presence of protease inhibitors to prevent ADAMTSL4 degradation. When analyzing conditioned media from cultured cells, concentration steps may be necessary due to the dilute nature of secreted proteins .

When studying glycosylation patterns of ADAMTSL4, enzymatic deglycosylation can provide valuable insights. Treatment with PNGase F removes N-linked glycans, while O-glycosidase treatment removes O-linked glycans . These treatments followed by Western blotting can reveal the contribution of glycosylation to the protein's apparent molecular weight.

How do you validate the specificity of ADAMTSL4 antibodies?

Rigorous validation of ADAMTSL4 antibodies is essential for accurate research results and should employ multiple complementary approaches:

Positive and negative controls: Test antibodies on tissues or cells known to express ADAMTSL4 (positive controls) such as lens epithelium, skeletal muscle, or liver versus tissues with minimal expression (negative controls) . ADAMTSL4-transfected versus non-transfected cell lines provide defined control systems with clear expression differences .

Peptide competition assays: Pre-incubation of the antibody with the immunogenic peptide should abolish specific signals in Western blot or immunostaining applications. For example, ADAMTSL4 antibody reactivity can be attenuated by blocking peptides (ratio 1:2 for Western blot, 1:5 for immunostaining) when incubated at 37°C for 40 minutes to 2.5 hours .

Multiple detection methods: Correlation between protein detection (immunoblotting/immunostaining) and mRNA expression (RT-PCR/in situ hybridization) in the same samples strengthens validation. RNAscope techniques have been successfully employed to verify ADAMTSL4 expression patterns in tissues that show antibody reactivity .

Molecular weight verification: In Western blot, ADAMTSL4 typically appears as a major species around 150 kDa with additional bands at higher or lower molecular weights reflecting glycosylation states or proteolytic processing . The observed molecular weight often differs from the predicted 116.5 kDa due to post-translational modifications .

Knockdown/knockout validation: siRNA-mediated knockdown or genetic knockout models provide definitive specificity controls, as specific antibody signals should be significantly reduced or absent in these systems .

What are the optimal conditions for detecting ADAMTSL4 in Western blot applications?

Successful Western blot detection of ADAMTSL4 requires optimization of several parameters:

Sample preparation: For cell lysates, extraction in buffers containing protease inhibitors is essential to prevent degradation. For detection of secreted ADAMTSL4, analyze both cell lysates and conditioned media, as ADAMTSL4 is primarily a secreted protein . Conditioned media from ADAMTSL4-transfected HEK293F cells shows strong immunoreactivity compared to vector-transfected controls .

Gel electrophoresis and transfer: Use 8-10% SDS-PAGE gels to achieve optimal resolution of ADAMTSL4, which can appear as multiple bands between 65-150 kDa. Longer transfer times or semi-dry transfer systems may improve transfer efficiency of higher molecular weight forms .

Antibody incubation: Dilutions ranging from 1:500 to 1:3000 have proven effective, with overnight incubation at 4°C often yielding the best results . Blocking with 5% non-fat milk or BSA in TBST typically provides adequate background reduction.

Band interpretation: Expect to observe multiple bands representing different forms of ADAMTSL4. A major species around 150 kDa is commonly detected, with additional minor species at approximately 240 kDa, 130 kDa, 90 kDa, or 65-70 kDa . These multiple bands reflect glycosylation states, proteolytic processing, or non-reducible complexes.

Troubleshooting: If detection is weak, concentrating conditioned media samples or using enhanced chemiluminescence detection systems can improve sensitivity. If multiple non-specific bands appear, more stringent washing or increasing antibody dilution may help reduce background .

How does glycosylation affect ADAMTSL4 detection by antibodies?

Glycosylation substantially impacts ADAMTSL4 detection across experimental platforms:

ADAMTSL4 contains multiple N- and O-glycosylation consensus sites that significantly influence its apparent molecular weight and detection characteristics . The predicted molecular weight of 116.5 kDa is typically observed as 150 kDa or higher in Western blot analysis due to these post-translational modifications . This glycosylation creates heterogeneity in the protein's mass, often manifesting as multiple bands or smears on Western blots.

To characterize glycosylation patterns, enzymatic deglycosylation experiments provide valuable insights. Treatment with PNGase F removes N-linked glycans, while O-glycosidase removes O-linked glycans, allowing researchers to determine the contribution of each glycosylation type to the protein's apparent size . When analyzing ADAMTSL4 samples treated with these enzymes, Western blotting can reveal shifts in molecular weight that confirm the presence and extent of glycosylation.

Glycosylation may also affect epitope accessibility for certain antibodies, particularly those raised against peptide sequences near glycosylation sites. This can result in differential detection efficiency between glycosylated and deglycosylated forms of the protein. In some cases, antibodies may preferentially recognize either native glycosylated forms or deglycosylated species, necessitating careful selection based on the experimental question.

When comparing ADAMTSL4 expression across different tissues or experimental conditions, researchers should consider that tissue-specific glycosylation patterns may influence antibody detection sensitivity and the apparent molecular weight of detected bands .

What is the relationship between ADAMTSL4 and fibrillin-1 microfibrils?

ADAMTSL4 has a critical functional relationship with fibrillin-1 microfibrils that can be studied using specific methodological approaches:

ADAMTSL4 directly binds to fibrillin-1 microfibrils and accelerates their biogenesis, suggesting a role in microfibril assembly or stabilization . This interaction is particularly important in the eye, where ADAMTSL4 is expressed in the equatorial lens epithelium at the insertion site of the ciliary zonule, which is composed primarily of fibrillin-1-rich microfibrils . The functional significance of this relationship is underscored by the fact that mutations in ADAMTSL4 result in isolated ectopia lentis, a disorder characterized by dislocation of the lens due to disruption of the zonular fibers .

To study this interaction experimentally, several approaches have proven effective:

• Co-immunofluorescence: Using anti-ADAMTSL4 and anti-fibrillin-1 monoclonal antibodies (such as antibody 1919) to investigate co-localization in cell cultures or tissues .

• Functional cell culture models: Evaluating fibrillin-1 deposition in the extracellular matrix of fetal bovine nuchal ligament cells after culture in ADAMTSL4-conditioned medium versus control medium, with assessment by fibrillin-1 immunofluorescence .

• Animal models: Adamtsl4 knockout mice exhibit zonular fiber detachment, providing in vivo evidence of the interdependence of these proteins in maintaining tissue integrity .

The relationship between these proteins has significant implications for understanding the pathogenesis of ectopia lentis and potentially other disorders involving microfibril dysfunction.

What are the methodological challenges in studying ADAMTSL4 in ocular tissues?

Investigating ADAMTSL4 in ocular tissues presents distinct methodological challenges requiring specialized approaches:

Tissue preservation and processing: The complex architecture of the eye necessitates careful handling during fixation and processing. For immunohistochemistry or immunofluorescence, overfixation can mask ADAMTSL4 epitopes while inadequate fixation compromises tissue morphology . The fibrillin-rich zonular fibers, where ADAMTSL4 plays a critical role, are particularly difficult to preserve intact during processing.

Microdissection techniques: Isolating specific ocular structures (lens epithelium, ciliary body, zonular fibers) requires precise microdissection to study region-specific ADAMTSL4 expression and function . Laser capture microdissection may be necessary for isolating small cellular populations like the equatorial lens epithelium for molecular analysis.

Glycosylation considerations: The high glycosylation state of ADAMTSL4 in ocular tissues affects antibody recognition, necessitating optimization of antigen retrieval protocols (typically using TE buffer at pH 9.0) . Deglycosylation experiments may be particularly informative when characterizing ocular ADAMTSL4.

mRNA expression studies: For detecting ADAMTSL4 transcripts in specific ocular cell types, techniques like RNAscope provide superior spatial resolution compared to conventional in situ hybridization. This approach has successfully localized Adamtsl4 mRNA to the equatorial lens epithelium in mouse eyes .

Three-dimensional context: Maintaining the 3D organization of ocular structures in ex vivo culture systems presents challenges when studying ADAMTSL4-fibrillin interactions. Organ culture systems that preserve the lens-zonule-ciliary body complex may be necessary for certain functional studies .

Cross-species considerations: When using animal models (particularly mice), researchers must account for anatomical differences between rodent and human eyes when interpreting ADAMTSL4 localization and function .

How can different isoforms of ADAMTSL4 be distinguished experimentally?

Distinguishing between ADAMTSL4 isoforms requires a multifaceted approach combining molecular and immunological techniques:

mRNA analysis: RT-PCR using isoform-specific primers can selectively amplify distinct splice variants. This approach has been employed for detecting ADAMTSL4 transcripts, using primers such as 5′-TCC TTT CAC CTG TCC CTT CAG-3′ (forward) and 5′-GAT GAC ATA CTG ATA GAA AAC ACC TGG-3′ (reverse) for bovine ADAMTSL4 . RNA sequencing provides a comprehensive view of all expressed isoforms and their relative abundance.

Isoform-specific antibodies: Generating antibodies targeting regions unique to specific isoforms represents the most direct approach. These can be developed by creating peptide immunogens from isoform-specific sequences and can be used in Western blot, immunoprecipitation, or immunostaining applications .

Electrophoretic separation: Western blot analysis can separate isoforms based on molecular weight differences, though interpretation is complicated by glycosylation. Two-dimensional gel electrophoresis followed by Western blotting can provide enhanced resolution by separating isoforms based on both molecular weight and isoelectric point.

Mass spectrometry: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated ADAMTSL4 can identify peptide sequences unique to specific isoforms with high sensitivity, allowing definitive isoform identification even in complex samples.

Recombinant expression systems: For functional studies, expression constructs encoding individual isoforms can be generated for transfection into cell models, allowing assessment of isoform-specific effects on processes like fibrillin-1 microfibril assembly .

CRISPR-based genome editing: Targeting isoform-specific exons provides an advanced approach to studying the function of individual ADAMTSL4 isoforms in cellular or animal models, allowing selective disruption of specific variants.

What techniques are effective for studying ADAMTSL4 interactions with other extracellular matrix proteins?

Investigating ADAMTSL4 interactions with extracellular matrix components requires specialized methodologies that preserve native conformations:

Co-immunoprecipitation (co-IP): This foundational approach uses antibodies against ADAMTSL4 to pull down interacting partners like fibrillin-1, which can then be identified by Western blotting. Crosslinking reagents may be necessary to stabilize transient interactions before cell lysis .

Proximity ligation assay (PLA): This in situ technique visualizes protein-protein interactions within tissues or cells with high sensitivity, detecting proteins within 40 nm of each other. PLA is particularly valuable for studying ADAMTSL4-fibrillin-1 interactions in their native tissue context.

Direct binding assays: Surface plasmon resonance (SPR) or bio-layer interferometry with purified ADAMTSL4 and candidate binding partners can determine binding kinetics and affinity constants. Solid-phase binding assays, where one protein (e.g., fibrillin-1) is immobilized and binding of ADAMTSL4 is detected using specific antibodies, provide an alternative approach.

Cell culture models: Fetal bovine nuchal ligament cells have been successfully used to evaluate fibrillin-1 deposition in the extracellular matrix after culture in ADAMTSL4-conditioned medium versus control medium . This functional assay demonstrates ADAMTSL4's ability to enhance microfibril formation.

High-resolution microscopy: Confocal or super-resolution microscopy with dual immunofluorescence labeling can visualize co-localization of ADAMTSL4 with other extracellular matrix components. For ADAMTSL4 and fibrillin-1 co-localization, researchers have successfully employed anti-ADAMTSL4 antibodies alongside anti-fibrillin-1 monoclonal antibody MAB1919 (diluted 1:100) .

Proteomics approaches: BioID or APEX proximity labeling can identify proteins in close proximity to ADAMTSL4 in living cells, providing unbiased discovery of potential interaction partners in the cellular context.

In vivo models: Adamtsl4-deficient mice exhibit zonular fiber detachment, providing an in vivo system to study how ADAMTSL4 loss affects the organization and stability of fibrillin-1 and other extracellular matrix components .

What are the emerging applications of ADAMTSL4 antibodies in kidney disease research?

ADAMTSL4 antibodies are finding increasing applications in kidney disease research, particularly in studying chronic kidney disease (CKD) progression:

Recent studies have demonstrated ADAMTSL4 expression in various kidney compartments, with expression patterns changing in correlation with disease states . Immunohistochemistry using ADAMTSL4 antibodies has revealed expression in interstitial tissue (INT) and peritubular capillaries (PTC), with increased expression associated with higher stages of interstitial fibrosis (ci > 1 and IFTA > 1) . This suggests ADAMTSL4 may play a role in the fibrotic processes characteristic of CKD progression.

For these applications, methodological considerations include:

Tissue immunostaining optimization: Antigen retrieval with TE buffer pH 9.0 or citrate buffer pH 6.0 is typically recommended to optimize ADAMTSL4 detection in kidney tissues . Scoring systems for ADAMTSL4 expression in different kidney compartments (glomeruli, tubules, interstitium, vessels) can be developed to correlate with disease parameters.

Plasma quantification: ELISA assays using validated ADAMTSL4 antibodies (such as Human ADAMTS-4 DuoSet ELISA DY4307-05 from R&D Systems) enable quantification of the protein in plasma samples from CKD patients . Research has shown that plasma ADAMTSL4 concentration varies significantly across CKD stages, being highest in CKD stages 2 and 3 compared to other groups (p = 0.0064) .

Dialysis monitoring: Differences in ADAMTSL4 levels have been observed between patients on different renal replacement therapies, with hemodialysis patients showing higher concentrations than those on peritoneal dialysis (p < 0.00001) . This suggests potential utility in monitoring treatment efficacy.

Co-localization studies: Dual-labeling immunofluorescence approaches can co-localize ADAMTSL4 with markers of specific kidney structures or cell types, providing insights into the cellular sources of ADAMTSL4 and its relationship to extracellular matrix changes in kidney fibrosis .

As this field evolves, ADAMTSL4 antibodies may become valuable tools for patient stratification, prognosis assessment, and therapeutic monitoring in kidney disease, particularly as kidney biopsy analysis remains an important diagnostic procedure in nephrology.