SMOC1 Antibody

What is SMOC1 and why is it significant in research?

SMOC1 (SPARC-related modular calcium-binding protein 1) is a secreted glycoprotein involved in cell-matrix interactions, osteoblast differentiation, embryonic development, and homeostasis. The protein contains two EF-hand domains, a Kazal-like domain, and two thyroglobulin type-1 domains, which contribute to its structural integrity and functional versatility. SMOC1 is present in the basement membrane zones of various organs during embryonic development, including brain, blood vessels, skin, skeletal muscle, lung, heart, liver, pancreas, ovary, intestine, and kidney . Recent research has identified SMOC1 as one of the most significantly dysregulated proteins in Alzheimer's disease, with elevated levels in brain tissue, cerebrospinal fluid, and plasma, making it a promising biomarker for early disease detection .

What types of SMOC1 antibodies are available for research applications?

SMOC1 antibodies are available in multiple formats to suit different experimental needs. Common types include:

Most commercial SMOC1 antibodies are validated for detecting SMOC1 in mouse, rat, and human samples . When selecting an antibody, consider the specific host species, clonality (monoclonal vs. polyclonal), and validated applications for your experimental design.

What experimental applications are SMOC1 antibodies commonly used for?

SMOC1 antibodies have been validated for multiple applications including:

• Western blotting (WB): For detecting SMOC1 protein expression levels in tissue or cell lysates

• Immunoprecipitation (IP): For isolating SMOC1 and its binding partners

• Immunofluorescence (IF): For visualizing SMOC1 cellular localization

• Enzyme-linked immunosorbent assay (ELISA): For quantitative measurement of SMOC1 in biological fluids

• Immunohistochemistry (IHC): For detecting SMOC1 in tissue sections, particularly useful in studying its colocalization with pathological features

How can I validate a SMOC1 antibody for my specific application?

A standardized validation approach for SMOC1 antibodies includes:

• Cell line selection: Identify cell lines with adequate SMOC1 expression (HeLa cells are recommended based on proteomics databases like PaxDB and DepMap)

• Knockout controls: Compare antibody performance between wild-type and SMOC1 knockout cell lines

• Secretion analysis: Since SMOC1 is a secreted protein, collect and concentrate culture media for Western blot analysis

• Multiple antibody comparison: Test several commercial antibodies side-by-side under identical conditions

• Application-specific validation: Perform validation specific to your intended application (WB, IP, IF, etc.)

This systematic approach ensures that any observed signals are specific to SMOC1 rather than non-specific binding or artifacts .

What are the recommended protocols for SMOC1 detection in cell culture samples?

For detecting secreted SMOC1 in cell culture:

• Culture cells to 70-80% confluence

• Wash cells three times with PBS 1x

• Starve cells in serum-free media for approximately 18 hours

• Collect culture media and centrifuge at 500 x g for 10 minutes to remove cells

• Further centrifuge at 4500 x g for 10 minutes to remove smaller contaminants

• Concentrate media using Amicon Ultra-15 Centrifugal Filter Units (10 kDa NMWL)

• Supplement with protease inhibitor cocktail

• Analyze by Western blot using 4-20% Tris-Glycine polyacrylamide gels

• Transfer to nitrocellulose membranes

• Block with 5% milk for 1 hour

• Incubate with primary SMOC1 antibody overnight at 4°C

• Wash and incubate with appropriate secondary antibody

This protocol is particularly effective for detecting secreted SMOC1 in conditioned media.

How can I optimize co-immunoprecipitation experiments to study SMOC1 protein interactions?

For effective SMOC1 co-immunoprecipitation:

• Select an antibody validated for IP applications (such as mouse monoclonal IgG2a SMOC1 antibody)

• Start with concentrated conditioned media from cells expressing SMOC1

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

• Incubate cleared lysates with SMOC1 antibody (typically 2-5 μg per sample)

• Add protein A/G beads and rotate overnight at 4°C

• Wash extensively (at least 4-5 washes) with IP buffer containing mild detergent

• Elute bound proteins with sample buffer and analyze by Western blot

• Include appropriate controls: IgG control IP, input sample, and when possible, SMOC1 knockout samples

This methodology has been successfully used to demonstrate interactions between SMOC1 and Aβ in Mild Cognitive Impairment (MCI) and Alzheimer's disease brain tissue, as well as with phosphorylated tau in AD brain tissue .

What are the recommended procedures for studying SMOC1 colocalization with AD pathology markers?

For studying SMOC1 colocalization with AD pathology:

• Tissue preparation:

  • Fix brain tissue in 10% neutral buffered formalin

  • Process and embed in paraffin

  • Section at 5-7 μm thickness

• Dual immunohistochemistry/immunofluorescence:

  • Perform heat-induced epitope retrieval

  • Block with serum-free protein block

  • Incubate with primary antibodies (SMOC1 and AD pathology markers like Aβ or phosphorylated tau)

  • Use differently labeled secondary antibodies for visualization

  • Counterstain nuclei with DAPI

• Quantitative analysis:

  • Capture images using a confocal microscope

  • Quantify colocalization using specialized software

  • Calculate percentage of plaques or tangles positive for SMOC1

Research has shown that SMOC1 strongly colocalizes with a subpopulation of amyloid plaques in AD (43.8 ± 2.4%), MCI (32.8 ± 5.4%), and preclinical AD (28.3 ± 6.4%), and also colocalizes with a subpopulation of phosphorylated tau aggregates in AD (9.6 ± 2.6%) .

How does SMOC1 relate to AD progression and can it serve as a biomarker?

SMOC1 has emerged as one of the most significant biomarkers of early Alzheimer's disease. Research findings indicate:

• SMOC1 levels in cerebrospinal fluid increase many years before symptom onset, with significant increases detected up to 29 years before symptoms in autosomal dominant AD

• SMOC1 levels in brain tissue positively correlate with amyloid plaque load across all disease stages, from preclinical AD to advanced disease

• The percentage of SMOC1-positive plaques increases with disease progression:

  • Preclinical AD: 28.3 ± 6.4%

  • Mild Cognitive Impairment: 32.8 ± 5.4%

  • Advanced AD: 43.8 ± 2.4%

These findings suggest SMOC1 could be a valuable early biomarker for AD, potentially helping identify at-risk individuals decades before clinical manifestation, making it useful for both research and potentially for clinical applications in the future .

What methods are used to investigate SMOC1's effect on amyloid beta aggregation?

To study SMOC1's effect on Aβ aggregation:

• Thioflavin-T (ThT) aggregation assays:

  • Prepare monomeric Aβ peptide solutions

  • Add recombinant SMOC1 at various concentrations

  • Include ThT as a fluorescent reporter of β-sheet formation

  • Monitor fluorescence at 440nm excitation/485nm emission over time

  • Compare aggregation kinetics with and without SMOC1

• Electron microscopy:

  • Allow Aβ to aggregate with or without SMOC1

  • Apply samples to carbon-coated grids

  • Negative stain with uranyl acetate

  • Image using transmission electron microscopy

  • Analyze fibril morphology and dimensions

These methods have demonstrated that SMOC1 significantly delays Aβ aggregation in a dose-dependent manner, and that Aβ fibrils formed in the presence of SMOC1 show altered morphology . This suggests SMOC1 may play a role in modulating Aβ pathology in AD.

What are common challenges when working with SMOC1 antibodies and how can they be addressed?

When working with SMOC1 antibodies, researchers commonly encounter:

• Low signal in Western blots:

  • Solution: Concentrate conditioned media as SMOC1 is a secreted protein

  • Use serum-free media to reduce background

  • Consider using agarose-conjugated antibodies for immunoprecipitation followed by Western blot

• Non-specific bands:

  • Solution: Always include a knockout control when possible

  • Use antibodies validated through standardized protocols

  • Optimize antibody concentration and incubation conditions

• Inconsistent results across experiments:

  • Solution: Standardize cell culture conditions

  • Maintain consistent protocols for media collection and concentration

  • Include both positive and negative controls in each experiment

Always validate antibodies using multiple techniques and controls to ensure specificity for your application.

How can I design experiments to study SMOC1's functional roles in development and disease?

To investigate SMOC1's functional roles:

• Knockdown/knockout approaches:

  • Use CRISPR/Cas9 to generate SMOC1 knockout cell lines

  • Employ siRNA or shRNA for transient knockdown

  • Validate knockdown efficiency by Western blot and qPCR

• Overexpression studies:

  • Create stable cell lines overexpressing SMOC1

  • Use inducible expression systems for temporal control

  • Express tagged versions (His, FLAG, GFP) for easier detection

• Functional assays:

  • Cell migration and invasion assays to assess ECM interactions

  • Calcium signaling assays to investigate the role of calcium-binding domains

  • Protein-protein interaction studies using co-IP or proximity ligation assays

  • Osteoblast differentiation assays to study bone development functions

• Animal models:

  • Conditional SMOC1 knockout mice to study tissue-specific functions

  • Evaluate developmental phenotypes

  • Assess neurological function in AD model mice with modified SMOC1 expression

These approaches can provide comprehensive insights into SMOC1's diverse biological functions and potential therapeutic implications.