TMEM131L Antibody, Biotin conjugated

What is TMEM131L and why is it important for research?

TMEM131L (Transmembrane protein 131-like) is a membrane-associated protein with several critical biological functions. It plays significant roles in antagonizing canonical Wnt signaling by triggering lysosome-dependent degradation of Wnt-activated LRP6 and regulates thymocyte proliferation . Additionally, recent research has demonstrated TMEM131L's potential impact on the occurrence and prognosis of glioblastoma multiforme (GBM) and low-grade glioma (LGG) via mechanisms related to nerve conduction and information transfer . The TMEM131 family proteins have evolutionarily conserved roles in collagen recruitment and secretion across various species, from C. elegans to humans, making them important targets for extracellular matrix research .

What are the key specifications of commercially available TMEM131L antibodies, Biotin conjugated?

Currently available TMEM131L antibodies with biotin conjugation typically have the following specifications:

How does TMEM131L antibody detection compare with detection of other TMEM family proteins?

While TMEM131L antibodies target the transmembrane protein 131-like specifically, other TMEM family proteins have distinct antibody specifications and applications. For instance, TMEM119 antibodies are often used as microglia markers in neuroinflammation research, with applications in flow cytometry and immunohistochemistry . TMEM132A antibodies typically have applications in ELISA, IHC-P, and IHC-F with predicted reactivity across multiple species including human, mouse, rat, and others .

What are the optimal conditions for using TMEM131L antibody, Biotin conjugated, in ELISA applications?

For optimal ELISA performance with biotin-conjugated TMEM131L antibody:

• Dilution optimization: Start with a 1:500-1000 dilution range and optimize based on signal-to-noise ratio .

• Buffer system: Use a blocking buffer containing 1-3% BSA in PBS with 0.05% Tween-20 to minimize non-specific binding.

• Detection system: Utilize a streptavidin-HRP conjugate system, which binds with high affinity to the biotin conjugation. This provides superior sensitivity compared to traditional secondary antibody detection methods.

• Incubation conditions:

  • Primary antibody (biotin-conjugated TMEM131L): 1-2 hours at room temperature or overnight at 4°C

  • Streptavidin-HRP: 30-60 minutes at room temperature

  • Development using TMB substrate: 5-30 minutes (monitor color development)

• Controls: Include both positive controls (recombinant TMEM131L protein) and negative controls (unrelated proteins) to validate specificity.

The methodology should be adjusted based on specific experimental conditions and the nature of the samples being analyzed.

How can I optimize flow cytometry protocols using TMEM131L antibody, Biotin conjugated?

For flow cytometry applications using biotin-conjugated TMEM131L antibody:

• Cell preparation:

  • Use single-cell suspensions at 1×10^6 cells per 100μl

  • Fix cells with 2-4% paraformaldehyde if intracellular staining is required

  • For intracellular staining, permeabilize cells with 0.1% saponin or 0.1% Triton X-100

• Antibody staining protocol:

  • Block cells with 5% normal serum in PBS for 30 minutes

  • Incubate with biotin-conjugated TMEM131L antibody (1:100-1:200 dilution) for 30-60 minutes at 4°C

  • Wash cells 3 times with PBS containing 1% BSA

  • Incubate with fluorophore-conjugated streptavidin (such as Alexa Fluor 488 or Quantum dot 655) at 1:5000 dilution for 30 minutes at 4°C

  • Wash cells 3 times and analyze by flow cytometer

• Gating strategy:

  • Use forward and side scatter to identify intact cells

  • Apply appropriate compensation when using multiple fluorophores

  • Analyze TMEM131L expression based on fluorescence intensity compared to isotype controls

• Data analysis considerations:

  • Comparison of mean/median fluorescence intensity between experimental groups

  • Analysis of percentage of TMEM131L-positive cells within different populations

This protocol can be adapted for various cell types, including those from glioma tissues, where TMEM131L has demonstrated significant expression and prognostic value .

What is the recommended protocol for Western blotting using TMEM131L antibody, Biotin conjugated?

For Western blotting with biotin-conjugated TMEM131L antibody:

• Sample preparation:

  • Extract proteins using RIPA or NP-40 buffer with protease inhibitors

  • Quantify using BCA or Bradford assay

  • Load 20-50μg of total protein per lane

• Electrophoresis and transfer:

  • Use 8-10% SDS-PAGE (TMEM131L is approximately 195-210 kDa)

  • Transfer to PVDF membrane at 100V for 90-120 minutes or overnight at 30V

• Blocking and antibody incubation:

  • Block with 5% non-fat milk or 3-5% BSA in TBST for 1 hour

  • Incubate with biotin-conjugated TMEM131L antibody at 1:1000 dilution overnight at 4°C

  • Wash 3 times with TBST, 5-10 minutes each

  • Incubate with streptavidin-HRP (1:5000-1:10,000) for 1 hour at room temperature

  • Wash 3 times with TBST, 5-10 minutes each

• Detection:

  • Use ECL substrate for visualization

  • Expected band size: ~195-210 kDa for full-length TMEM131L

• Controls and validation:

  • Use GAPDH or β-actin as loading controls

  • Consider including lysates from cells with known TMEM131L expression levels

  • For validation, compare with cells treated with TMEM131L shRNA knockdown

This protocol is adaptable based on specific experimental requirements and should be optimized for each cell or tissue type being studied.

How does TMEM131L expression correlate with clinical outcomes in glioma patients?

Research has revealed significant correlations between TMEM131L expression and clinical outcomes in glioma patients:

These findings suggest that TMEM131L antibodies can be valuable tools for prognostic research in glioma, potentially informing patient stratification and treatment decisions.

What is known about the functional role of TMEM131L in collagen trafficking and secretion?

TMEM131L belongs to the TMEM131 family, which plays critical roles in collagen processing and secretion:

• Structural domains and their functions:

  • N-terminal PapD chaperone-like domains: Recruit premature collagen monomers for proper assembly and secretion

  • C-terminal domains: Interact with TRAPPC8, a component of the TRAPP tethering complex

• Cellular trafficking mechanism:

  • TMEM131 proteins localize to the endoplasmic reticulum (ER)

  • They facilitate collagen cargo trafficking from ER to the Golgi

  • This function is mediated through interaction with TRAPPC8 via the C-terminal TRAPID (TRAPP III–interacting domain)

• Evolutionary conservation:

  • These roles in collagen recruitment and secretion are evolutionarily conserved across species:

    • C. elegans

    • Drosophila

    • Humans

• Loss-of-function effects:

  • Deficiency of TMEM131 leads to:

    • Impaired collagen production

    • Activation of ER stress response

    • Reduction in collagen abundance in relevant tissues

This fundamental role in collagen biology makes TMEM131L a potential target for research in extracellular matrix disorders, tissue engineering, and fibrosis-related pathologies.

What methodologies are available for studying TMEM131L interactions with other proteins?

Several methodologies have been employed to study TMEM131L protein interactions:

• Yeast Two-Hybrid (Y2H) screening:

  • Successfully used to identify TRAPPC8 as an interaction partner of TMEM131L

  • The C-terminal region of TMEM131L (particularly amino acids 1741-1883) has been shown to be critical for this interaction

  • Mutagenesis studies have revealed that the WRD motif in TRAPPC8 is important for binding to TMEM131L

• Co-immunoprecipitation (Co-IP):

  • Can be performed using anti-TMEM131L antibodies to pull down protein complexes

  • Biotin-conjugated antibodies can be advantageous for pull-down experiments using streptavidin beads

  • Detection of interacting partners can be done through Western blotting or mass spectrometry

• Proximity ligation assay (PLA):

  • Allows visualization of protein-protein interactions in situ

  • Particularly useful for membrane-associated proteins like TMEM131L

  • Requires pairs of antibodies against TMEM131L and suspected interaction partners

• CRISPR-Cas9 gene editing:

  • Has been used to generate precise deletions of functional domains in TMEM131 proteins

  • For example, deletion of the cytoplasmic domain (amino acids 1287-1808) demonstrated its importance for interaction with TRAPPC8

• Fluorescence resonance energy transfer (FRET):

  • Can detect protein interactions in living cells

  • Requires fluorescently tagged versions of TMEM131L and interaction partners

These methodologies can be combined to comprehensively characterize TMEM131L interactions in various cellular contexts and disease models.

What are the common challenges when using biotin-conjugated antibodies, and how can they be addressed?

When working with biotin-conjugated antibodies like those targeting TMEM131L, researchers may encounter several challenges:

• High background signal:

  • Cause: Endogenous biotin in tissues or cell samples

  • Solution: Pre-block endogenous biotin using avidin/biotin blocking kits before applying the biotin-conjugated primary antibody

  • Alternative approach: Use a biotin-free detection system for tissues with high endogenous biotin content

• Weak signal intensity:

  • Cause: Suboptimal antibody concentration or insufficient biotin conjugation

  • Solutions:

    • Titrate antibody concentration (try 1:100, 1:500, 1:1000 dilutions)

    • Use signal amplification systems (e.g., TSA system)

    • Consider detection with quantum dots for enhanced sensitivity

• Non-specific binding:

  • Cause: Cross-reactivity with other proteins or insufficient blocking

  • Solutions:

    • Increase blocking time/concentration (5% BSA or 10% normal serum)

    • Include 0.1-0.3% Triton X-100 in the antibody diluent

    • Validate specificity using TMEM131L knockdown controls

• Inconsistent results between experiments:

  • Cause: Variability in sample preparation or antibody performance

  • Solutions:

    • Standardize all protocols

    • Use the same lot of antibody when possible

    • Include positive and negative controls in each experiment

• Storage-related issues:

  • Cause: Antibody degradation due to improper storage

  • Solution: Store at recommended temperatures (-20°C to -80°C) and avoid repeated freeze-thaw cycles

How can I design experiments to study the role of TMEM131L in glioma progression using the biotin-conjugated antibody?

A comprehensive experimental design to study TMEM131L in glioma progression could include:

• Expression analysis in patient samples:

  • Technique: Immunohistochemistry using biotin-conjugated TMEM131L antibody

  • Samples: Glioma tissue microarrays with corresponding clinical data

  • Controls: Normal brain tissue, isotype control antibodies

  • Analysis: Scoring of staining intensity and percentage of positive cells

  • Correlation: With clinical parameters including tumor grade, IDH status, and 1p/19q codeletion

• Functional studies in glioma cell lines:

  • TMEM131L knockdown: Using shRNA or CRISPR-Cas9

  • TMEM131L overexpression: Using lentiviral constructs

  • Phenotypic assays:

    • Proliferation (MTT, BrdU incorporation)

    • Migration and invasion (transwell assays)

    • Colony formation

    • Apoptosis (Annexin V/PI staining)

  • Validation: Western blot with biotin-conjugated TMEM131L antibody

• Signaling pathway analysis:

  • Technique: Western blotting for Wnt signaling components (given TMEM131L's role in antagonizing canonical Wnt signaling)

  • Key proteins to analyze: LRP6, β-catenin, GSK3β, phosphorylated forms

  • Validation: Rescue experiments with Wnt pathway modulators

• Tumor microenvironment studies:

  • Flow cytometry: Using biotin-conjugated TMEM131L antibody with fluorescent streptavidin

  • Analysis of immune cell populations: Correlating with TMEM131L expression

  • Cytokine profiling: Using multiplex ELISA

  • Spatial analysis: Using multiplexed immunofluorescence with TMEM131L and immune cell markers

• In vivo studies:

  • Orthotopic xenograft models: Using TMEM131L-modified glioma cells

  • Analysis: Tumor growth, survival, invasion

  • Immunohistochemistry: Using biotin-conjugated TMEM131L antibody on tumor sections

  • Correlation: With tumor progression and immune infiltration

This comprehensive approach would provide insights into both the prognostic value and functional significance of TMEM131L in glioma biology.

What considerations should be made when using TMEM131L antibody, Biotin conjugated, for single-cell analysis techniques?

When adapting biotin-conjugated TMEM131L antibody for single-cell analysis, several important considerations should be addressed:

• Flow cytometry optimization:

  • Cell fixation/permeabilization: Optimize based on TMEM131L's subcellular localization (membrane-associated and cytoplasmic)

  • Antibody concentration: Typically higher concentrations (1:50-1:100) are needed for single-cell detection

  • Fluorophore selection: Choose streptavidin conjugates compatible with your cytometer and other markers

    • Quantum dot 655 offers superior brightness and photostability for rare populations

  • Controls: Include fluorescence-minus-one (FMO) controls to set accurate gates

• Mass cytometry (CyTOF) considerations:

  • Metal-conjugated streptavidin: Select appropriate metal tags that don't overlap with other markers

  • Panel design: Place TMEM131L in context with lineage markers, functional markers, and other proteins of interest

  • Signal optimization: Titrate antibody concentration to maximize signal-to-noise ratio

  • Batch effects: Include barcoding strategies for multi-sample experiments

• Single-cell imaging techniques:

  • Immunofluorescence microscopy:

    • Use confocal microscopy for subcellular localization

    • Consider super-resolution techniques for detailed localization studies

  • Imaging flow cytometry:

    • Combines advantages of flow cytometry with cellular imaging

    • Useful for confirming membrane vs. cytoplasmic localization

• Single-cell sequencing integration:

  • CITE-seq approaches:

    • Can couple antibody detection with transcriptomics

    • Requires oligo-tagged streptavidin rather than fluorescent streptavidin

  • Validation: Correlate protein expression with TMEM131L mRNA levels

• Technical challenges:

  • Signal amplification: May be necessary for low-abundance expression

  • Antibody penetration: Critical for tissues and 3D cultures

  • Multiplexing: Consider cyclic immunofluorescence or sequential staining approaches

  • Quantification: Standardize using calibration beads for flow cytometry

Addressing these considerations will enable robust single-cell analysis of TMEM131L expression, particularly in heterogeneous samples like glioma tissues where cell-to-cell variation may have biological and clinical significance .

How might TMEM131L antibodies be utilized in developing prognostic tools for glioma patients?

Based on current evidence of TMEM131L's prognostic significance in glioma , several approaches could be developed:

These approaches could ultimately lead to the development of a clinical-grade test for routine prognostic assessment in glioma patients.

What are the potential applications of studying TMEM131L in the context of collagen-related disorders?

Given the established role of TMEM131 family proteins in collagen trafficking and secretion , several promising research directions emerge:

• Fibrotic disease research:

  • Investigate TMEM131L expression and function in:

    • Liver fibrosis

    • Pulmonary fibrosis

    • Kidney fibrosis

    • Cardiac fibrosis

  • Analyze correlation between TMEM131L levels and disease severity

  • Explore potential as a therapeutic target to modulate collagen secretion

• Connective tissue disorders:

  • Study TMEM131L dysregulation in:

    • Ehlers-Danlos syndromes

    • Marfan syndrome

    • Osteogenesis imperfecta

  • Analyze how mutations in TMEM131L might affect collagen processing

  • Develop cellular models using patient-derived cells

• Tissue engineering applications:

  • Manipulate TMEM131L expression to optimize collagen production in:

    • Artificial skin constructs

    • Bioengineered cartilage

    • Vascular grafts

  • Monitor extracellular matrix quality using biotin-conjugated TMEM131L antibodies

• Cancer metastasis research:

  • Investigate how TMEM131L-mediated collagen remodeling affects:

    • Tumor cell invasion

    • Metastatic niche formation

    • Treatment resistance

  • Develop potential therapeutic strategies targeting this pathway

• Aging-related matrix degradation:

  • Study how TMEM131L function changes with age

  • Investigate potential interventions to maintain proper collagen processing

  • Examine correlations with age-related conditions

These research directions could significantly advance our understanding of extracellular matrix biology and potentially identify novel therapeutic targets for collagen-related disorders.

How could advanced proteomics methods be combined with TMEM131L antibody studies to elucidate its interactome?

Advanced proteomics approaches can significantly enhance our understanding of TMEM131L's protein interaction network:

• Proximity-dependent biotin identification (BioID):

  • Fuse a biotin ligase (BirA*) to TMEM131L

  • Allow proximal proteins to be biotinylated in living cells

  • Capture biotinylated proteins using streptavidin

  • Identify interacting partners by mass spectrometry

  • Validate key interactions using biotin-conjugated TMEM131L antibodies

• Quantitative interactomics:

  • SILAC or TMT labeling: Compare TMEM131L interactome under different conditions:

    • Normal vs. disease states

    • With/without stress conditions

    • Different subcellular compartments

  • Data analysis: Apply bioinformatics to identify condition-specific interactions

• Cross-linking mass spectrometry (XL-MS):

  • Use chemical cross-linkers to stabilize protein-protein interactions

  • Perform immunoprecipitation with biotin-conjugated TMEM131L antibody

  • Identify cross-linked peptides by mass spectrometry

  • Map interaction interfaces at amino acid resolution

• Thermal proximity co-aggregation (TPCA):

  • Analyze co-aggregation patterns of proteins during thermal denaturation

  • Identify proteins that aggregate with TMEM131L, suggesting physical interaction

  • Validate using traditional approaches with biotin-conjugated antibodies

• In situ proximity labeling:

  • Apply enzyme-catalyzed proximity labeling in tissue sections

  • Visualize TMEM131L interaction partners in their native context

  • Particularly valuable for studying interactions in disease-relevant tissues

• Integrative multi-omics approach:

  • Combine interactome data with:

    • Transcriptomics (RNA-seq)

    • Phosphoproteomics

    • Glycoproteomics

  • Create comprehensive models of TMEM131L-centered pathways

These approaches would significantly expand our understanding of TMEM131L's functional network beyond the currently known interactions with TRAPPC8 and collagen-related proteins .