TMEM131L Antibody, HRP conjugated

What is TMEM131L and what are its known biological functions?

TMEM131L (also known as KIAA0922) is a transmembrane protein that belongs to the TMEM131 family. Based on current research, TMEM131L functions as a membrane-associated protein that antagonizes canonical Wnt signaling by triggering lysosome-dependent degradation of Wnt-activated LRP6 . It has been demonstrated to regulate thymocyte proliferation and plays an important role in several cellular processes . Recent studies have also identified TMEM131L as a potential prognostic marker in glioma, with significant expression in glioma tissues correlating with poor patient outcomes .

The TMEM131 family proteins, including TMEM131L, contain bacterial PapD chaperone-like domains in their amino termini, which are involved in recruiting premature collagen monomers for proper assembly and secretion . The carboxy termini interact with TRAPPC8, a component of the TRAPP tethering complex, to facilitate collagen cargo trafficking from the endoplasmic reticulum to the Golgi apparatus .

What are the key specifications of commercially available TMEM131L antibody with HRP conjugation?

The TMEM131L antibody with HRP conjugation is typically a polyclonal antibody produced in rabbit hosts. Its specifications include:

Information sourced from product data sheets

Why choose an HRP-conjugated TMEM131L antibody over unconjugated versions?

HRP (Horseradish peroxidase) conjugated antibodies offer several methodological advantages in research applications:

• Enhanced signal amplification: HRP conjugation significantly amplifies detection signals, increasing assay sensitivity for detecting low abundance proteins like TMEM131L .

• Reduced experimental steps: Direct conjugation eliminates the need for secondary antibody incubation steps, streamlining experimental protocols and reducing potential sources of variability .

• Lower background noise: HRP-conjugated primary antibodies can provide higher specificity with lower background compared to two-antibody detection systems, particularly valuable when investigating novel proteins like TMEM131L with limited characterization .

• Versatile detection options: HRP conjugates can be detected using multiple substrates (chemiluminescent, colorimetric, or fluorescent), allowing researchers to optimize visualization based on equipment availability and experimental needs .

This conjugation is particularly valuable when investigating proteins expressed at low levels or when sample volume is limited, as is often the case in early TMEM131L research .

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

For optimal ELISA performance with HRP-conjugated TMEM131L antibody, consider the following methodological guidelines:

• Antigen coating concentration: For recombinant TMEM131L protein, use 1-10 μg/ml in carbonate buffer (pH 9.6) or PBS (pH 7.4) for coating microplate wells.

• Blocking solution: Use 3-5% BSA or 5% non-fat dry milk in PBS with 0.05% Tween-20 for 1-2 hours at room temperature to minimize non-specific binding.

• Antibody dilution: The recommended dilution range for TMEM131L antibody, HRP conjugated is typically 1:20 to 1:50, though optimal concentration should be determined empirically for each lot .

• Incubation conditions: Incubate the primary HRP-conjugated antibody for 1-2 hours at room temperature or overnight at 4°C for maximum sensitivity.

• Washing protocol: Perform 4-5 washes with PBS containing 0.05% Tween-20 between steps to reduce background.

• Substrate selection: TMB (3,3',5,5'-Tetramethylbenzidine) is recommended for HRP detection, with reaction stopped using 2N H₂SO₄ after appropriate color development (typically 5-30 minutes).

• Controls: Include positive controls (known TMEM131L-expressing samples), negative controls (samples lacking TMEM131L), and blank wells (no primary antibody) to validate results.

• Signal measurement: Read absorbance at 450nm with 620nm as reference wavelength.

When troubleshooting, ensure that the antigen is properly immobilized and accessible, as TMEM131L's transmembrane nature may affect epitope presentation.

How should researchers optimize western blotting protocols when using HRP-conjugated TMEM131L antibody?

While the primary documented application for TMEM131L antibody with HRP conjugation is ELISA , researchers can adapt it for western blotting with the following optimization strategies:

• Sample preparation: Use RIPA buffer supplemented with protease inhibitors for efficient extraction of membrane-associated TMEM131L. Consider using phosphatase inhibitors if investigating phosphorylation states.

• Protein loading: Load 20-50 μg of total protein per lane for cell lysates or 10-25 μg for tissue extracts.

• Gel selection: Use 8-10% SDS-PAGE gels for optimal resolution of TMEM131L, which has a molecular weight of approximately 131 kDa.

• Transfer conditions: Transfer to PVDF membrane (preferred over nitrocellulose for transmembrane proteins) at 30V overnight at 4°C for complete transfer of high molecular weight proteins.

• Blocking optimization: Test both 5% non-fat dry milk and 3-5% BSA in TBST to determine optimal blocking for your specific application.

• Antibody dilution: Begin with a 1:20 to 1:50 dilution in blocking buffer, adjusting based on signal-to-noise ratio .

• Incubation parameters: Incubate with primary antibody overnight at 4°C with gentle agitation.

• Stringent washing: Perform 5-6 washes with TBST, 5 minutes each, to reduce background associated with HRP-conjugated antibodies.

• Detection system: Use enhanced chemiluminescence (ECL) substrates, with standard sensitivity for abundant samples or high sensitivity variants for low expression levels.

• Exposure optimization: Begin with short exposures (30 seconds) and increase as needed to optimize signal detection without saturation.

Include positive controls such as cell lines known to express TMEM131L (e.g., U2OS bone osteosarcoma cells) to validate antibody specificity.

What controls should be included when validating TMEM131L antibody specificity?

Proper validation of TMEM131L antibody specificity is critical for generating reliable research data. Implement the following control strategy:

• Positive expression controls: Include samples from tissues/cells with known TMEM131L expression, such as:

  • Human glioma tissues, which show significant TMEM131L expression

  • Bone osteosarcoma U2OS cell lines, which express TMEM131 family proteins

  • Thymocytes, as TMEM131L regulates their proliferation

• Negative expression controls:

  • Cell lines with TMEM131L knockdown using validated shRNAs

  • Tissues known to have minimal TMEM131L expression

  • Competitive peptide blocking with the immunizing peptide (amino acids 963-978 of human TMEM131L)

• Technical controls:

  • Secondary antibody-only control (for two-step detection methods)

  • Substrate-only control to assess endogenous peroxidase activity

  • Isotype control antibody (rabbit IgG) to identify non-specific binding

• Orthogonal validation:

  • Correlation with mRNA expression levels via RT-PCR

  • Comparison with alternative antibodies targeting different TMEM131L epitopes

  • Recombinant expression of tagged TMEM131L followed by parallel detection

• Knockout/knockdown validation:

  • CRISPR/Cas9-mediated knockout or siRNA/shRNA knockdown of TMEM131L to confirm signal specificity

  • Test against the five different validated shRNA sequences targeting TMEM131L that have shown correlation between knockdown efficiency and phenotypic effects

These rigorous controls will help distinguish specific signals from artifacts and validate the TMEM131L antibody's performance across different experimental contexts.

How can TMEM131L antibody be used to investigate its role in collagen secretion pathways?

TMEM131L and related family proteins play critical roles in collagen secretion through their PapD chaperone-like domains and interaction with trafficking machinery. Researchers can employ HRP-conjugated TMEM131L antibody to investigate these pathways through:

• Co-immunoprecipitation assays: Use the antibody to pull down TMEM131L protein complexes, followed by western blotting or mass spectrometry to identify interacting partners, particularly:

  • Collagen proteins, especially COL1A2, which has been shown to interact with the PapD-L domain

  • TRAPPC8 and other components of the TRAPP tethering complex

  • Components of the lysosomal degradation pathway involved in Wnt signaling

• Immunofluorescence co-localization: Though not directly using the HRP conjugate (which is optimized for enzymatic detection), the same antibody clone can be used in immunofluorescence to:

  • Visualize co-localization with ER and Golgi markers

  • Track TMEM131L distribution during collagen trafficking

  • Assess changes in localization upon disruption of trafficking pathways

• ELISA-based interaction studies: Using the HRP-conjugated antibody to:

  • Detect TMEM131L binding to immobilized collagen proteins

  • Quantify TMEM131L-collagen complex formation under various conditions

  • Screen for compounds that modulate TMEM131L-collagen interactions

• Functional rescue experiments: In cells with TMEM131L knockdown (which show decreased extracellular type I collagen fibers) , measure restoration of collagen secretion after reconstitution with:

  • Full-length TMEM131L

  • TMEM131L lacking the PapD-L domain

  • TMEM131L with mutations in the C-terminal region that interacts with TRAPPC8

These approaches can help delineate TMEM131L's precise role in the evolutionarily conserved collagen trafficking pathway from ER to Golgi and beyond.

What methodologies can be employed to investigate TMEM131L's prognostic value in glioma using HRP-conjugated antibodies?

Recent research has identified TMEM131L as a potential prognostic marker in glioma . Researchers can leverage HRP-conjugated TMEM131L antibodies to further investigate this relationship through:

• Tissue microarray (TMA) analysis: Apply immunohistochemistry (IHC) with HRP-conjugated TMEM131L antibody to:

  • Quantify TMEM131L expression across large cohorts of glioma patient samples

  • Correlate expression patterns with clinicopathological features like:

    • Histological type (GBM vs. LGG)

    • IDH mutation status

    • 1p/19q codeletion status

    • Patient age and primary therapy outcomes

  • Develop standardized scoring systems for TMEM131L expression

• Multiplex IHC approaches: Combine TMEM131L detection with markers for:

  • Oxidative stress (given TMEM131L's association with oxidative stress phenotype)

  • Immune infiltration markers to validate correlation with immune and stromal scores

  • CD151, PHF20, ROS1, and SOX2 (positively correlated with TMEM131L)

  • GFAP and MGMT (negatively correlated with TMEM131L)

• Survival analysis methodologies:

  • Kaplan-Meier survival analysis comparing patients with high vs. low TMEM131L expression

  • Cox regression models incorporating TMEM131L expression with other clinical variables

  • Development of nomogram prediction models including TMEM131L status

• Functional validation in patient-derived models:

  • Correlate TMEM131L protein levels with invasiveness in patient-derived glioma cell lines

  • Assess impact of TMEM131L knockdown on glioma cell proliferation, migration, and therapy resistance

  • Investigate potential mechanisms connecting TMEM131L to poor prognosis

The diagnostic efficacy of TMEM131L for glioma appears promising with reported area under curve (AUC) values of 0.858 and confidence interval (CI) of 0.841–0.874 for GBM and LGG , warranting further exploration of its clinical utility.

How can researchers investigate the relationship between TMEM131L and the Wnt signaling pathway using HRP-conjugated antibodies?

TMEM131L has been implicated in antagonizing canonical Wnt signaling by triggering lysosome-dependent degradation of Wnt-activated LRP6 . To explore this relationship, researchers can employ the following methodological approaches with HRP-conjugated TMEM131L antibody:

• Biochemical interaction studies:

  • Co-immunoprecipitation followed by western blotting to detect associations between TMEM131L and:

    • LRP6 (particularly phosphorylated forms)

    • Other Wnt pathway components (β-catenin, GSK3β, Axin)

    • Lysosomal proteins involved in LRP6 degradation

  • ELISA-based quantification of TMEM131L-LRP6 complexes under Wnt stimulation and inhibition conditions

• Pathway activity assays:

  • TOP/FOP luciferase reporter assays to measure Wnt pathway activity while modulating TMEM131L levels

  • Quantification of β-catenin nuclear translocation in relation to TMEM131L expression

  • Analysis of Wnt target gene expression (e.g., AXIN2, cMYC) in response to TMEM131L manipulation

• Degradation kinetics assessment:

  • Pulse-chase experiments to track LRP6 half-life in the presence/absence of TMEM131L

  • Lysosomal inhibitor studies to confirm the degradation pathway

  • Western blot analysis of LRP6 phosphorylation status in relation to TMEM131L levels

• Structure-function analysis:

  • Mutational studies to identify TMEM131L domains critical for Wnt antagonism

  • Investigation of whether the PapD-L domains implicated in collagen trafficking also participate in Wnt regulation

  • Deletion constructs to separate TMEM131L's roles in collagen secretion from its Wnt regulatory functions

These approaches can help delineate the mechanistic details of how TMEM131L regulates Wnt signaling and potentially identify novel therapeutic targets for conditions with aberrant Wnt pathway activation.

What are common challenges when using TMEM131L antibody, HRP conjugated, and how can they be resolved?

Researchers working with HRP-conjugated TMEM131L antibodies may encounter several technical challenges. Here are methodological solutions for common issues:

• High background signal:

  • Cause: Insufficient blocking, excessive antibody concentration, or non-specific binding

  • Solution: Increase blocking time to 2 hours, optimize antibody dilution (try 1:50 initially then adjust), use casein-based blockers instead of BSA, or add 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

• Weak or no signal:

  • Cause: Low TMEM131L expression, epitope masking, or antibody degradation

  • Solution: Increase sample concentration, optimize antigen retrieval (for IHC), try alternative lysis buffers for membrane protein extraction, verify antibody activity with a positive control, or reduce washing stringency

• Non-specific bands in Western blotting:

  • Cause: Cross-reactivity with related proteins or degradation products

  • Solution: Increase blocking stringency, add 0.1% SDS to antibody dilution buffer, perform peptide competition assays, or optimize transfer conditions for large proteins (>100 kDa)

• Signal variability between replicates:

  • Cause: Inconsistent technique or sample preparation

  • Solution: Standardize all procedures, prepare fresh working solutions for each experiment, control incubation temperatures precisely, and use automated washing systems if available

• Rapid signal fading in HRP detection:

  • Cause: Photobleaching or substrate depletion

  • Solution: Minimize light exposure, use signal enhancers, optimize substrate volume, or switch to more stable HRP substrates

• Limited reactivity with certain sample types:

  • Cause: Species differences or isoform specificity

  • Solution: Verify TMEM131L sequence homology across species of interest, test antibodies targeting different epitopes, or validate with genetic knockdown controls

The HRP-conjugated TMEM131L antibody is optimized for ELISA applications , so additional optimization may be required when adapting it for other techniques like western blotting or immunoprecipitation.

How can researchers assess and compare the performance of different TMEM131L antibodies for specific applications?

Systematic evaluation of different TMEM131L antibodies is essential for selecting the optimal reagent for specific research applications. Implement this methodological framework for antibody assessment:

• Multi-parameter comparison matrix:
  Create a standardized evaluation table comparing key parameters:

  Parameter	Antibody A	Antibody B	Antibody C
  Epitope region	N-terminal	PapD-L domain	C-terminal
  Host species	Rabbit	Mouse	Rabbit
  Clonality	Polyclonal	Monoclonal	Polyclonal
  Conjugate	HRP	Unconjugated	FITC
  Validated applications	ELISA	WB, IF	FACS, IP
  Species reactivity	Human	Human, Mouse	Human
  Sensitivity (LOD)	0.5 ng/mL	2 ng/mL	1 ng/mL
  Specificity (cross-reactivity)	Low	High	Medium
  Lot-to-lot consistency	Variable	Consistent	Variable
  Cost-effectiveness	High	Medium	Low

• Side-by-side application testing:

  • Western blotting: Compare antibodies using identical samples, concentrations, and detection methods; quantify band intensity and background

  • ELISA: Generate standard curves with recombinant TMEM131L protein; calculate EC50 values and dynamic ranges

  • Immunofluorescence: Assess staining patterns, signal-to-noise ratios, and co-localization with established markers

• Validation in genetic models:

  • Test antibody specificity against TMEM131L knockdown samples using validated shRNAs targeting different regions

  • Compare signal reduction across antibodies targeting different epitopes

  • Evaluate detection of overexpressed tagged TMEM131L constructs

• Cross-validation with orthogonal techniques:

  • Correlate antibody signals with TMEM131L mRNA levels via qPCR

  • Compare protein detection results with mass spectrometry data

  • Validate localization patterns with fluorescently tagged TMEM131L

• Application-specific optimization matrix:
  Document optimal conditions for each antibody across applications:

  Condition	ELISA	Western Blot	IHC
  Optimal dilution	1:20-1:50	1:100-1:500	1:50-1:200
  Blocking agent	3% BSA	5% milk	10% goat serum
  Incubation time	2h RT	Overnight 4°C	3h RT
  Detection system	TMB substrate	ECL	DAB

• Reproducibility assessment:

  • Test inter-lot variability by comparing multiple lots of the same antibody

  • Evaluate reproducibility across different users and laboratories when possible

  • Assess stability over time with repeated freeze-thaw cycles

This systematic approach enables objective comparison of different TMEM131L antibodies and selection of the optimal reagent for specific research applications.

How might TMEM131L antibodies contribute to understanding the protein's role in immune cell regulation?

TMEM131L has been implicated in regulating thymocyte proliferation and shows correlation with immune infiltration in cancer contexts . Researchers can use HRP-conjugated TMEM131L antibodies to explore its immunological functions through these methodological approaches:

• Immune cell profiling:

  • Develop flow cytometry panels (using the same antibody clone in non-HRP format) to assess TMEM131L expression across immune cell populations

  • Correlate TMEM131L levels with activation states and functional markers

  • Compare expression in healthy versus disease-associated immune cells

• Mechanistic studies in thymocyte development:

  • Use ELISA with HRP-conjugated antibodies to quantify TMEM131L during stages of thymocyte maturation

  • Assess how TMEM131L expression changes during positive and negative selection

  • Correlate TMEM131L levels with key developmental markers and transcription factors

• Tumor immune microenvironment analysis:

  • Apply multiplex IHC to analyze TMEM131L expression in relation to:

    • Infiltrating immune cell subtypes (using the CIBERSORT algorithm for quantification)

    • Immune checkpoint molecules that correlate with TMEM131L expression

    • Matrix, immunity, and ESTIMATE scores in various tumor types

• Functional immune assays:

  • Investigate how TMEM131L modulation affects:

    • T cell proliferation and cytokine production

    • Macrophage polarization and function

    • Interaction between immune and tumor cells

• Targeted pathway investigations:

  • Explore TMEM131L's relationship with specific immune signaling pathways:

    • Connection to Wnt signaling in immune contexts

    • Potential roles in inflammasome activation

    • Involvement in cytokine receptor trafficking or stability

• Therapeutic implications:

  • Develop strategies to target TMEM131L for immunomodulation

  • Assess whether TMEM131L status predicts response to immunotherapy

  • Explore TMEM131L as a biomarker for immune-related adverse events

These research directions could establish TMEM131L as an important regulator of immune function with implications for autoimmunity, cancer immunotherapy, and inflammatory disorders.

What methodological approaches would best investigate the relationship between TMEM131L and oxidative stress in cancer contexts?

TMEM131L has been linked to oxidative stress phenotypes in glioma , suggesting an important functional relationship that warrants further investigation. Researchers can employ these methodological approaches using HRP-conjugated TMEM131L antibodies:

• Correlation analysis with oxidative stress biomarkers:

  • Use ELISA to quantify TMEM131L in relation to:

    • Lipid peroxidation products (MDA, 4-HNE)

    • Protein carbonylation levels

    • GSH/GSSG ratio

    • SOD and catalase activities

  • Create multiparameter correlation matrices across patient samples

• Functional modulation studies:

  • Manipulate TMEM131L expression and assess impact on:

    • Cellular ROS levels using fluorescent probes

    • Mitochondrial membrane potential

    • Antioxidant response element (ARE) activation

    • NRF2 pathway regulation

• Targeted transcriptomic analysis:

  • Following TMEM131L knockdown or overexpression, analyze expression changes in:

    • Oxidative stress response genes (SOD1/2, CAT, GPX)

    • ROS-generating enzymes (NOX family)

    • Mitochondrial genes related to redox balance

    • Antioxidant pathway components

• Co-expression network analysis:

  • Expand on the established correlations with oxidative stress markers:

    • CD151, PHF20, ROS1, and SOX2 (positively correlated with TMEM131L)

    • Create protein-protein interaction networks centered on TMEM131L

    • Identify hub genes connecting TMEM131L to oxidative stress processes

• Structure-function studies:

  • Investigate whether specific domains of TMEM131L are required for its effects on oxidative stress

  • Determine if the PapD-L domains participate in ROS regulation

  • Assess post-translational modifications of TMEM131L under oxidative conditions

• Therapeutic targeting approaches:

  • Screen for compounds that modulate TMEM131L-dependent oxidative stress phenotypes

  • Test antioxidant interventions in models with altered TMEM131L expression

  • Investigate potential synthetic lethality between TMEM131L status and oxidative stress inducers

The established risk prediction model for oxidative stress-related TMEM131L co-expression genes provides a valuable foundation for these investigations, potentially revealing new therapeutic vulnerabilities in cancers like glioma.

How can researchers integrate TMEM131L antibody-based detection with multi-omics approaches to understand its function?

Modern multi-omics approaches can significantly enhance our understanding of TMEM131L function when integrated with antibody-based detection methods. Researchers can implement the following methodological framework:

• Integrative proteomics approaches:

  • Use HRP-conjugated TMEM131L antibodies for immunoprecipitation followed by mass spectrometry to:

    • Identify novel interaction partners

    • Map the TMEM131L protein interactome under different cellular conditions

    • Detect post-translational modifications that regulate function

  • Compare results with publicly available protein-protein interaction databases

• Proteogenomic correlation:

  • Correlate TMEM131L protein levels (detected by antibody-based assays) with:

    • mRNA expression from RNA-seq data

    • Copy number variations affecting the TMEM131L locus

    • Methylation status of TMEM131L promoter regions

  • Investigate mechanisms of post-transcriptional regulation

• Spatial multi-omics integration:

  • Combine immunohistochemistry using TMEM131L antibodies with:

    • Spatial transcriptomics to map expression patterns in tissue contexts

    • Multiplexed ion beam imaging (MIBI) for simultaneous detection of multiple proteins

    • Digital spatial profiling for regional quantification of TMEM131L and related pathway components

• Single-cell multi-modal analysis:

  • Integrate single-cell protein detection (using flow cytometry) with:

    • scRNA-seq to correlate TMEM131L protein and mRNA at single-cell resolution

    • CITE-seq for simultaneous detection of cell surface markers

    • Metabolic profiling to connect TMEM131L to cellular metabolic states

• Functional genomics integration:

  • Combine CRISPR screens targeting TMEM131L with antibody-based detection to:

    • Identify genetic dependencies related to TMEM131L function

    • Map synthetic lethal interactions

    • Discover new pathway connections

• Clinical multi-omics applications:

  • Develop prognostic and predictive signatures incorporating:

    • TMEM131L protein levels measured by standardized assays

    • Genomic alterations affecting TMEM131L or related pathways

    • Transcriptomic profiles of TMEM131L-associated genes

    • Methylation signatures correlating with TMEM131L expression