TMEM2 Antibody, Biotin conjugated

What is TMEM2 and what cellular functions does it perform?

TMEM2 (Transmembrane Protein 2) is a type II transmembrane protein that functions as a cell surface hyaluronidase. It mediates the initial cleavage of extracellular high-molecular-weight hyaluronan (HMW-HA) into intermediate-size fragments of approximately 5 kDa before internalization and degradation in lysosomes. TMEM2 plays critical roles in regulating angiogenesis and heart morphogenesis through mediating the degradation of extracellular hyaluronan, thereby influencing VEGF signaling. It shows remarkable substrate specificity for hyaluronan and cannot cleave other glycosaminoglycans such as chondroitin sulfate or dermatan sulfate . Recent research has also identified TMEM2 as a member of the interferon-induced transmembrane protein superfamily with antiviral activity through activation of the JAK/STAT signaling pathway .

What are the structural features of TMEM2 protein?

The human TMEM2 protein consists of:

• An 82-residue cytoplasmic N-terminal domain

• A single transmembrane domain

• A 1278-residue extracellular C-terminal domain

The extracellular domain contains :

• One G8 domain

• One GG domain

• Three PbH1 repeats (structural features present in polysaccharide lyase family)

The protein's active site contains conserved residues including Arg265, Asp273, and Asp286 that are critical for its hyaluronidase activity .

What are the specifications of the TMEM2 Antibody, Biotin conjugated?

The TMEM2 Antibody, Biotin conjugated (e.g., ABIN7147331) has the following specifications :

How is the subcellular localization of TMEM2 determined using immunostaining techniques?

To determine TMEM2's subcellular localization, researchers typically employ:

• Surface biotinylation assays: These confirm cell-surface localization of TMEM2. When performed on TMEM2-transfected cells (like MG-63 and 293T cells), TMEM2 is detected in the surface fraction but not in culture supernatants, confirming its membrane-anchored nature .

• Live cell immunostaining: Using anti-FLAG antibodies against epitope-tagged TMEM2 constructs has demonstrated that TMEM2 is indeed expressed on the cell surface with its C-terminus oriented outside the cell .

• Immunofluorescence microscopy: Studies using fluorescently tagged TMEM2 (e.g., mCherry-tagged mouse TMEM2) reveal that the protein localizes to the plasma membrane and shows overlapping colocalization with focal adhesion proteins like vinculin . Additional staining patterns indicate presence in nucleoli and cytosol in certain cell lines .

What controls should be included when using TMEM2 Antibody, Biotin conjugated for immunohistochemistry?

When conducting immunohistochemistry experiments with TMEM2 Antibody, Biotin conjugated, include these critical controls:

• Negative controls:

  • Isotype control: Use biotin-conjugated rabbit IgG at the same concentration

  • No primary antibody control: Perform the staining procedure omitting the primary antibody

  • Peptide competition/blocking: Pre-incubate the antibody with the immunizing peptide (AA 734-866) to confirm specificity

• Positive controls:

  • Tissues known to express TMEM2 (based on search results, lymph nodes show strong TMEM2 expression, particularly in subcapsular sinus regions)

  • Cell lines overexpressing TMEM2 compared to knockdown cells

• Validation controls:

  • Compare staining with another TMEM2 antibody targeting a different epitope

  • Parallel RNA detection (e.g., RNAscope ISH) to correlate protein staining with mRNA expression patterns

• Endogenous biotin blocking: Since the antibody is biotin-conjugated, endogenous biotin blocking steps are essential to reduce background, particularly in tissues like liver, kidney, and brain that contain high levels of endogenous biotin.

How can I optimize fixation conditions when using TMEM2 Antibody, Biotin conjugated for immunofluorescence?

For optimal immunofluorescence results with the TMEM2 Antibody, Biotin conjugated:

• Fixation protocol optimization:

  • For immunocytochemistry/immunofluorescence applications, a combination of paraformaldehyde (PFA) and Triton X-100 is recommended

  • Standard protocol: Fix cells with 4% PFA for 10-15 minutes at room temperature, followed by permeabilization with 0.1-0.5% Triton X-100 for 5-10 minutes

  • For membrane proteins like TMEM2, consider gentler permeabilization with 0.1% saponin or digitonin if Triton X-100 produces high background

  • Avoid methanol fixation which may disrupt epitope recognition in the AA 734-866 region

• Antigen retrieval considerations:

  • For tissue sections, test heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Monitor retrieval times carefully, as over-retrieval may damage tissue architecture

• Concentration optimization:

  • Start with the recommended concentration range (0.25-2 μg/ml)

  • Perform a titration experiment with serial dilutions to determine optimal signal-to-noise ratio

  • For double immunolabeling, adjust concentrations to achieve balanced signals from both targets

What are the best approaches for quantifying TMEM2 expression levels in tissue samples?

Multiple complementary approaches can be used to quantify TMEM2 expression:

• Western blot analysis:

  • Allows semi-quantitative comparison of protein levels between samples

  • Include housekeeping proteins (β-actin, GAPDH) as loading controls

  • Use densitometry software to normalize TMEM2 signal intensity to loading controls

  • In mouse models, this approach revealed decreased TMEM2 protein expression in Graves' orbitopathy tissues compared to non-GO tissues

• qRT-PCR for mRNA quantification:

  • Enables precise quantification of TMEM2 transcript levels

  • Design primers spanning exon-exon junctions to avoid genomic DNA amplification

  • Normalize to stable reference genes (validated for your tissue type)

  • Absolute quantification methods have shown that Tmem2 mRNA is expressed ubiquitously in adult mouse tissues at higher levels than other hyaluronidases

• Immunohistochemistry with scoring systems:

  • Develop a scoring system based on staining intensity (0-3) and percentage of positive cells

  • Use digital image analysis software for unbiased quantification

  • Compare staining patterns between experimental groups (e.g., GO vs. non-GO tissues)

• Flow cytometry:

  • For cell suspensions derived from tissues or cell cultures

  • Use biotin-conjugated TMEM2 antibody with streptavidin-fluorophore secondary detection

  • Quantify mean fluorescence intensity across different experimental conditions

How can I use TMEM2 Antibody, Biotin conjugated to study the functional interaction between TMEM2 and hyaluronan in focal adhesions?

To investigate TMEM2's role in focal adhesions:

• Dual immunofluorescence microscopy:

  • Co-stain cells with biotin-conjugated TMEM2 antibody and antibodies against focal adhesion proteins (vinculin, paxillin, FAK)

  • Use streptavidin-conjugated fluorophores with minimal spectral overlap with other fluorescent channels

  • Analyze colocalization using confocal microscopy and quantitative colocalization analysis (Pearson's coefficient, Manders' overlap coefficient)

  • Research has shown that mCherry-tagged TMEM2 exhibits overlapping colocalization with vinculin-positive puncta and sites of HA removal

• In situ hyaluronan degradation assay:

  • Plate cells on fluorescently labeled hyaluronan substrates

  • Visualize focal adhesions using anti-vinculin antibodies

  • Monitor local HA degradation using the biotin-conjugated TMEM2 antibody

  • Analyze the spatial relationship between TMEM2 localization and HA degradation patterns

  • Previous studies have demonstrated TMEM2 hyaluronidase activity in association with focal adhesions at sites of in situ HA degradation

• Proximity ligation assay (PLA):

  • Use biotin-conjugated TMEM2 antibody with streptavidin-conjugated oligonucleotides

  • Pair with antibodies against focal adhesion components or hyaluronan binding proteins

  • Quantify interaction signals at different cellular locations and under different conditions

What strategies can be employed to analyze the role of TMEM2 in inflammatory conditions using the biotin-conjugated antibody?

To investigate TMEM2's role in inflammation:

• Immunohistochemical analysis of inflamed tissues:

  • Compare TMEM2 expression between normal and inflamed tissues (e.g., Graves' orbitopathy model)

  • Correlate TMEM2 staining with inflammatory markers (CD3 for T lymphocytes, etc.)

  • Research has shown that TMEM2 protein and mRNA expression levels are significantly decreased in GO tissue samples compared to non-GO tissue samples

• Flow cytometric analysis of immune cells:

  • Isolate immune cell populations from inflamed tissues

  • Analyze TMEM2 expression on different immune cell subsets using the biotin-conjugated antibody

  • Compare expression levels between homeostatic and inflammatory states

• Functional recovery experiments:

  • Overexpress TMEM2 in inflammatory models (e.g., via adeno-associated virus-mediated delivery)

  • Assess the effects on inflammatory markers, adipogenesis, and fibrosis

  • In GO mouse models, TMEM2 overexpression reduced inflammation, adipogenesis, and fibrosis

• Signaling pathway analysis:

  • Use the antibody to immunoprecipitate TMEM2 from cells treated with inflammatory stimuli

  • Analyze associated proteins to identify signaling complexes

  • Focus on JAK/STAT pathway components, as TMEM2 has been shown to activate this pathway with downstream effects on inflammation

How can TMEM2 Antibody, Biotin conjugated be utilized in studying hyaluronan metabolism in development and disease?

To investigate hyaluronan metabolism:

• Developmental studies:

  • Use the antibody to track TMEM2 expression patterns during embryonic development

  • Correlate with hyaluronan deposition patterns

  • TMEM2 acts as a regulator of heart morphogenesis by mediating degradation of extracellular hyaluronan

• Disease model analysis:

  • Compare TMEM2 expression in normal versus diseased tissues

  • In knockout mouse models, TMEM2 ablation leads to pronounced accumulation of HA in circulating blood and various organs, reaching levels as high as 40-fold above control mice

  • Analyze the molecular weight distribution of hyaluronan using agarose gel electrophoresis with Stains-All dye to detect differences in HA processing

• Tissue HA turnover assessment:

  • Inject fluorescent HA tracers into lymphatic and vascular systems

  • Use the biotin-conjugated TMEM2 antibody to monitor TMEM2 expression in tissues responsible for HA clearance

  • Studies have shown that HA degradation in the lymphatic system and liver is significantly impaired in TMEM2 knockout mice

• Cell-specific expression analysis:

  • Perform cell sorting (e.g., magnetic cell sorting of CD31-positive endothelial cells)

  • Analyze TMEM2 expression in specific cell populations

  • RNAscope ISH technique has revealed strong TMEM2 expression in lymph node stromal components, particularly in cells of the subcapsular sinus

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

Common challenges include:

• High background due to endogenous biotin:

  • Problem: Tissues like liver, kidney, and brain contain high levels of endogenous biotin

  • Solution: Use commercial biotin blocking kits before applying the primary antibody

  • Alternative approach: Consider avidin-biotin blocking steps (incubate with avidin, wash, then incubate with biotin)

• Cross-reactivity with biotin-containing proteins:

  • Problem: Non-specific binding to naturally biotinylated proteins

  • Solution: Increase blocking time and concentration (5% BSA or 10% normal serum)

  • Validation: Include appropriate negative controls (isotype control, blocking peptide)

• Signal amplification issues:

  • Problem: Over-amplification leading to high background or insufficient signal

  • Solution: Titrate streptavidin-reporter conjugate concentrations

  • Alternative: Use tyramide signal amplification for enhanced sensitivity with controlled amplification

• Tissue autofluorescence interfering with detection:

  • Problem: Autofluorescence masking specific signals

  • Solution: Use autofluorescence quenching reagents or spectral unmixing during image acquisition

  • Alternative: Choose fluorophores with emission spectra distinct from tissue autofluorescence

How can I validate the specificity of TMEM2 Antibody, Biotin conjugated results to ensure accurate interpretation?

To validate antibody specificity:

• Genetic approaches:

  • Compare staining between wild-type samples and TMEM2 knockout or knockdown samples

  • In induced global Tmem2 knockout mice, TMEM2 staining should be significantly reduced

  • Use siRNA-mediated knockdown of endogenous TMEM2 in cell culture models

• Peptide competition assays:

  • Pre-incubate the antibody with excess immunizing peptide (the AA 734-866 region of TMEM2)

  • If staining is specific, the pre-absorbed antibody should show significantly reduced or absent staining

• Correlation with transcript levels:

  • Perform parallel analysis of TMEM2 protein (using the antibody) and mRNA (using qRT-PCR or RNAscope)

  • Confirm concordance between protein and mRNA expression patterns

  • Studies have shown correlation between decreased TMEM2 protein and mRNA levels in GO tissues

• Multiple antibody validation:

  • Compare staining patterns with other TMEM2 antibodies targeting different epitopes

  • Consistent patterns across different antibodies support specificity

• Functional correlation:

  • Correlate TMEM2 staining with functional assays (e.g., hyaluronidase activity, HA degradation patterns)

  • In TMEM2-expressing cells, hyaluronan degradation should be observed in a contact-dependent manner

What considerations should be made when interpreting TMEM2 expression in disease models using immunohistochemistry?

Important considerations include:

• Quantitative interpretation challenges:

  • Even modest changes in TMEM2 expression (e.g., 20% downregulation) may have functional significance

  • Studies in GO mouse models showed that relatively small changes in TMEM2 expression correlated with significant phenotypic effects

  • Consider that biological systems often exhibit non-linear relationships between protein expression and functional outcomes

• Cell type-specific expression patterns:

  • TMEM2 shows differential expression across cell types

  • In lymph nodes, TMEM2 expression is primarily in stromal components, particularly subcapsular sinus cells, with negligible expression in lymphoid follicles

  • Use dual immunolabeling with cell type-specific markers for accurate interpretation

• Species differences:

  • While the antibody is specific for human TMEM2, consider species differences when translating findings between human and animal models

  • Use species-specific antibodies for comparative studies

  • Domain structures and functional activities may vary across species

• Context-dependent regulation:

  • TMEM2 expression may be dynamically regulated under different physiological and pathological conditions

  • Consider the influence of local microenvironment (inflammation, hypoxia, etc.)

  • Temporal aspects of expression changes may be critical for interpretation

• Relationship to hyaluronan metabolism:

  • Correlate TMEM2 expression with hyaluronan content and distribution

  • In TMEM2 knockout mice, undigested high molecular weight HA accumulates, suggesting impaired hyaluronan processing

  • Use agarose gel electrophoresis with Stains-All dye to analyze HA molecular weight distribution alongside TMEM2 expression analyses

How might TMEM2 Antibody, Biotin conjugated be used to explore the potential role of TMEM2 in cancer progression?

Emerging research directions include:

• Expression profiling across cancer types:

  • TMEM2 has been associated with bladder, breast, and pancreatic cancer

  • Use the biotin-conjugated antibody for tissue microarray analysis across tumor types and stages

  • Correlate expression patterns with clinical outcomes and molecular subtypes

• Investigation of TMEM2 in tumor microenvironment remodeling:

  • Analyze TMEM2 expression in tumor-associated stromal cells

  • Study the relationship between TMEM2 activity and hyaluronan accumulation in tumors

  • High molecular weight hyaluronan is associated with cancer progression and therapeutic resistance

• Functional studies in metastasis models:

  • Examine TMEM2 expression at sites of tumor cell invasion and metastasis

  • Investigate association with focal adhesion dynamics and cell migration

  • TMEM2 hyaluronidase activity in focal adhesions suggests potential roles in cell migration and invasion

• Therapeutic targeting assessment:

  • Use the antibody to monitor TMEM2 expression changes following cancer treatments

  • Investigate TMEM2 as a biomarker for response to therapies targeting the tumor microenvironment

  • Explore correlations between TMEM2 expression and efficacy of hyaluronan-targeting therapies

What experimental approaches could be developed to study the interplay between TMEM2 and JAK/STAT signaling using the biotin-conjugated antibody?

Advanced experimental approaches include:

• Proximity-based protein interaction studies:

  • Use biotin-conjugated TMEM2 antibody in proximity ligation assays (PLA) with antibodies against JAK/STAT components

  • Investigate direct or indirect interactions between TMEM2 and JAK/STAT pathway proteins

  • TMEM2 has been shown to activate the JAK/STAT signaling pathway

• Phosphorylation state analysis:

  • Perform dual immunofluorescence with biotin-conjugated TMEM2 antibody and phospho-specific antibodies for JAK/STAT pathway components

  • Analyze correlation between TMEM2 expression and phosphorylation status of STAT proteins

  • Use flow cytometry for quantitative single-cell analysis of these relationships

• Transcriptional regulation studies:

  • Combine ChIP-seq analysis of STAT binding with TMEM2 expression profiling

  • Investigate whether TMEM2 is a target gene of STAT transcription factors

  • Use reporter assays to determine if TMEM2 promoter activity is regulated by JAK/STAT signaling

• Therapeutic intervention models:

  • Use the antibody to monitor TMEM2 expression following JAK inhibitor treatment

  • Investigate whether JAK/STAT modulation affects TMEM2-mediated hyaluronan degradation

  • Combine with functional assays for inflammation, adipogenesis, and fibrosis endpoints

What novel techniques could be developed to simultaneously visualize TMEM2 localization and hyaluronidase activity in living systems?

Innovative approaches include:

• Dual-function reporter systems:

  • Combine biotin-tagged TMEM2 antibody fragments (e.g., scFv) with hyaluronan-binding peptides

  • Develop FRET-based biosensors that report on both TMEM2 localization and nearby HA degradation

  • Use split fluorescent protein complementation assays where one fragment is linked to TMEM2 and another to HA-binding domains

• Live cell enzymatic activity visualization:

  • Develop fluorogenic or FRET-based hyaluronan substrates that change spectral properties upon cleavage

  • Use in combination with labeled TMEM2 antibodies or fluorescently tagged TMEM2 constructs

  • Building on established assays showing TMEM2-expressing cells can degrade substrate-bound HA in a contact-dependent manner

• Advanced intravital microscopy approaches:

  • Adapt biotinylated TMEM2 antibodies for in vivo imaging using streptavidin-conjugated near-infrared fluorophores

  • Combine with injectable fluorescent HA tracers to monitor degradation in real-time

  • Previous studies have used lymphatic and vascular injection of fluorescent HA tracers to demonstrate impaired HA degradation in TMEM2-deficient mice

• Engineered animal models:

  • Develop knock-in mice expressing tagged TMEM2 that preserves enzymatic function

  • Create conditional expression systems to study tissue-specific TMEM2 functions

  • Combine with in vivo HA visualization techniques to correlate TMEM2 expression with local HA processing