TMEM87B Antibody

What is TMEM87B and what cellular functions is it associated with?

TMEM87B (transmembrane protein 87B) is a member of the GOLD-domain seven-transmembrane helix protein family. Based on structural and functional analyses of related proteins, TMEM87B has been associated with several critical cellular processes including protein transport to and from the Golgi apparatus, mechanosensitive cation channel activity, and cardiac functions . The protein is encoded by the TMEM87B gene (gene ID: 84910) in humans and represents an important target for studying membrane protein biology and cellular transport mechanisms.

The protein likely shares structural similarities with TMEM87A, which features an extracellular beta-sandwich domain positioned above a G-protein coupled receptor (GPCR)-like seven-transmembrane (7TM) domain . This structural arrangement suggests potential roles in signaling pathways and cellular communication that warrant further investigation in various physiological and pathological contexts.

What applications is the TMEM87B antibody suitable for?

TMEM87B antibodies, such as the commercially available HPA035183 antibody, are validated for several key research applications:

• Immunohistochemistry (IHC): Recommended dilution range of 1:50-1:200

• Western blotting (immunoblotting): Effective concentration range of 0.04-0.4 μg/mL

• Immunofluorescence (IF): Optimal concentration range of 0.25-2 μg/mL

These applications enable researchers to investigate TMEM87B expression patterns in tissues, protein levels in cell lysates, and subcellular localization within cellular compartments. The antibody has been tested through initiatives like the Human Protein Atlas project, which systematically characterizes antibodies against human proteins in tissue samples and cellular contexts .

What is the immunogen sequence used for TMEM87B antibody production?

The immunogen sequence used for generating the TMEM87B antibody (HPA035183) is:

NLDCNSDSQVFPSLNNKELINIRNVSNQERSMDVVARTQKDGFHIFIVSIKTENTDASWNLNVSLSMIGPHGYISAS

This sequence represents a specific region of the human TMEM87B protein that was used to immunize host animals (typically rabbits) for antibody production. Understanding this immunogen sequence is crucial for researchers to:

• Evaluate potential cross-reactivity with other proteins containing similar sequences

• Design appropriate blocking peptides for specificity controls

• Interpret results when the antibody fails to detect certain protein variants or isoforms that might lack this sequence

How should TMEM87B antibody be stored and handled for optimal performance?

Proper storage and handling of TMEM87B antibody is essential for maintaining its functionality and specificity. The antibody is typically:

• Shipped on wet ice to prevent degradation during transport

• Recommended to be stored at -20°C for long-term preservation

• Supplied in a buffered aqueous glycerol solution that helps maintain stability

For optimal performance in experiments, researchers should:

• Minimize freeze-thaw cycles by aliquoting the antibody upon first thawing

• Avoid contamination by using sterile technique when handling

• Consider including protease inhibitors in working solutions if extended handling times are required

• Validate each new lot of antibody before use in critical experiments

How can researchers validate the specificity of TMEM87B antibody in their experimental systems?

Validating antibody specificity is critical for generating reliable research data. For TMEM87B antibody, a multi-faceted validation approach is recommended:

• Genetic validation: Utilize CRISPR/Cas9 knockout or siRNA knockdown of TMEM87B to demonstrate loss of signal in Western blot or immunostaining applications.

• Peptide competition assay: Pre-incubate the antibody with the immunogen peptide before application to samples. Specific binding should be blocked by the peptide.

• Multiple antibodies approach: Compare results using antibodies targeting different epitopes of TMEM87B.

• Cross-species validation: If the antibody is reported to recognize TMEM87B from multiple species, testing reactivity across these species can provide confidence in specificity.

• Recombinant expression: Overexpress tagged TMEM87B in a cellular system with low endogenous expression and confirm co-localization of antibody signal with the tag.

This rigorous validation strategy follows best practices similar to those used in high-quality antibody validation studies, which carefully assess expression levels, purity, thermal stability, and other biophysical properties .

What expression systems are most effective for studying TMEM87B protein?

Based on successful approaches with related membrane proteins like TMEM87A, researchers should consider these expression systems for TMEM87B studies:

• Insect cell expression (Sf9 cells): This system has been successfully used for the related protein TMEM87A and provides advantages for membrane protein expression including proper folding and post-translational modifications .

• Mammalian cell expression: For functional studies and when human-like glycosylation is important, mammalian expression systems are preferable. The expression protocol might include:

  • Cloning TMEM87B into a vector with appropriate tags (e.g., GFP, His-tag)

  • Transient transfection or stable cell line generation in HEK293 or CHO cells

  • Validation of expression through fluorescence (if using fluorescent tags) or Western blotting

• Cell-free expression systems: For rapid protein production and when post-translational modifications are less critical.

The choice of expression system should align with the research goals - structural studies may require higher protein yields obtainable from insect cells, while functional studies might benefit from mammalian expression systems that provide a more native-like environment.

What biophysical techniques are most informative for characterizing TMEM87B antibody quality?

Advanced biophysical characterization of antibodies provides crucial insights into their quality and performance potential. For TMEM87B antibody characterization, researchers should consider these techniques:

• Thermal stability analysis: Measure the melting temperature (Tm) of the antibody's Fab region to assess stability. High-quality antibodies typically show Tm values around 70-83°C, similar to what has been observed for well-characterized therapeutic antibodies .

• Size exclusion chromatography (SEC): Evaluate the monomer content of purified antibody preparations. High-quality antibodies should demonstrate >95% monomer content after purification .

• Hydrophobicity assessment: Techniques like HIC (hydrophobic interaction chromatography) can help assess the antibody's hydrophobic properties, which influence non-specific binding.

• Self-association measurements: Methods like CS-SINS (cross-interaction chromatography self-interaction nanoparticle spectroscopy) can quantify antibody self-association tendencies, with low scores (<0.2) indicating favorable properties .

The table below summarizes typical biophysical parameter ranges for high-quality antibodies, based on data from experimental antibody characterization studies:

Data derived from experimental antibody characterization studies .

How does post-translational modification affect TMEM87B detection by antibodies?

Post-translational modifications (PTMs) can significantly impact antibody detection of TMEM87B. Researchers should consider:

• Glycosylation effects: Like many membrane proteins, TMEM87B may undergo N-linked or O-linked glycosylation. These modifications can:

  • Mask epitopes, preventing antibody recognition

  • Cause molecular weight shifts in Western blot analyses

  • Create heterogeneous banding patterns

• Phosphorylation and other modifications: TMEM87B may undergo regulatory phosphorylation, ubiquitination, or other modifications that alter antibody recognition.

• Sample preparation considerations:

  • Treatment with glycosidases may be necessary to reduce heterogeneity

  • Phosphatase treatment might be required if phosphorylation affects epitope recognition

  • Different lysis buffers may preserve certain PTMs better than others

When selecting TMEM87B antibodies, consider whether they target unmodified or modified forms of the protein. The HPA035183 antibody, for example, is reported to target unmodified TMEM87B , which means that heavily modified forms of the protein might show reduced detection.

What are the optimal protocols for immunohistochemistry with TMEM87B antibody?

For successful immunohistochemistry using TMEM87B antibody, researchers should follow these methodology guidelines:

• Tissue preparation:

  • For formalin-fixed paraffin-embedded (FFPE) tissues, use standard deparaffinization and rehydration protocols

  • Consider heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • For frozen sections, fixation with 4% paraformaldehyde for 10-15 minutes is recommended

• Antibody application:

  • Optimal dilution range for HPA035183: 1:50-1:200

  • Incubation at 4°C overnight generally yields superior results compared to shorter incubations

  • Use a humidified chamber to prevent section drying

• Detection system:

  • For brightfield microscopy, an HRP-polymer detection system offers good sensitivity

  • For fluorescence, appropriate fluorophore-conjugated secondary antibodies matching the TMEM87B antibody host species (rabbit for HPA035183)

• Controls:

  • Positive control: Tissues known to express TMEM87B (consult the Human Protein Atlas)

  • Negative control: Either omission of primary antibody or use of isotype control

  • Blocking peptide control: Pre-incubation of antibody with immunogen peptide

This protocol aligns with the approaches used by the Human Protein Atlas project, which has systematically characterized antibodies against human proteins in hundreds of tissue samples .

What are the best approaches for optimizing Western blot detection of TMEM87B?

Western blot optimization for TMEM87B detection requires attention to several methodological details:

• Sample preparation:

  • For membrane proteins like TMEM87B, use lysis buffers containing 1-2% non-ionic detergents (NP-40, Triton X-100) or stronger detergents (SDS, sodium deoxycholate) for more stringent extraction

  • Include protease inhibitors to prevent degradation

  • Consider using membrane fraction enrichment protocols

• Gel electrophoresis:

  • Use gradient gels (4-15% or 4-20%) to better resolve membrane proteins

  • Load appropriate protein amounts (typically 10-30 μg of total protein)

  • Include positive control lysates from cells/tissues known to express TMEM87B

• Transfer conditions:

  • For large membrane proteins, semi-dry transfer may be less effective than wet transfer

  • Consider using lower methanol concentrations in transfer buffer (5-10% instead of 20%)

  • Longer transfer times (overnight at low voltage) may improve transfer efficiency

• Antibody incubation:

  • Recommended concentration for HPA035183: 0.04-0.4 μg/mL

  • Optimize blocking conditions (5% BSA often performs better than milk for phospho-specific antibodies)

  • Consider overnight primary antibody incubation at 4°C

• Detection:

  • Enhanced chemiluminescence (ECL) systems with varying sensitivity are available

  • Fluorescently-labeled secondary antibodies can provide more quantitative results

These optimization strategies have been successfully employed in antibody validation studies and can be adapted for TMEM87B detection .

How can researchers perform quantitative analysis of TMEM87B expression across multiple samples?

For rigorous quantitative analysis of TMEM87B expression, researchers should employ these methodological approaches:

• Western blot quantification:

  • Use housekeeping proteins (β-actin, GAPDH) or total protein staining (Ponceau S, REVERT) for normalization

  • Include a standard curve of recombinant TMEM87B or positive control lysate

  • Employ technical replicates (minimum triplicate) and biological replicates

  • Use image analysis software with background subtraction capabilities

• qRT-PCR for mRNA expression:

  • Design primers specific to TMEM87B with efficiency testing

  • Validate using melt curve analysis and sequencing of PCR products

  • Use multiple reference genes for normalization

  • Apply the 2^(-ΔΔCt) method for relative quantification

• Immunohistochemistry quantification:

  • Use digital image analysis software for objective quantification

  • Establish consistent thresholds for positive staining

  • Consider H-score, Allred score, or percentage positive cells as quantification methods

  • Blinded scoring by multiple observers improves reliability

• Flow cytometry:

  • For cell surface TMEM87B detection if applicable

  • Include fluorescence-minus-one (FMO) controls

  • Use median fluorescence intensity (MFI) for quantification

Each method has strengths and limitations; combining multiple approaches provides more comprehensive and reliable quantification of TMEM87B expression levels.

What are common challenges in TMEM87B antibody applications and how can they be addressed?

Researchers frequently encounter these challenges when working with TMEM87B antibody:

• High background in immunostaining:

  • Increase blocking time/concentration (try 5-10% normal serum from secondary antibody host species)

  • Optimize antibody concentration through titration experiments

  • Add 0.1-0.3% Triton X-100 to antibody diluent to reduce non-specific binding

  • Pre-absorb antibody with tissue powder from species being examined

• Multiple bands in Western blot:

  • Could represent isoforms, degradation products, or post-translational modifications

  • Compare with expected molecular weight (~67-73 kDa for human TMEM87B)

  • Test different sample preparation methods (various lysis buffers, denaturation conditions)

  • Validate using knockout/knockdown controls

• Weak or absent signal:

  • For membrane proteins like TMEM87B, extraction efficiency is critical; try various detergent combinations

  • Extend antibody incubation time or increase concentration

  • For IHC/IF, test different antigen retrieval methods (heat vs. enzymatic, pH variations)

  • Consider signal amplification systems (tyramine signal amplification, HRP-polymer systems)

• Variability between experiments:

  • Standardize all protocols with detailed SOPs

  • Prepare larger batches of working solutions

  • Include consistent positive controls in each experiment

  • Consider automated systems for critical steps (staining, washing)

These troubleshooting approaches are based on general antibody optimization strategies and should be adapted specifically for TMEM87B detection based on experimental outcomes.

How can structural information about TMEM87 family proteins inform experimental design?

Understanding the structural features of TMEM87 family proteins provides valuable insights for experimental design:

• Epitope accessibility considerations:

  • TMEM87 proteins have seven transmembrane domains, making certain epitopes inaccessible in native conformations

  • The extracellular beta-sandwich domain may be more accessible for antibody binding in non-denaturing conditions

  • Consider different fixation/permeabilization approaches based on epitope location

• Protein topology mapping:

  • Combine antibodies targeting different domains (extracellular, intracellular) to confirm protein orientation

  • Use protease protection assays in conjunction with domain-specific antibodies

• Structural homology considerations:

  • TMEM87A and TMEM87B share structural similarities, requiring careful specificity validation

  • The GPCR-like seven-transmembrane domain suggests potential involvement in signaling pathways that could be investigated

• Protein-protein interaction studies:

  • The extracellular domain structure suggests potential interaction sites

  • Consider proximity labeling approaches (BioID, APEX) to identify interaction partners

  • Design co-immunoprecipitation experiments accounting for membrane protein solubilization challenges

Leveraging structural insights from the TMEM87 family can significantly enhance experimental design and interpretation of results when working with TMEM87B antibodies.

What emerging technologies can enhance TMEM87B research?

Several cutting-edge technologies can advance TMEM87B research beyond traditional antibody applications:

• Advanced imaging technologies:

  • Super-resolution microscopy (STORM, PALM, SIM) for nanoscale localization

  • Lattice light-sheet microscopy for dynamic studies in living cells

  • Correlative light and electron microscopy (CLEM) to combine functional and ultrastructural information

  • Expansion microscopy for improved spatial resolution of conventional microscopes

• Proximity-based protein interaction methods:

  • BioID or TurboID for biotinylation of proteins in proximity to TMEM87B

  • APEX2-based proximity labeling for electron microscopy compatibility

  • Split-protein complementation assays for direct interaction studies

• CRISPR-based technologies:

  • CRISPRi/CRISPRa for modulating TMEM87B expression without genetic modification

  • CRISPR-Cas9 knock-in of fluorescent tags at endogenous loci

  • Base editing for introducing specific mutations to study structure-function relationships

• Single-cell technologies:

  • Single-cell RNA-seq to examine expression heterogeneity

  • Mass cytometry (CyTOF) for high-dimensional protein expression profiling

  • Spatial transcriptomics to connect expression patterns with tissue architecture

• Computational approaches:

  • Deep learning models for antibody design and optimization, similar to those used in antibody development studies

  • AlphaFold2 and related tools for structural prediction to guide experimental design

  • Systems biology approaches to place TMEM87B in broader cellular networks

These emerging technologies can complement traditional antibody-based approaches and provide deeper insights into TMEM87B biology and function.