TMEM87B Antibody, HRP conjugated

What is TMEM87B and why is it a significant research target?

TMEM87B (Transmembrane protein 87B) is a highly conserved transmembrane protein involved in diverse cellular functions including cell proliferation, migration, and signal transduction . It has emerged as an important research target due to its potential role in multiple physiological and pathological conditions. Recent studies have identified TMEM87B's involvement in cardiac development, with zebrafish models showing that its depletion affects cardiac development and leads to cardiac hypoplasia . Additionally, TMEM87B has been found to interact with human papillomavirus 18 E6 oncoprotein, suggesting a possible role in carcinogenesis . These findings highlight TMEM87B as a promising target for research in cancer biology, developmental biology, and neurobiology.

How does HRP conjugation enhance TMEM87B antibody functionality?

Horseradish Peroxidase (HRP) conjugation provides a sensitive enzymatic detection method for antibodies. When conjugated to TMEM87B antibodies, HRP enables:

• Enhanced signal detection in Western blotting through chemiluminescent reactions

• Improved sensitivity in immunohistochemistry applications

• Quantitative analysis in ELISA assays

• Compatibility with various substrates (DAB, TMB, ECL)

HRP-conjugated secondary antibodies provide strong signal with minimal background and virtually complete elimination of lot-to-lot variation associated with polyclonal secondary antibodies . This conjugation is particularly valuable for detecting low-abundance proteins like TMEM87B in complex biological samples.

What are the optimal storage conditions for TMEM87B antibodies?

For maximum stability and performance of TMEM87B antibodies:

Use a manual defrost freezer and avoid repeated freeze-thaw cycles to maintain antibody integrity . Most antibodies are stable in glycerol-containing buffers with preservatives such as 0.03% Proclin 300, similar to the storage buffer used for TMEM87B antibodies (50% Glycerol, 0.01M PBS, pH 7.4) .

What are the recommended applications and dilutions for TMEM87B antibodies?

Based on validated research protocols, the following applications and dilutions are recommended for TMEM87B antibodies:

These recommendations are based on polyclonal TMEM87B antibodies such as PACO60849, which have been validated for these applications . When using HRP-conjugated antibodies, either direct conjugates or via secondary antibodies, these dilutions may need optimization depending on your specific experimental system.

How should I optimize immunohistochemistry protocols for TMEM87B detection?

For optimal IHC detection of TMEM87B in tissue samples:

• Perform antigen retrieval: Use high-pressure treatment in citrate buffer (pH 6.0) to unmask epitopes

• Block effectively: Treat sections with 10% normal goat serum for 30 minutes at room temperature

• Primary antibody incubation: Apply TMEM87B antibody at 1:200-1:400 dilution

• Secondary antibody selection: For non-conjugated primaries, use appropriate HRP-conjugated secondary antibodies (e.g., mouse anti-goat IgG-HRP for goat primaries)

• Signal development: Use DAB substrate for visualization

• Counterstain: Apply hematoxylin for nuclear visualization

This methodology has been successfully applied for detecting TMEM87B in human prostate tissue samples . Ensure adequate controls are included in each experiment, including negative controls with isotype-matched antibodies.

What methods are most effective for validating TMEM87B antibody specificity?

To ensure TMEM87B antibody specificity:

• Western blot analysis: Verify a single band at approximately 90-95 kDa

• TMEM87B knockdown controls: Compare staining in wild-type vs. TMEM87B-depleted samples

• Recombinant protein testing: Use purified TMEM87B protein as a positive control

• Cross-reactivity assessment: Test against similar proteins (e.g., TMEM87A)

• Peptide competition assay: Pre-incubate antibody with immunizing peptide to block specific binding

• Multiple antibody validation: Compare results using antibodies targeting different TMEM87B epitopes

Proper validation is critical as TMEM87B shares sequence homology with TMEM87A, requiring careful specificity testing to avoid cross-reactivity .

How can I detect TMEM87B interactions with viral oncoproteins?

To investigate TMEM87B interactions with viral oncoproteins such as HPV18 E6:

• Yeast two-hybrid system: This has been successfully used to identify TMEM87B as an HPV18 E6 binding partner

• Co-immunoprecipitation: Use anti-TMEM87B antibodies to pull down potential interacting partners

• Proximity ligation assay: Visualize protein interactions in situ within cells

• FRET/BRET analysis: Measure real-time protein interactions in living cells

• Mass spectrometry: Identify complexes containing TMEM87B after immunoprecipitation

Research has demonstrated that TMEM87B interacts with HPV18 E6 oncoprotein, suggesting its potential involvement in cervical carcinogenesis . These methods can help elucidate the molecular mechanisms and functional consequences of such interactions.

What approaches can resolve contradictory TMEM87B expression data?

When encountering inconsistent TMEM87B expression results:

• Compare protein vs. mRNA expression: Validate protein detection with transcriptomic data

• Use multiple detection methods: Combine Western blot, IHC, and flow cytometry

• Validate with different antibodies: Test antibodies recognizing distinct TMEM87B epitopes

• Consider post-translational modifications: Phosphorylation states may affect antibody binding

• Examine subcellular localization: TMEM87B may have differential compartmental expression

• Account for isoform expression: Check for alternative splicing variants

• Implement CRISPR/Cas9 knockout controls: Generate definitive negative controls

Bioinformatics analysis has identified that TMEM87B contains many phosphorylation sites and functional motifs that may affect detection depending on cellular context and experimental conditions .

How can I investigate TMEM87B's role in cardiac development and pathology?

To study TMEM87B in cardiac development and disease:

• Knockout/knockdown models: Assess cardiac phenotypes in TMEM87B-depleted systems

• Mutation analysis: Evaluate the impact of variants like p.Asn456Asp on cardiac function

• Expression profiling: Compare TMEM87B levels across cardiac developmental stages

• Patient sample analysis: Examine TMEM87B expression in cardiomyopathy tissues

• Protein interaction studies: Identify cardiac-specific binding partners

• Zebrafish models: Utilize established models where TMEM87B depletion affects cardiac development

Research has shown that a potentially deleterious variant in TMEM87B (c.1366A>G, p.Asn456Asp) may be associated with restrictive cardiomyopathy, and TMEM87B depletion in zebrafish embryos affected cardiac development . These findings suggest TMEM87B plays a crucial role in cardiac physiology that warrants further investigation.

How do I troubleshoot weak or absent signal when using HRP-conjugated TMEM87B antibodies?

When experiencing detection issues:

• Antibody concentration: Test a range of dilutions (1:100 to 1:5000)

• Antigen retrieval optimization: Try different pH buffers and retrieval methods

• Protein loading: Increase sample concentration for low-abundance targets

• Blocking optimization: Test different blocking agents (BSA, milk, serum)

• Incubation conditions: Extend primary antibody incubation to overnight at 4°C

• Detection system: Ensure fresh HRP substrate; consider enhanced chemiluminescence

• Transfer efficiency: Verify protein transfer with reversible staining

• Secondary antibody compatibility: Ensure proper species reactivity

For Western blot applications, PVDF membranes often provide better results than nitrocellulose for transmembrane proteins like TMEM87B .

How can I distinguish between TMEM87B and the similar TMEM87A protein?

To differentiate these similar transmembrane proteins:

• Specific epitopes: Select antibodies targeting unique regions not conserved between the proteins

• Molecular weight differences: TMEM87A appears at approximately 98 kDa while TMEM87B is typically 90-95 kDa

• Expression patterns: Utilize cell lines with differential expression profiles

• siRNA validation: Perform selective knockdown of each protein

• Mass spectrometry: Identify unique peptides following immunoprecipitation

• Isoform-specific PCR: Confirm at the mRNA level

Human TMEM87A and TMEM87B share sequence homology but have distinct functional domains and expression patterns that can be exploited for differential detection .

What are the key considerations when selecting secondary antibodies for TMEM87B detection?

When selecting secondary antibodies for TMEM87B primary antibodies:

• Host species compatibility: Match to the species of your primary antibody (e.g., mouse anti-goat IgG-HRP for goat primary antibodies)

• Class and subclass specificity: Ensure recognition of the correct immunoglobulin class

• Cross-adsorption: Use highly cross-adsorbed secondaries to minimize background

• Conjugate selection: Choose HRP for Western blot and IHC; fluorophores for IF

• Sensitivity requirements: Consider signal amplification systems for low-abundance targets

High-quality secondary antibodies, such as mouse anti-goat IgG-HRP (sc-2354), provide strong signal with minimal background and virtually complete elimination of lot-to-lot variation associated with polyclonal secondary antibodies .