TMEM87B Antibody, Biotin conjugated

What is TMEM87B and what cellular functions does it perform?

TMEM87B (Transmembrane protein 87B) is a multi-pass membrane protein that plays significant roles in various cellular processes, including cell proliferation, cell migration, and signal transduction. Most notably, it appears to be involved in retrograde transport from endosomes to the trans-Golgi network (TGN), suggesting its importance in intracellular trafficking pathways . The protein belongs to the LU7TM family, TMEM87 subfamily, and has two isoforms produced by alternative splicing in humans. It is encoded by a gene located on chromosome 2q13 .

What are the optimal application protocols for TMEM87B antibody in immunohistochemistry?

For optimal immunohistochemistry (IHC) applications with TMEM87B antibody, follow this validated protocol:

• Dewax and hydrate paraffin-embedded tissue sections according to standard procedures

• Perform antigen retrieval using high-pressure citrate buffer (pH 6.0)

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

• Dilute the TMEM87B antibody to 1:200-1:500 (with 1:400 being optimal for many tissues) in 1% BSA solution

• Incubate sections with primary antibody overnight at 4°C

• Detect using a biotinylated secondary antibody and visualize with an HRP-conjugated SP system

This protocol has been validated on human prostate tissue using the Leica BondTM system . For proper signal development, ensure adequate incubation time and thorough washing between steps.

What are the recommended dilutions for different experimental applications?

The following dilutions have been experimentally validated for TMEM87B antibody applications:

These dilutions should be optimized for your specific experimental conditions, tissue type, and detection system . For biotin-conjugated antibodies specifically, lower dilutions may be required when used with streptavidin detection systems.

How does antibody selection impact TMEM87B localization studies in subcellular compartments?

When designing experiments to study TMEM87B subcellular localization, epitope selection is critical. TMEM87B antibodies targeting different regions have distinct advantages:

• C-terminal antibodies (AA 484-513): Provide robust detection in Western blotting applications and are effective for detecting native protein in cell lysates . These antibodies are particularly useful when studying protein trafficking and processing.

• Mid-region antibodies (AA 451-554): Offer superior performance in immunohistochemistry applications and can maintain reactivity even after formalin fixation and paraffin embedding processes .

For subcellular localization studies, biotin-conjugated antibodies provide advantages when used with avidin-based amplification systems, resulting in enhanced signal detection in confocal microscopy. When designing co-localization experiments, consider using TMEM87B antibodies in conjunction with trans-Golgi network markers and endosomal compartment markers to accurately assess its retrograde transport function .

What validation approaches confirm TMEM87B antibody specificity in experimental systems?

Comprehensive antibody validation requires multiple complementary approaches:

• Peptide competition assays: Pre-incubate antibody with the immunizing peptide (in this case, recombinant human TMEM87B protein fragments AA 451-554 or AA 484-513) before application to samples. Signal elimination confirms specificity .

• Knockout/knockdown controls: Compare staining patterns between TMEM87B-expressing samples and those with TMEM87B genetically depleted.

• Cross-reactivity assessment: Test antibody against recombinant TMEM87A (the closest paralog) to ensure no cross-reactivity occurs.

• Immunoprecipitation-mass spectrometry: Confirm that immunoprecipitated proteins match the expected molecular weight (~63.4 kDa) and peptide sequence of TMEM87B.

• Multiple antibody concordance: Compare staining patterns between different antibodies targeting distinct TMEM87B epitopes; concordant patterns support specificity .

For biotin-conjugated antibodies specifically, additional controls using streptavidin-only staining are necessary to exclude endogenous biotin interference.

How can researchers optimize double immunolabeling with TMEM87B biotin-conjugated antibodies?

When designing double immunolabeling experiments with TMEM87B biotin-conjugated antibodies, consider these critical optimization steps:

• Sequential detection protocol:

  • Apply primary antibody against the non-TMEM87B target

  • Detect with appropriate fluorophore-conjugated secondary antibody

  • Block remaining binding sites with excess unconjugated secondary antibody

  • Apply biotin-conjugated TMEM87B antibody

  • Detect with streptavidin-conjugated fluorophore with minimal spectral overlap

• Blocking optimization: Add avidin/biotin blocking steps (commercially available kits) before applying the biotin-conjugated TMEM87B antibody to reduce background, especially in biotin-rich tissues like liver or kidney.

• Signal amplification considerations: For low-abundance TMEM87B detection, use tyramide signal amplification systems compatible with biotinylated antibodies, but carefully titrate reaction times to prevent signal bleeding.

• Control experiments: Include single-label controls for each antibody to verify signal specificity and absence of bleed-through .

How can researchers address weak or absent signal when using TMEM87B biotin-conjugated antibodies?

When encountering weak or absent signals with TMEM87B biotin-conjugated antibodies, implement this systematic troubleshooting approach:

• Antigen retrieval optimization:

  • Test multiple antigen retrieval methods (citrate buffer pH 6.0, EDTA buffer pH 9.0, enzymatic retrieval)

  • Extend retrieval time or increase temperature if appropriate for sample type

  • For IHC applications specifically, high-pressure retrieval in citrate buffer (pH 6.0) has shown superior results

• Antibody concentration adjustment:

  • Increase antibody concentration to 1:100-1:200 for challenging samples

  • Extend primary antibody incubation time to overnight at 4°C

• Detection system enhancement:

  • Switch to high-sensitivity detection systems (e.g., SuperSignal™ for WB, TSA™ for IHC/IF)

  • Use streptavidin conjugates with brighter fluorophores for fluorescence applications

  • Consider using HRP-polymer detection systems with DAB enhancement for chromogenic detection

• Sample-specific considerations:

  • Check for appropriate positive control tissues (human prostate tissue shows reliable TMEM87B expression)

  • Verify sample storage conditions haven't compromised antigenicity

  • For cell cultures, confirm TMEM87B expression levels in your specific cell type

What strategies effectively reduce background when using biotin-conjugated TMEM87B antibodies?

High background is a common challenge with biotin-conjugated antibodies. Implement these targeted approaches:

• Endogenous biotin blocking: Apply avidin-biotin blocking kit before antibody incubation, particularly critical for biotin-rich tissues like liver, kidney, and brain.

• Optimized blocking solutions:

  • For IHC/ICC: Use 10% normal serum from the same species as the secondary antibody plus 1% BSA

  • For Western blot: 5% non-fat dry milk in TBST often provides superior background reduction compared to BSA

• Buffer optimization:

  • Include 0.1-0.3% Triton X-100 in antibody diluent for better penetration

  • Add 0.05% Tween-20 to all wash buffers to reduce non-specific binding

• Dilution adjustments:

  • Prepare antibody dilutions in buffer containing 1% BSA and 10% normal serum

  • For particularly challenging samples, prepare antibody dilution 24 hours in advance and store at 4°C to allow non-specific binding components to precipitate

• Storage buffer consideration: The presence of 50% glycerol and 0.03% Proclin 300 in the antibody storage buffer helps maintain stability but may contribute to background at very low dilutions; additional washing steps can help mitigate this effect .

How do fixation methods affect TMEM87B epitope detection in immunocytochemistry/immunohistochemistry?

Fixation methods significantly impact TMEM87B epitope accessibility and detection sensitivity:

How should researchers design experiments to study TMEM87B's role in retrograde transport?

To effectively investigate TMEM87B's role in retrograde transport from endosomes to the trans-Golgi network, implement these experimental approaches:

• Colocalization studies:

  • Design triple labeling experiments using biotin-conjugated TMEM87B antibody alongside markers for:

    • Trans-Golgi network (e.g., TGN46, Golgin-97)

    • Early endosomes (e.g., EEA1, Rab5)

    • Late endosomes (e.g., Rab7, Rab9)

  • Analyze colocalization coefficients quantitatively using Pearson's or Mander's correlation coefficients

• Functional transport assays:

  • Utilize cargo proteins known to undergo retrograde transport (e.g., STxB, TGN38)

  • Compare transport kinetics between control and TMEM87B-depleted cells

  • Track cargo movement using live-cell imaging with fluorescently-labeled markers

• Interaction studies:

  • Perform co-immunoprecipitation using TMEM87B antibodies to identify binding partners

  • Validate interactions with known retrograde transport machinery components (GARP complex proteins, SNARE proteins)

  • Use proximity ligation assays to confirm interactions in situ

• Loss-of-function approaches:

  • Generate TMEM87B knockdown/knockout models using siRNA or CRISPR-Cas9

  • Evaluate impacts on retrograde transport pathways

  • Perform rescue experiments with wild-type and mutant TMEM87B constructs

• Cargo tracking:

  • Use pulse-chase experiments with endocytosed markers

  • Quantify arrival kinetics at the TGN in the presence/absence of functional TMEM87B

What considerations are important when interpreting TMEM87B cellular distribution data?

When interpreting TMEM87B cellular distribution data, researchers should consider several critical factors:

• Expression level artifacts:

  • Overexpression systems may cause mislocalization of TMEM87B

  • Compare endogenous staining patterns with tagged expression constructs

  • Validate observations across multiple cell types to distinguish authentic from artifactual localization

• Epitope accessibility variations:

  • Different antibodies (targeting AA 451-554 versus AA 484-513) may yield varying localization patterns due to epitope masking in specific compartments

  • C-terminal epitopes may be obscured in protein complexes

  • Compare results from multiple antibodies targeting distinct epitopes

• Dynamic localization considerations:

  • TMEM87B localization may change during cell cycle progression

  • Trafficking between compartments may result in distinct populations

  • Synchronize cells when comparing treatments/conditions

• Fixation and permeabilization artifacts:

  • Membrane protein localization is particularly sensitive to fixation methods

  • Overly harsh permeabilization can disrupt membrane architecture

  • Compare results across multiple fixation/permeabilization protocols

• Resolution limitations:

  • Standard confocal microscopy may not resolve closely associated compartments

  • Consider super-resolution approaches (STED, STORM, SIM) for detailed localization studies

  • Complement optical approaches with biochemical fractionation data

How can researchers quantitatively assess TMEM87B protein levels across different experimental conditions?

For rigorous quantitative assessment of TMEM87B protein levels across experimental conditions, implement these methodological approaches:

• Western blot quantification:

  • Establish linear detection range for TMEM87B antibody using recombinant standards

  • Normalize to appropriate loading controls (β-actin for whole cell lysates, compartment-specific markers for fractionation studies)

  • Use fluorescent secondary antibodies rather than chemiluminescence for wider linear range

  • Calculate relative expression using densitometry with appropriate software (ImageJ, Image Studio Lite)

• ELISA-based quantification:

  • Develop sandwich ELISA using capture and detection antibodies targeting different TMEM87B epitopes

  • For biotin-conjugated antibodies (1:2000-1:10000 dilution), use streptavidin-HRP for detection

  • Generate standard curves using recombinant TMEM87B protein

  • Typical detection range: 50-5000 pg/mL

• Flow cytometry quantification:

  • Use permeabilization protocols optimized for transmembrane proteins

  • Calibrate using quantitative beads to establish molecules of equivalent soluble fluorochrome (MESF)

  • Control for autofluorescence and non-specific binding

  • For biotin-conjugated antibodies, use streptavidin conjugated to bright fluorophores (e.g., PE, APC)

• Immunoprecipitation and mass spectrometry:

  • Use TMEM87B antibodies for immunoprecipitation

  • Quantify using label-free or isotope-labeled approaches

  • Monitor TMEM87B-specific peptides for absolute quantification

• Image-based quantification:

  • Establish consistent acquisition parameters (exposure, gain, offset)

  • Implement automated analysis pipelines to reduce bias

  • Report integrated intensity per cell rather than maximum intensity

  • Use automated cell segmentation for high-throughput analysis

How can TMEM87B biotin-conjugated antibodies be incorporated into proximity-based protein interaction studies?

TMEM87B biotin-conjugated antibodies offer valuable advantages in proximity-based protein interaction studies through these methodological approaches:

• Proximity Ligation Assay (PLA) applications:

  • Pair biotin-conjugated TMEM87B antibody with antibodies against suspected interaction partners

  • Detect using streptavidin-conjugated DNA oligonucleotides and complementary oligonucleotides conjugated to secondary antibodies against the partner protein

  • Rolling circle amplification then generates fluorescent spots only when proteins are within ~40nm proximity

  • This approach has advantages over traditional co-IP for detecting transient or weak interactions

• BioID/TurboID proximity labeling:

  • Compare proximity labeling results with biotin-conjugated antibody detection to validate interactions

  • Use biotin-conjugated TMEM87B antibodies to immunoprecipitate the protein complex after proximity labeling

  • Apply two-color detection to distinguish endogenous biotinylated proteins from proximity-labeled proteins

• APEX2-based proximity labeling:

  • Use biotin-conjugated TMEM87B antibodies to verify APEX2 fusion protein localization

  • Implement multi-label imaging to correlate TMEM87B localization with biotinylated proximity proteins

• Cross-linking Mass Spectrometry (XL-MS):

  • Use biotin-conjugated TMEM87B antibodies for affinity purification after crosslinking

  • Exploit biotin-streptavidin interaction for stringent purification of TMEM87B complexes

  • Optimize elution conditions to maintain crosslinks while releasing protein complexes

• FRET-based approaches:

  • Combine biotin-conjugated TMEM87B antibodies with fluorophore-conjugated streptavidin as FRET acceptors

  • Use fluorescently-labeled antibodies against interaction partners as FRET donors

  • Measure energy transfer to identify proteins in close proximity to TMEM87B

What are the best approaches for studying TMEM87B in the context of tissue-specific expression patterns?

For comprehensive analysis of TMEM87B tissue-specific expression patterns, implement these methodological strategies:

• Multiplex immunohistochemistry optimization:

  • Use biotin-conjugated TMEM87B antibody at 1:200-1:500 dilution alongside cell type-specific markers

  • Implement spectral unmixing for multi-color analysis

  • Consider tyramide signal amplification for detection of low-abundance expression

  • Human prostate tissue serves as a reliable positive control for TMEM87B expression patterns

• Tissue microarray screening:

  • Apply standardized IHC protocols across diverse tissue types

  • Use digital pathology approaches for quantitative comparison

  • Develop scoring systems that account for both intensity and distribution patterns

  • Cross-validate findings with publicly available transcriptomic datasets

• Single-cell analysis approaches:

  • Optimize tissue dissociation protocols to maintain epitope integrity

  • Implement flow cytometry or mass cytometry (CyTOF) with TMEM87B antibodies

  • Correlate protein expression with scRNA-seq data in parallel samples

  • Analyze co-expression patterns with lineage-specific markers

• Spatial transcriptomics integration:

  • Combine TMEM87B immunohistochemistry with spatial transcriptomics

  • Register protein and RNA localization data

  • Analyze spatial correlation between TMEM87B protein and mRNA

  • Identify regions with post-transcriptional regulation

• Developmental and disease-state profiling:

  • Compare TMEM87B expression across developmental stages

  • Analyze expression changes in disease contexts

  • Implement quantitative image analysis for objective comparisons

  • Correlate with functional changes in retrograde transport

How can researchers effectively study post-translational modifications of TMEM87B protein?

To effectively characterize post-translational modifications (PTMs) of TMEM87B, implement these specialized analytical approaches:

• Phosphorylation analysis:

  • Immunoprecipitate TMEM87B using biotin-conjugated antibodies followed by phospho-specific detection

  • Use phosphatase treatment as control to confirm specificity

  • Employ mass spectrometry to identify specific phosphorylation sites

  • Design experiments with phosphatase inhibitors to preserve transient modifications

• Glycosylation characterization:

  • Treat samples with glycosidases (PNGase F, Endo H) before Western blotting

  • Compare mobility shifts to identify N-linked glycosylation

  • Use lectins alongside TMEM87B antibodies for co-localization studies

  • Assess glycosylation status in different subcellular compartments

• Ubiquitination detection:

  • Co-immunoprecipitate using TMEM87B antibodies followed by ubiquitin detection

  • Use proteasome inhibitors to accumulate ubiquitinated species

  • Distinguish between mono- and poly-ubiquitination patterns

  • Analyze ubiquitination kinetics during protein trafficking

• SUMO modification analysis:

  • Implement denaturing immunoprecipitation to preserve SUMO modifications

  • Use SUMO-specific antibodies alongside TMEM87B detection

  • Identify SUMO-interacting proteins in the TMEM87B pathway

  • Assess impacts of SUMO-site mutations on TMEM87B function

• Integrated PTM analysis:

  • Combine multiple enrichment strategies for comprehensive PTM profiling

  • Use biotin-conjugated TMEM87B antibodies for initial purification

  • Apply advanced MS/MS approaches for PTM mapping

  • Correlate modifications with trafficking dynamics and protein interactions

For all PTM studies, controls are critical - always compare results across multiple antibodies and validate findings using complementary approaches to avoid epitope masking artifacts or detection biases .