TMEM267 Antibody

What is TMEM267 and what is known about its structure and function?

TMEM267 (Transmembrane Protein 267) is a human transmembrane protein formerly known as C5orf28 (Chromosome 5 open reading frame 28). It is identified by the UniProt Primary Accession Q0VDI3, with secondary accessions B2RDA6 and Q9H6Z2 . The protein contains multiple predicted membrane domains, suggesting it is predominantly localized to the plasma membrane, similar to other TMEM family proteins like TMEM263 which has been experimentally confirmed to localize to the plasma membrane .

While specific functions of TMEM267 remain under investigation, research on related TMEM family proteins suggests potential roles in cellular signaling pathways. For instance, TMEM263 has been implicated in the GH/IGF-1 axis regulation as demonstrated by knockout studies in mice . TMEM267's exact biological role requires further characterization, but its transmembrane nature suggests potential involvement in membrane transport, cell signaling, or receptor function.

What types of TMEM267 antibodies are available for research?

Currently available TMEM267 antibodies include:

The FITC-conjugated polyclonal antibody is derived from rabbits immunized with recombinant human Transmembrane protein C5orf28 protein (amino acids 1-76) . This antibody has excitation/emission wavelengths of 499/515 nm and is compatible with the 488 nm laser line commonly used in flow cytometry and fluorescence microscopy applications .

For validation experiments, researchers can use the TMEM267 control fragment (amino acids 32-55) recombinant protein for blocking experiments with corresponding antibodies .

What are the optimal storage conditions for TMEM267 antibodies?

For maximum stability and performance of TMEM267 antibodies, follow these evidence-based storage protocols:

• Store antibodies in aliquots at -20°C to minimize freeze-thaw cycles

• Protect FITC-conjugated antibodies from light exposure, as fluorophores are light-sensitive

• Avoid repeated freeze/thaw cycles which can degrade antibody performance

• TMEM267 antibodies are typically provided in a buffer containing 0.01 M PBS, pH 7.4, 0.03% Proclin-300, and 50% Glycerol for stability

• When stored properly, antibodies typically maintain reactivity for at least 12 months from date of receipt

The inclusion of glycerol in the storage buffer helps prevent freeze-thaw damage, while Proclin-300 serves as an antimicrobial preservative. These storage recommendations are consistent with best practices for maintaining antibody integrity and performance in research applications .

What are the recommended protocols for using TMEM267 antibodies in immunofluorescence studies?

When utilizing TMEM267 antibodies for immunofluorescence applications, researchers should implement the following protocol based on established methods for transmembrane protein detection:

Sample Preparation:

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.1% Triton X-100 for 10 minutes (for intracellular domains)

• Block with 5% normal serum (from species other than antibody host) for 1 hour

Antibody Incubation:

• Dilute TMEM267 antibody according to manufacturer's recommendations (typically 1:100 to 1:500)

• Incubate overnight at 4°C in a humidified chamber

• Wash 3x with PBS containing 0.1% Tween-20

Detection and Visualization:

• For non-conjugated primary antibodies, incubate with appropriate secondary antibody

• For FITC-conjugated antibodies (499/515 nm excitation/emission), proceed directly to nuclear counterstaining

• Counterstain nuclei with DAPI (if applicable)

• Mount with anti-fade mounting medium

Controls:

• Include no-primary antibody control

• Consider peptide blocking control using TMEM267 (aa 32-55) control fragment

• Include positive and negative cell lines if known

Based on studies with related transmembrane proteins, TMEM267 would be expected to show predominantly membrane-associated staining patterns, potentially with some cytoplasmic distribution .

How can I optimize TMEM267 antibody performance in Western blot analyses?

Optimizing Western blot analysis for TMEM267 requires specific considerations for transmembrane proteins:

Sample Preparation:

• Perform subcellular fractionation to separate plasma membrane, cytosolic, and nuclear fractions, as different TMEM protein isoforms may localize to different cellular compartments

• Use a membrane protein extraction buffer containing 1% NP-40 or Triton X-100

• Add protease inhibitor cocktail to prevent degradation

• Avoid boiling samples (heat to 37-50°C instead) to prevent aggregation of transmembrane domains

Gel Electrophoresis and Transfer:

• Use 10-12% SDS-PAGE gels for optimal resolution

• Consider using gradient gels (4-20%) if multiple isoforms are expected

• Transfer at low voltage (30V) overnight at 4°C for efficient transfer of membrane proteins

Antibody Incubation:

• Block with 5% non-fat milk or BSA in TBST

• Use primary antibody at manufacturer's recommended dilution

• Consider longer incubation times (overnight at 4°C)

• Include the recombinant TMEM267 control fragment in a parallel blocking experiment to confirm specificity

Detection:

• For chemiluminescent detection, use extended exposure times if signal is weak

• For fluorescent detection with FITC-conjugated antibodies, use appropriate imaging systems with 488nm excitation capabilities

Based on studies with related TMEM proteins, researchers should be prepared to detect potential glycosylated and non-glycosylated forms of TMEM267, as studies of TMEM26 revealed multiple protein isoforms with different molecular weights (40kDa, 44kDa, and 53kDa) .

What controls should I include when working with TMEM267 antibodies?

Rigorous experimental design for TMEM267 antibody applications should include the following controls:

Positive Controls:

• Recombinant TMEM267 protein

• Cell lines known to express TMEM267 (based on RNA expression databases)

• Tissues with documented TMEM267 expression

Negative Controls:

• Antibody diluent only (no primary antibody)

• Isotype control (rabbit IgG at equivalent concentration)

• Pre-absorption with TMEM267 (aa 32-55) control fragment

• TMEM267 knockdown/knockout samples if available

Specificity Validation Controls:

• Peptide competition assay: Pre-incubate antibody with recombinant TMEM267 control fragment

• Reprobing with antibody to another protein (e.g., Elf-1) after peptide blocking to confirm specificity of blocking effect

• siRNA knockdown of TMEM267 to demonstrate specificity

Technical Controls:

• Loading control (β-actin, GAPDH)

• Fractionation quality controls (e.g., Na/K ATPase for membrane fraction)

• Molecular weight marker to confirm expected protein size

Implementing these controls will significantly enhance data reliability and interpretation when working with TMEM267 antibodies across different experimental platforms .

How do post-translational modifications affect TMEM267 detection with antibodies?

Research on related transmembrane proteins suggests that TMEM267 likely undergoes post-translational modifications (PTMs) that can significantly impact antibody recognition and experimental outcomes:

N-Glycosylation:
Studies on related TMEM proteins indicate that N-glycosylation can affect protein localization and antibody accessibility. For instance, treatment of cells with the N-glycosylation inhibitor tunicamycin has been shown to substantially alter the plasma membrane localization of TMEM26 . This suggests that:

• TMEM267 may exist in both glycosylated and non-glycosylated forms

• Glycosylation may be critical for proper membrane localization

• Antibodies may show differential reactivity to glycosylated and non-glycosylated forms

Experimental Approaches:

• Treat samples with glycosidases (PNGase F) to remove N-linked glycans

• Compare antibody reactivity before and after deglycosylation

• Use tunicamycin to inhibit N-glycosylation in cell culture (5μg/ml for 24-120 hours)

• Examine multiple cellular fractions to detect different TMEM267 isoforms

Phosphorylation:
The PI3K/AKT pathway has been shown to regulate other TMEM proteins post-transcriptionally , suggesting that TMEM267 might also be regulated by phosphorylation events:

• Examine changes in TMEM267 detection following treatment with PI3K/AKT pathway activators (e.g., insulin) or inhibitors

• Use phosphatase treatment of samples prior to immunoblotting

• Consider using phospho-specific antibodies if phosphorylation sites are identified

Understanding how these PTMs affect TMEM267 will improve experimental design and interpretation of results when using TMEM267 antibodies .

What are the best approaches for studying TMEM267 interactions with other proteins?

To investigate protein-protein interactions involving TMEM267, researchers should consider these methodological approaches:

Co-immunoprecipitation (Co-IP):

• Use TMEM267 antibodies for pulldown experiments followed by mass spectrometry

• Perform reciprocal Co-IPs with antibodies against suspected interaction partners

• Include appropriate controls (IgG control, lysates from TMEM267 knockdown cells)

• Consider using crosslinking agents to stabilize transient interactions

Proximity Ligation Assay (PLA):

• Combine TMEM267 antibody with antibodies against potential interaction partners

• Visualize interactions as fluorescent dots when proteins are in close proximity (<40nm)

• Quantify interaction signals using appropriate imaging software

FRET/BRET Analysis:

• Generate fluorescent protein-tagged TMEM267 constructs

• Co-express with tagged versions of candidate interaction partners

• Measure energy transfer as evidence of protein proximity

Membrane Protein Interaction Considerations:

• Use mild detergents (0.5-1% NP-40, digitonin) to preserve membrane protein complexes

• Consider using DTSSP or other membrane-impermeable crosslinkers

• Perform studies in native membrane environments when possible

Validation Approaches:

• Confirm interactions using multiple independent techniques

• Demonstrate functional significance through mutation of interaction domains

• Show co-localization using super-resolution microscopy techniques

These approaches are particularly relevant for membrane proteins like TMEM267, as their transmembrane nature presents unique challenges for interaction studies .

How can I study TMEM267 expression regulation in different physiological conditions?

To investigate the regulation of TMEM267 expression under different physiological conditions, consider these experimental approaches:

Transcriptional Regulation:

• Analyze TMEM267 mRNA levels using qRT-PCR across different:

  • Cell lines and tissue types

  • Treatment conditions (hormones, growth factors, stress inducers)

  • Disease states

• Perform promoter analysis and reporter assays to identify key regulatory elements

• Use ChIP assays to identify transcription factors binding to the TMEM267 promoter

Post-transcriptional Regulation:

• Examine protein levels using Western blotting with TMEM267 antibodies

• Compare mRNA vs. protein levels to identify discrepancies suggesting post-transcriptional regulation

• Investigate microRNA targeting using prediction algorithms and functional assays

Signaling Pathway Impact:
Given that related TMEM proteins are regulated by various signaling pathways, examine TMEM267 expression following:

• PI3K/AKT pathway modulation (e.g., insulin treatment, PI3K inhibitors)

• Hormone receptor signaling (e.g., estrogen receptor modulation with fulvestrant)

• Growth factor signaling (e.g., IGF-1 pathway components)

Example Experimental Design:

This comprehensive approach will help elucidate the complex regulation of TMEM267 expression and localization under various physiological and pathological conditions .

What are common challenges in detecting TMEM267 and how can they be addressed?

Based on research with related transmembrane proteins, researchers may encounter several challenges when working with TMEM267 antibodies:

Challenge: Low Signal Intensity
Solutions:

• Optimize antibody concentration through titration experiments

• Extend primary antibody incubation time (overnight at 4°C)

• Use signal amplification systems (TSA, polymer-based detection)

• Ensure proper antigen retrieval for fixed tissues (citrate buffer pH 6.0 or EDTA buffer pH 9.0)

• Test different fixation methods if working with cells or tissues

Challenge: Multiple Bands in Western Blot
Solutions:

• Perform subcellular fractionation to separate different TMEM267 isoforms

• Use peptide competition assays to confirm specificity of bands

• Include deglycosylation treatment to identify glycosylated forms

• Compare patterns across multiple cell lines to identify consistent bands

Challenge: Variable Results Across Experiments
Solutions:

• Standardize protein extraction methods

• Use fresh antibody aliquots for each experiment

• Implement rigorous positive and negative controls

• Maintain consistent experimental conditions (time, temperature, reagents)

Challenge: Inconsistent Immunofluorescence Staining
Solutions:

• Optimize fixation and permeabilization protocols

• Include membrane markers for co-localization studies

• Compare staining patterns in multiple cell types

• Validate patterns using alternative detection methods

Challenge: Cross-Reactivity Concerns
Solutions:

• Perform specificity validation using the TMEM267 (aa 32-55) control fragment

• Include TMEM267 knockdown controls if possible

• Compare staining patterns with alternative TMEM267 antibodies if available

• Verify expression using orthogonal methods (mRNA detection)

Addressing these challenges will improve detection reliability and data interpretability when working with TMEM267 antibodies .

How should I design experiments to study the functional role of TMEM267?

To investigate TMEM267 function, implement a multi-faceted experimental approach:

Genetic Manipulation Strategies:

• CRISPR/Cas9-mediated knockout

• siRNA or shRNA-mediated knockdown

• Overexpression of wild-type TMEM267

• Expression of mutant forms lacking key domains or PTM sites

Functional Readouts to Assess:

• Cell proliferation, migration, and invasion capabilities

• Signaling pathway activation (particularly PI3K/AKT)

• Membrane transport activities

• Cell-cell or cell-matrix interactions

• Protein localization using TMEM267 antibodies before and after perturbations

Interaction Network Analysis:

• Perform IP-MS to identify the TMEM267 interactome

• Validate key interactions using TMEM267 antibodies in co-IP experiments

• Use proximity labeling approaches (BioID, APEX) to identify neighborhood proteins

Physiological Context Studies:

• Examine expression patterns across tissues and development using TMEM267 antibodies

• Investigate regulation under stress conditions or disease states

• Study phenotypic consequences of TMEM267 depletion in model systems

Suggested Experimental Workflow:

By implementing this comprehensive approach, researchers can systematically uncover the functional roles of TMEM267 in various cellular contexts .

What are the considerations for multiplexed detection of TMEM267 with other proteins?

When designing multiplexed detection experiments involving TMEM267 antibodies, consider these critical factors:

Antibody Compatibility:

• Select antibodies raised in different host species to avoid cross-reactivity

• If using multiple rabbit antibodies, consider sequential detection with thorough stripping

• Validate each antibody individually before multiplexing

• Ensure TMEM267 antibody works in your specific application (the FITC-conjugated rabbit anti-TMEM267 has excitation/emission at 499/515nm)

Fluorophore Selection for Imaging:

• Choose fluorophores with minimal spectral overlap

• For the FITC-conjugated TMEM267 antibody (499/515nm), pair with far-red fluorophores (e.g., Cy5)

• Consider using spectral unmixing for closely overlapping fluorophores

• Account for autofluorescence by including appropriate controls

Suggested Fluorophore Combinations with FITC-TMEM267:

Flow Cytometry Considerations:

• The FITC-conjugated TMEM267 antibody is compatible with 488nm laser excitation

• Design compensation controls for multi-color flow experiments

• Include FMO (fluorescence minus one) controls

• Validate staining using imaging flow cytometry if available

Protocol Optimizations:

• Test sequential vs. simultaneous antibody incubation

• Optimize concentrations of each antibody individually

• Include blocking steps to minimize cross-reactivity

• Perform peptide blocking controls with TMEM267 (aa 32-55) fragment

By carefully designing multiplexed detection experiments, researchers can effectively study TMEM267 in the context of other cellular components and signaling pathways .

What are emerging research areas involving TMEM267?

While specific studies on TMEM267 are still emerging, research on related transmembrane proteins suggests several promising directions:

• Structure-Function Relationships: Determining the three-dimensional structure of TMEM267 and mapping functional domains will be crucial for understanding its biological role.

• Disease Associations: Investigating potential links between TMEM267 expression/mutations and human diseases, as other TMEM family members have been implicated in various pathologies. For instance, TMEM263 deletion causes dwarfism through disruption of the GH/IGF-1 axis .

• Signaling Pathway Integration: Exploring how TMEM267 interfaces with established signaling networks, particularly the PI3K/AKT pathway that has been shown to regulate other TMEM proteins .

• Post-Translational Regulation: Characterizing the glycosylation and other PTMs of TMEM267 and their impact on protein function and cellular localization .

• Development of Novel Antibodies and Tools: Creating additional TMEM267-specific research tools, including monoclonal antibodies targeting different epitopes, tagged constructs for live imaging, and domain-specific reagents.

Future experimental approaches combining CRISPR-based genome editing, advanced imaging techniques, and proteomics will likely yield significant insights into TMEM267 biology and potential therapeutic applications.

How can I stay updated on new developments in TMEM267 research?

To remain current with TMEM267 research advances:

• Set up citation alerts for key TMEM267 papers through Google Scholar, PubMed, or Web of Science

• Create saved searches with terms like "TMEM267," "C5orf28," "Q0VDI3"

• Follow research groups working on transmembrane proteins and cellular signaling

• Monitor antibody company technical resources for new TMEM267 products and protocols

• Join research communities focused on membrane proteins and cellular signaling

• Attend conferences covering membrane biology, cell signaling, and protein trafficking