TMEM150A Antibody

What is TMEM150A and why is it important for research?

TMEM150A (Transmembrane Protein 150A/TM6P1/damage-regulated autophagy modulator 5) is a member of the TMEM150/DRAM family of proteins that possesses 6 transmembrane domains with both termini positioned within the cytoplasm . It plays crucial roles in regulating phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] production at the plasma membrane by modifying the composition of the phosphatidylinositol 4-kinase enzyme complex . TMEM150A is particularly important for research into immune regulation, as knockdown studies demonstrate it affects cytokine production both in basal conditions and after immune stimulation with lipopolysaccharide (LPS) .

What is the cellular localization of TMEM150A?

TMEM150A primarily localizes to the plasma membrane, with only minor presence on intracellular structures . Topological studies using GFP-tagged TMEM150A have confirmed that its C-terminal tail is oriented toward the cytosol, while its Loop 1 region is exposed to the extracellular medium . This plasma membrane localization aligns with its function in regulating phosphoinositide production through interaction with the PI4KIIIα complex .

What applications are TMEM150A antibodies commonly used for?

TMEM150A antibodies are primarily used for:

• Western blotting (typical dilution range: 1:750)

• Immunoprecipitation for interaction studies

• Immunofluorescence microscopy to determine subcellular localization

• Validation of siRNA knockdown experiments

• Investigation of protein-protein interactions, particularly with components of the PI4KIIIα complex

How should I validate a TMEM150A antibody for my research?

For rigorous TMEM150A antibody validation:

• Perform Western blot analysis comparing control and TMEM150A-knockdown samples (using siRNA) to confirm specific band disappearance

• Compare antibody reactivity in cells overexpressing TMEM150A-GFP or other tagged constructs

• Verify expected molecular weight (~25-30 kDa) and band pattern

• Include appropriate positive controls (tissues/cells known to express TMEM150A, such as liver samples)

• Test reactivity in multiple applications if using for different techniques

• Conduct cross-reactivity testing against related family members (TMEM150B, TMEM150C) if specificity is crucial

How do I choose between different commercial TMEM150A antibodies?

When selecting a TMEM150A antibody:

• Consider the epitope location - antibodies targeting different regions of the protein may have varying accessibility depending on your application

• Evaluate validation data provided by manufacturers, especially knockdown/knockout validation

• For studying protein interactions with PI4KIIIα or EFR3, choose antibodies targeting regions not involved in these interactions

• Check species reactivity if working with non-human models

• Review literature citations for the specific antibody (e.g., NBP1-81885 from Novus Biologicals has been successfully used in TMEM150A research)

• Consider polyclonal versus monoclonal options based on your experimental needs

What is the optimal protocol for Western blotting with TMEM150A antibodies?

Based on published methodologies:

• Sample preparation:

  • Extract proteins using standard lysis buffers (RIPA or NP-40 based)

  • Include protease inhibitors to prevent degradation

• Gel electrophoresis and transfer:

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

  • Transfer to PVDF membranes (as used in successful studies)

• Blocking and antibody incubation:

  • Block membranes in Tris-buffered-saline with 0.1% Tween (TBST) containing 5% skim milk (TBSTM) for 1 hour at room temperature

  • Incubate with anti-TMEM150A antibody (e.g., NBP1-81885) at 1:750 dilution in TBSTM overnight at 4°C

  • Wash with TBST

  • Incubate with appropriate HRP-conjugated secondary antibody in TBSTM for 1 hour at room temperature

• Detection:

  • Develop using ECL substrate (e.g., BioRad Clarity ECL)

  • Image using digital imaging systems (e.g., ChemiDoc)

How can I detect TMEM150A in immunofluorescence applications?

For optimal immunofluorescence detection of TMEM150A:

• Fixation and permeabilization considerations:

  • Standard paraformaldehyde fixation (4%) for 15-20 minutes

  • Permeabilization is essential for accessing the C-terminal cytosolic epitopes

  • For epitopes in extracellular loops (e.g., Loop 1), cell-surface staining can be performed without permeabilization

• Optimal antibody dilutions:

  • Start with 1:100-1:500 dilution range and optimize

  • Include membrane markers (e.g., wheat germ agglutinin) for co-localization studies

• Controls:

  • TMEM150A-knockdown cells as negative controls

  • TMEM150A-GFP transfected cells as positive controls

Why might I be getting non-specific bands in my TMEM150A Western blot?

Non-specific bands in TMEM150A Western blots can occur for several reasons:

• Cross-reactivity with TMEM150 family members:

  • TMEM150B and TMEM150C share sequence homology with TMEM150A

  • Solution: Use antibodies specifically validated against these family members

• Post-translational modifications:

  • Multiple bands may represent different glycosylation states

  • Solution: Treat samples with glycosidases to confirm

• Degradation products:

  • Solution: Use fresh samples and ensure complete protease inhibition

• Optimization issues:

  • Solution: Adjust antibody concentration, blocking conditions, or washing stringency

  • Consider longer blocking times (2+ hours) or alternative blocking agents

• Validation approach:

  • Compare band patterns between control and TMEM150A-knockdown samples

  • Expected result: The specific TMEM150A band should be significantly reduced in knockdown samples

How can I improve co-immunoprecipitation efficiency when studying TMEM150A interactions?

For enhanced co-immunoprecipitation of TMEM150A and its binding partners:

• Lysis buffer optimization:

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

  • Include phosphatase inhibitors to maintain phosphorylation-dependent interactions

• Cross-linking considerations:

  • Consider reversible cross-linking for transient interactions

  • DSP (dithiobis(succinimidyl propionate)) works well for membrane protein complexes

• Tag position considerations:

  • For studies involving PI4KIIIα interactions, C-terminal tags on TMEM150A may interfere less with binding than N-terminal tags

• Control experiments:

  • Include GFP-only controls when using GFP-tagged TMEM150A

  • Compare TMEM150A-GFP with TMEM150B-GFP to confirm specificity

• Detection strategy:

  • When studying the TMEM150A-PI4KIIIα-EFR3 complex, be aware that this complex appears mutually exclusive with TTC7B presence

  • Optimize antibody combinations to detect all components simultaneously

How can I investigate the role of TMEM150A in TLR4-mediated signaling?

To investigate TMEM150A's role in TLR4 signaling:

• Experimental cell models:

  • Use HEK293 cells stably expressing TLR4/MD2/CD14 (HEK-TLR4) for a defined TLR4 pathway

  • Consider H292 lung epithelial cells for studying broader cytokine responses

• TMEM150A knockdown approach:

  • Transfect cells with validated siRNAs targeting TMEM150A (5 pmol with Lipofectamine RNAiMax)

  • Validate knockdown efficiency by RT-qPCR and Western blot

• LPS stimulation protocol:

  • Challenge cells with LPS concentration series (30-300 ng/mL)

  • Collect supernatants for cytokine ELISAs

  • Harvest RNA for transcript analysis

• Readout measurements:

  • Primary readouts: CXCL8, IL6, and CCL5 secretion by ELISA

  • Secondary readouts: Broader cytokine profiling (IL7, IL10, IL12B, IFN-γ, TNF) using Milliplex technology

  • Confirm protein changes with transcript analysis via RT-qPCR

How can I study TMEM150A's interaction with the PI4KIIIα complex?

For detailed investigation of TMEM150A's interaction with the PI4KIIIα complex:

• Protein domain mapping approach:

  • Create chimeric constructs swapping domains between TMEM150A and non-interacting TMEM150B

  • Focus on the C-terminal cytosolic tail, which is critical for PI4KIIIα interaction

  • Generate truncation mutants to identify minimal interaction domains

• Co-expression system:

  • Co-express tagged versions of TMEM150A, PI4KIIIα, EFR3B, and TTC7B

  • Use different tags (e.g., GFP, mCherry, HA, FLAG) to allow selective immunoprecipitation

  • Perform sequential immunoprecipitation to isolate specific complexes

• Competition experiments:

  • Investigate the mutually exclusive relationship between TMEM150A and TTC7B in the complex

  • Perform titration experiments with increasing amounts of one component while monitoring displacement of the other

• Functional readouts:

  • Monitor PI(4,5)P₂ recovery following depletion as a functional readout

  • Use PI(4,5)P₂ biosensors to assess kinetics in live cells

What approaches can be used to study TMEM150A's topology and membrane orientation?

To investigate TMEM150A topology and membrane orientation:

• Accessibility assays:

  • Express TMEM150A with tags in predicted extracellular and intracellular domains

  • Perform antibody accessibility experiments in non-permeabilized cells

  • Only extracellular epitopes will be detected without permeabilization

• Protease protection assays:

  • Prepare membrane fractions expressing tagged TMEM150A

  • Treat with proteases in the presence or absence of detergents

  • Analyze protection patterns of various domains

• Glycosylation mapping:

  • Introduce glycosylation sites at various positions

  • Assess N-glycosylation status (which occurs in the ER lumen)

  • Only extracellular/luminal domains will be glycosylated

• Fluorescence approaches:

  • Use split-GFP complementation to determine cytosolic vs. extracellular localization

  • Compare antibody staining of GFP-tagged TMEM150A versus antibody to extracellular HA-tag in Loop 1

How might TMEM150A antibodies be used to study its role in autophagy regulation?

While TMEM150A's role in autophagy is less characterized than other DRAM family members, several approaches can investigate this connection:

• Co-localization studies:

  • Use TMEM150A antibodies with autophagosome markers (LC3) under various conditions

  • Investigate changes during nutrient starvation (fasting conditions increase TMEM150A in liver)

• Autophagy flux assessment:

  • Compare autophagy markers (p62, LC3-II) in TMEM150A knockdown vs. control cells

  • Use chloroquine to block autophagosome-lysosome fusion and assess differences

• Specific research questions to address:

  • Does TMEM150A relocalize during autophagy induction?

  • How does TMEM150A's regulation of PI(4,5)P₂ connect to autophagosome formation?

  • Is TMEM150A regulated by autophagy-inducing signals similar to other DRAM family members?

• Connection to PI4KIIIα pathway:

  • Investigate how TMEM150A's interaction with PI4KIIIα may influence membrane dynamics during autophagosome formation

What are the best approaches for studying cell-type specific expression patterns of TMEM150A?

For comprehensive analysis of TMEM150A expression patterns:

• Transcriptional profiling:

  • Analyze TMEM150A expression across tissue and cell types

  • Compare with related family members (TMEM150B/C, DRAM1/2)

• Immunohistochemistry optimization:

  • Test multiple fixation protocols (paraformaldehyde, methanol)

  • Compare antigen retrieval methods for optimal epitope exposure

  • Validate antibody specificity in TMEM150A-knockdown tissues/cells

• Single-cell analysis:

  • Use flow cytometry with TMEM150A antibodies to quantify expression in heterogeneous populations

  • Correlate with functional markers of cell state

• Expression regulation studies:

  • Investigate changes during fasting conditions, which increase expression in liver

  • Examine expression changes during immune activation or TLR4 stimulation