TMEM165 Antibody

What is TMEM165 and why is it significant in research?

TMEM165 (Transmembrane Protein 165) is a highly conserved hydrophobic protein of 324 amino acids containing 7 transmembrane-spanning domains . It functions as a putative divalent cation:proton antiporter that exchanges calcium or manganese ions for protons across the Golgi membrane . TMEM165 is significant in research because:

• Mutations cause Congenital Disorders of Glycosylation (CDG)

• It's overexpressed in hepatocellular carcinoma (HCC) and associated with cancer aggressiveness

• It promotes invasion and growth in breast cancer

• Its deficiency leads to skeletal disorders characterized by major skeletal dysplasia and pronounced dwarfism

• It plays a role in maintaining lysosomal Ca²⁺ stores

How is TMEM165 involved in glycosylation?

TMEM165 plays a crucial role in proper glycosylation pathways. Studies have shown that TMEM165 deficiency causes Golgi glycosylation defects . Specifically, TMEM165 appears to be essential for the synthesis of glycosaminoglycan (GAG) chains, including both heparan sulfate (HS) and chondroitin sulfate (CS) chains of proteoglycans. In TMEM165-knockout cells, there is significant impairment in the elongation of these GAG chains, resulting in reduced detection of cell surface proteoglycans .

Which TMEM165 antibody applications are most effective for different experimental goals?

Based on the literature and commercial antibody information, different applications have varying effectiveness:

Choose applications based on your specific research questions and be aware of potential localization artifacts when using antibodies for TMEM165 detection.

How should I design experiments to study TMEM165 knockout/knockdown effects?

CRISPR/Cas9 has been successfully used to generate TMEM165 knockout models. Based on published methodologies:

• Design guide RNAs targeting exonic regions (e.g., exon 2) with appropriate cohesive ends for vector ligation

  • Example sequence: 5'-CACCGCTATAACCGGCTGACTGTGC-3' and 5'-AAACGCACAGTCAGCCGGTTATAGC-3'

• Clone into an appropriate vector system (e.g., pUC57-attbU6 sgRNA vector)

• Co-transfect with Cas9 expression vector and selection marker

• Screen clones by PCR and sequencing to confirm mutations

• Validate knockout at protein level using Western blot with anti-TMEM165 antibodies

• Assess phenotypic changes by examining:

  • Glycosylation patterns (lectin binding, GAG synthesis)

  • Signaling pathway alterations (TGF-β/BMP)

  • Cell morphology and cytoskeletal organization

  • Invasive capacity (for cancer cell models)

What controls should I include when using TMEM165 antibodies?

Essential controls for TMEM165 antibody experiments include:

• Positive controls:

  • Cell lines with known TMEM165 expression (e.g., MDAMB231, BT549, HS578T for high expression; T47D, MCF7 for low/no expression in breast cancer research)

  • Recombinant TMEM165 protein (if available)

• Negative controls:

  • TMEM165 knockout cells generated via CRISPR/Cas9

  • siRNA-mediated knockdown cells

  • Primary antibody omission control

• Specificity controls:

  • Peptide competition assay using the immunizing peptide

  • Testing multiple antibodies targeting different epitopes (e.g., N-terminal vs. internal domains)

• Subcellular localization controls:

  • Co-staining with compartment markers (GM130 for Golgi, LAMP2 for lysosomes)

  • Compare live-cell vs. fixed-cell vs. permeabilized-cell staining patterns

How do I investigate the differential localization of TMEM165 between Golgi and lysosomes?

The dual localization of TMEM165 presents a methodological challenge. Based on recent research findings:

• Live-cell imaging approach:

  • Use fluorescent protein-tagged TMEM165 (e.g., TMEM165-mCherry) in live cells

  • Co-express organelle markers (e.g., Golgi-EGFP for trans-Golgi)

  • Document localization patterns before fixation or permeabilization

• Biochemical fractionation approach:

  • Utilize magnetic isolation of lysosomes (e.g., iron-dextran loading followed by magnetic capture)

  • Analyze protein composition by Western blotting

  • Include markers for various organelles (LAMP2 for lysosomes, RCAS1/GORASP1 for Golgi, PDI for ER)

  • Consider V-ATPase inhibition (e.g., concanamycin A) during isolation to prevent cargo degradation

• Super-resolution microscopy:

  • Employ techniques like STORM or STED for higher resolution of membrane compartments

  • Use minimally disruptive fixation protocols that preserve native localization

What are the implications of TMEM165's role in cancer progression and how can antibodies help investigate this?

TMEM165 has emerged as a potential oncogenic factor in multiple cancers:

• Expression correlation analysis:

  • Use TMEM165 antibodies for tissue microarray analysis of patient samples

  • Correlate expression with clinical parameters (tumor stage, vascular invasion, survival)

  • Data indicates TMEM165 expression is associated with poor prognosis in HCC and breast cancer

• Mechanism investigation:

  • In HCC: TMEM165 promotes invasion via upregulation of MMP-2

  • In breast cancer: TMEM165 alters glycosylation of key proteins involved in epithelial-to-mesenchymal transition (EMT), including E-cadherin

  • Study both gain-of-function (overexpression) and loss-of-function (knockout) models

• Experimental approach:

  • Generate TMEM165 knockout cancer cell lines (e.g., using CRISPR/Cas9 in MDAMB231 cells)

  • Assess morphological changes (e.g., actin reorganization visualized with phalloidin staining)

  • Examine EMT marker expression (E-cadherin increases and vimentin decreases in TMEM165 knockout cells)

  • Perform functional assays (migration, invasion, tumor growth in vivo)

How does TMEM165 deficiency impact signaling pathways and how can this be studied?

TMEM165 deficiency significantly alters key developmental signaling pathways:

• TGF-β signaling pathway:

  • TMEM165-deficient cells show impaired TGF-β signaling

  • Use phospho-specific antibodies to assess Smad2 phosphorylation status

  • Analyze TGFβR2 expression (decreased at both mRNA and protein levels)

  • Examine expression of TGF-β antagonists like asporin (increased 4-fold in mutant cells)

• BMP signaling pathway:

  • TMEM165-deficient cells show increased BMP signaling

  • Assess Smad1/5/9 phosphorylation status

  • Analyze BMP receptor expression (increased BMPR1A, BMPR1B, BMPR2)

  • Examine expression of BMP antagonists like noggin (decreased in mutant cells)

• Experimental validation:

  • Reporter assays (e.g., BRE-Luc for BMP responsive elements showed 4-fold increase in knockout cells)

  • RT-qPCR for downstream target genes (e.g., Id1)

  • Rescue experiments with wild-type TMEM165 expression

Why might I observe different TMEM165 localization patterns in my immunofluorescence experiments?

The discrepancy in TMEM165 localization patterns is a documented phenomenon:

• Fixation/permeabilization effects:

  • TMEM165-mCherry localization dramatically changes from punctate lysosomal structures to sheet-like Golgi structures following permeabilization

  • Try different fixation methods (paraformaldehyde, methanol, glutaraldehyde)

  • Test different permeabilization agents (Triton X-100, saponin, digitonin at varying concentrations)

• Antibody epitope accessibility:

  • Different epitopes show different accessibility patterns

  • Sigma antibodies (recognizing aa176-229) detect a cytosolic epitope

  • Thermofischer antibodies (recognizing aa17-45) detect a different epitope

  • Try selective membrane permeabilization with low digitonin concentrations

• Recommended approach:

  • Compare live-cell imaging results with fixed-cell results

  • Use multiple antibodies targeting different epitopes

  • Consider biochemical fractionation as an alternative approach

How can I improve specificity when detecting endogenous TMEM165?

Improving specificity for TMEM165 detection:

• Antibody selection considerations:

  • Choose antibodies validated in knockout/knockdown models

  • Consider using antibodies against different epitopes (e.g., N-terminal region vs. internal domain)

  • Optimal dilution ranges: 1:500-2000 for WB, 1:50-300 for IHC-P, 1:2000-20000 for ELISA

• Technical optimization:

  • Increase blocking stringency (5% BSA/0.01% Tween 20 in PBS has been effective)

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

  • Include validation controls (knockout/knockdown samples)

• Signal enhancement approaches:

  • For low abundance detection, consider using more sensitive detection systems (e.g., chemiluminescent substrates like Clarity Western ECL)

  • For tissue sections, optimize antigen retrieval methods

How do I resolve contradictory findings between different TMEM165 antibodies?

When faced with contradictory results:

• Perform epitope mapping analysis:

  • Different antibodies target different regions (e.g., aa17-45, aa68-118, aa176-229)

  • Certain epitopes may be masked in specific cellular compartments

  • Some epitopes may be affected by post-translational modifications

• Validate with complementary approaches:

  • Compare antibody results with tagged TMEM165 constructs

  • Use biochemical fractionation to confirm protein presence in specific compartments

  • Employ proximity labeling techniques (BioID, APEX) to identify interactors and confirm localization

• Control experiments:

  • Test antibodies in TMEM165-knockout cells to confirm specificity

  • Perform peptide competition assays with immunizing peptides

  • Use multiple detection methods (Western blot, IF, IHC) to cross-validate findings