TMCC1 Antibody

What is TMCC1 and why is it important for cellular research?

TMCC1 is a member of the transmembrane and coiled-coil domain family that is evolutionarily conserved from nematode to human. It localizes primarily to the rough endoplasmic reticulum (ER) through its C-terminal transmembrane domains, with both N-terminal region and C-terminal tail residing in the cytoplasm. The protein contains predicted coiled-coil and transmembrane domains that are highly conserved across species, suggesting functional importance in most organisms .

TMCC1 has been found to be expressed in diverse human cell lines, with particularly high expression in epithelial cells (Hep G2, Caco-2, A549), neuroblastoma cells (SH-SY5Y), and glioblastoma cells (U87), while showing lower expression in leukemia and lymphoma cells . Its evolutionary conservation and expression pattern suggest important cellular functions, potentially in ER organization and calcium homeostasis.

What applications are TMCC1 antibodies validated for?

Current commercially available TMCC1 antibodies have been validated for several research applications:

Most TMCC1 antibodies show reactivity with human samples, and many are also cross-reactive with mouse and rat tissues, making them suitable for comparative studies across species . It's recommended that researchers titrate these antibodies in their specific testing systems to obtain optimal results for their particular application .

What are the key considerations when selecting a TMCC1 antibody for research?

When selecting a TMCC1 antibody for research, consider:

• Target epitope: Different antibodies target different regions of TMCC1. For example, some target N-terminal fragments (aa 1-100), while others target regions between residues 51-300 . The epitope can affect detection specificity and sensitivity.

• Reactivity: Verify that the antibody reacts with your species of interest. Some TMCC1 antibodies are reactive with human, mouse, and rat samples, while others may be limited to human samples only .

• Application compatibility: Ensure the antibody is validated for your specific application. Some antibodies work well for Western blotting but may not be optimal for immunofluorescence.

• Clonality: Most available TMCC1 antibodies are polyclonal, which can provide broader epitope recognition but potentially less specificity compared to monoclonal antibodies .

• Validation data: Review available validation data, including positive controls in relevant cell lines or tissues. For example, some TMCC1 antibodies have been validated in HCT 116 cells, MCF-7 cells, and brain tissues .

What are the recommended protocols for using TMCC1 antibodies in Western blotting?

For optimal Western blotting results with TMCC1 antibodies:

• Sample preparation: Prepare whole cell lysates from relevant cell lines. TMCC1 is highly expressed in epithelial cells, neuroblastoma, and glioblastoma cell lines, making these good positive controls .

• Expected molecular weight: TMCC1 has an observed molecular weight of approximately 72 kDa on SDS-PAGE gels .

• Dilution optimization: Start with a dilution of 1:500-1:1000 in 5% milk in TBST buffer. Incubate membranes with primary antibody overnight at 4°C .

• Secondary antibody: Use an appropriate HRP-conjugated secondary antibody (typically goat anti-rabbit at 1:10,000 dilution) and incubate for 2 hours at room temperature .

• Detection system: Standard ECL systems like Clarity ECL work well for TMCC1 detection .

• Controls: Include a known positive control (e.g., HeLa cells) and a negative control. THP-1 cells transduced with TMCC1-specific shRNA have been successfully used as negative controls in published research .

The expected result is a distinct immunoreactive band at approximately 72 kDa, which should be absent in knockdown samples, confirming antibody specificity .

How should TMCC1 antibodies be optimized for immunohistochemistry (IHC)?

For successful immunohistochemistry with TMCC1 antibodies:

• Tissue preparation: Both formalin-fixed, paraffin-embedded (FFPE) tissues and frozen sections can be used. Mouse embryo tissue has been validated for positive staining .

• Antigen retrieval: Two methods have shown success:

  • TE buffer pH 9.0 (recommended primary method)

  • Citrate buffer pH 6.0 (alternative method)

• Antibody dilution: Start with a dilution range of 1:50-1:500 and optimize based on signal-to-noise ratio .

• Incubation conditions: Typically overnight at 4°C or 1-2 hours at room temperature, depending on your specific protocol.

• Detection system: Use an appropriate secondary antibody and detection system compatible with your primary antibody host species (typically rabbit IgG for most commercially available TMCC1 antibodies).

• Controls: Include positive control tissues (like brain tissue) and negative controls (primary antibody omission or isotype controls).

What are effective strategies for troubleshooting weak or nonspecific TMCC1 antibody signals?

When encountering weak or nonspecific signals with TMCC1 antibodies:

• For weak signals:

  • Increase antibody concentration (reduce dilution)

  • Extend primary antibody incubation time

  • Optimize antigen retrieval conditions

  • Use signal amplification systems

  • Ensure TMCC1 is expressed in your sample type; check with positive control tissue/cells

  • Consider fresh antibody aliquots, as TMCC1 antibodies should be stored at -20°C and repeated freeze/thaw cycles should be avoided

• For nonspecific signals:

  • Increase antibody dilution

  • Reduce primary antibody incubation time

  • Optimize blocking conditions (try different blockers or longer blocking times)

  • Increase washing duration and number of wash steps

  • Validate specificity using TMCC1 knockdown samples as negative controls

  • Pre-absorb antibody with recombinant antigen if available

• Buffer optimization:

  • Most TMCC1 antibodies perform well in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

  • For immunofluorescence applications with FITC-conjugated antibodies, use 0.01 M PBS, pH 7.4, with 0.03% Proclin-300 and 50% Glycerol

How can TMCC1 antibodies be used to investigate TMCC1's role in protein-protein interactions?

To investigate TMCC1's role in protein-protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use TMCC1 antibodies to pull down TMCC1 and associated proteins

  • Research has shown that TMCC1 can dimerize or oligomerize with other TMCC family proteins through its coiled-coil domain

  • Protocol: Transfect cells with tagged TMCC1 constructs (e.g., FLAG-TMCC1 and GFP-TMCC1), prepare cell lysates, immunoprecipitate with anti-FLAG antibody, and detect interaction partners by Western blotting

• Domain-specific interaction studies:

  • The large coiled-coil domain (approximately residues 460-575) is critical for TMCC1 protein-protein interactions

  • Test different TMCC1 fragments to map interaction domains (e.g., fragments containing residues 460-575, 310-575, and 1-575 can pull down full-length TMCC1)

• Cross-family interactions:

  • TMCC1 can interact with other TMCC family members (TMCC2, TMCC3)

  • GFP-tagged TMCC2 and TMCC3 can co-immunoprecipitate with endogenous TMCC1

• Controls:

  • Use fragments lacking the coiled-coil domain (e.g., TMCC1(571-653)) as negative controls for interaction studies

  • Include IgG controls for immunoprecipitation specificity

These approaches can help elucidate TMCC1's functional role in cellular processes through its protein interaction network.

What approaches can be used to investigate TMCC1's subcellular localization and membrane topology?

To investigate TMCC1's subcellular localization and membrane topology:

• Immunofluorescence co-localization:

  • Use TMCC1 antibodies alongside markers for subcellular compartments

  • TMCC1 co-localizes with Sec61α (rough ER marker) and calnexin (ER marker)

  • Perform saponin extraction before fixing with methanol to enhance specificity of labeling for rough ER markers

• Domain-specific localization studies:

  • The C-terminal transmembrane domains (aa 571-653) are sufficient for ER targeting

  • TMCC1 lacking transmembrane domains (aa 1-575) localizes diffusely in the cytosol

• Membrane topology analysis:

  • Selective permeabilization: Use digitonin to selectively permeabilize the plasma membrane while leaving the ER membrane intact

  • GFP-tagged TMCC1 constructs with tags at either N- or C-terminus can reveal the orientation of these regions relative to the ER membrane

  • Both N-terminal region and C-terminal tail of TMCC1 reside in the cytoplasm

• Protease protection assays:

  • Permeabilize cells with digitonin and treat with trypsin

  • TMCC1 is digested by trypsin (unlike lysosomal proteins protected by organelle membranes), confirming its cytoplasmic exposure

• Subcellular fractionation:

  • TMCC1 distributes similarly to rough ER proteins CLIMP-63 and RPL4 in sucrose gradient fractionation

These approaches together establish TMCC1 as a rough ER protein with both N-terminal region and C-terminal tail in the cytoplasm, providing insights into its potential functions.

How can TMCC1 antibodies be applied to investigate the protein's role in pathological conditions?

TMCC1 antibodies can be utilized to investigate the protein's role in various pathological conditions:

• Neurodegenerative disorders:

  • Given that circular RNA Tmcc1 improves astrocytic glutamate metabolism and spatial memory via NF-κB and CREB signaling, TMCC1 antibodies can be used to examine protein expression levels in models of neurodegenerative diseases

  • Immunohistochemistry or Western blotting of brain tissues from disease models (e.g., hepatic encephalopathy models like bile duct ligation) can reveal changes in TMCC1 expression

• Congenital disorders:

  • Since the TMCC1 locus has been implicated in hereditary congenital facial palsy, antibodies can be used to examine TMCC1 expression in relevant tissues from affected individuals or animal models

  • Compare TMCC1 expression patterns between affected and unaffected tissues using immunohistochemistry

• Cancer research:

  • Given TMCC1's differential expression across cell types (high in epithelial, neuroblastoma, and glioblastoma cells; low in leukemia and lymphoma cells), investigate its expression in tumor versus normal tissues

  • Perform tissue microarray analysis with TMCC1 antibodies to correlate expression levels with clinical outcomes

• Mechanistic studies:

  • Investigate TMCC1's association with the NF-κB p65-CREB transcriptional complex in disease models

  • Examine how TMCC1 regulates the expression of astrocyte transporter EAAT2 and affects glutamate metabolism in neurological disorders

What are the key controls required when using TMCC1 antibodies for quantitative analyses?

For rigorous quantitative analyses using TMCC1 antibodies:

• Antibody specificity controls:

  • Positive control: Include cell lines with known high TMCC1 expression (e.g., HeLa, HepG2, SH-SY5Y, or U87 cells)

  • Negative control: Use TMCC1 knockdown samples (siRNA or shRNA) to confirm antibody specificity. THP-1 cells expressing TMCC1-specific shRNA have been successfully used as negative controls

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

  • Secondary antibody only: Omit primary antibody to check for non-specific binding of secondary antibody

• Loading controls for Western blots:

  • Use housekeeping proteins (e.g., GAPDH, β-actin, IQGAP) to normalize TMCC1 signals

  • For subcellular fractions, use compartment-specific markers (e.g., BAP31 for ER, CLIMP-63 for rough ER)

• Normalization for immunofluorescence:

  • Co-stain with organelle markers (e.g., calnexin, Sec61α for ER)

  • Include nuclear staining (DAPI) for cell counting

  • Use standardized exposure settings across samples

• Concentration standards:

  • For quantitative comparisons, include a concentration gradient of recombinant TMCC1 protein (if available)

  • Create a standard curve for densitometric analyses

• Technical replicates: Perform at least three technical replicates for statistical robustness

• Biological replicates: Use multiple independent samples or cell preparations to account for biological variability

How can researchers design experiments to investigate TMCC1's functional roles using antibody-based approaches?

To investigate TMCC1's functional roles using antibody-based approaches:

• Protein dynamics studies:

  • Use TMCC1 antibodies to monitor protein levels after cellular stress (ER stress, calcium dysregulation)

  • Examine TMCC1 expression in time-course experiments following treatments that affect ER function

  • Combine with phospho-specific antibodies (if available) to monitor post-translational modifications

• Interaction network analysis:

  • Perform co-immunoprecipitation with TMCC1 antibodies followed by mass spectrometry to identify novel interaction partners

  • Use proximity ligation assays to confirm direct interactions in situ

  • Investigate co-localization with proteins involved in ER organization and calcium homeostasis

• Loss-of-function studies:

  • Combine TMCC1 knockdown or knockout with antibody staining of potential downstream targets

  • Monitor changes in ER morphology and organization using antibodies against ER structural proteins

  • Examine effects on calcium signaling pathways using phospho-specific antibodies

• Gain-of-function studies:

  • Overexpress wild-type or mutant TMCC1 and analyze effects on protein localization and interactions

  • Use domain-specific antibodies (if available) to track the localization of specific TMCC1 regions

• Disease model investigations:

  • Compare TMCC1 expression and localization in normal versus disease models (e.g., neurodegeneration models)

  • Investigate TMCC1's role in astrocytic glutamate metabolism by examining its relationship with the NF-κB p65-CREB transcriptional complex and EAAT2 expression

How can researchers resolve contradictory results when using different TMCC1 antibodies?

When faced with contradictory results using different TMCC1 antibodies:

• Epitope mapping analysis:

  • Compare the immunogens used to generate each antibody (e.g., N-terminal regions like aa 1-100 versus aa 51-300)

  • Different epitopes may be differentially accessible depending on protein conformation or interactions

  • Some epitopes may be masked by protein-protein interactions or post-translational modifications

• Validation with multiple methods:

  • Use orthogonal techniques to confirm results (e.g., mass spectrometry, RNA expression analysis)

  • Combine antibody detection with genetic approaches (siRNA/shRNA knockdown, CRISPR knockout)

  • If possible, validate with an antibody against a different epitope of TMCC1

• Isoform-specific detection:

  • Check if antibodies recognize different TMCC1 isoforms or related family members (TMCC2, TMCC3)

  • Review the literature for reported TMCC1 splice variants or post-translational modifications

  • Design experiments to confirm isoform specificity (e.g., isoform-specific knockdown)

• Technical considerations:

  • Optimize each antibody independently (dilution, incubation conditions, buffers)

  • Test different sample preparation methods (lysis buffers, fixation protocols)

  • Evaluate antibody performance in different applications (WB vs. IHC vs. IF)

• Control experiments:

  • Test antibodies on overexpressed tagged TMCC1 to confirm detection

  • Use TMCC1 knockout or knockdown samples as negative controls

  • Include cross-reactivity tests with related proteins

How might TMCC1 antibodies be utilized in investigating novel mechanisms in ER calcium regulation?

Given the potential role of TMCC1 in ER calcium regulation, researchers can utilize TMCC1 antibodies to:

• Investigate TMCC1's relationship with calcium channels:

  • Perform co-immunoprecipitation experiments with TMCC1 antibodies to identify interactions with known ER calcium channels or regulators

  • Use proximity ligation assays to detect in situ interactions between TMCC1 and calcium-handling proteins

  • Analyze co-localization of TMCC1 with calcium sensors under normal and stressed conditions

• Examine TMCC1's role in calcium homeostasis:

  • Monitor TMCC1 expression and localization changes during calcium perturbation experiments

  • Combine calcium imaging techniques with TMCC1 immunofluorescence to correlate localization with functional calcium dynamics

  • Investigate if TMCC1 forms or regulates calcium-selective channels in the ER, similar to what's been reported for TMCO1

• Explore potential structural changes:

  • Use conformation-specific antibodies (if available) to detect structural changes in TMCC1 during calcium fluxes

  • Investigate whether TMCC1 undergoes oligomerization in response to ER calcium overloading, similar to TMCO1's assembly into homotetramers

• Examine potential post-translational modifications:

  • Use phospho-specific antibodies to monitor calcium-dependent modifications of TMCC1

  • Investigate if calcium-dependent enzymes (like CaMKII) interact with or modify TMCC1

What emerging techniques could enhance the utility of TMCC1 antibodies in understanding ER organization?

Emerging techniques that could enhance TMCC1 antibody applications include:

• Super-resolution microscopy:

  • Apply techniques like STORM, PALM, or STED microscopy with TMCC1 antibodies to visualize nanoscale organization of TMCC1 within the ER

  • Investigate TMCC1's spatial relationship with other ER structural proteins at nanometer resolution

  • Examine potential TMCC1 clustering or organization into functional domains

• Live-cell imaging with intrabodies:

  • Develop cell-permeable TMCC1 antibody fragments for live-cell imaging

  • Monitor dynamic changes in TMCC1 localization during cellular processes

  • Combine with fluorescent ER markers to track ER reorganization events

• Expansion microscopy:

  • Apply physical expansion of cellular structures to visualize TMCC1 organization at enhanced resolution with standard microscopes

  • Investigate the spatial relationship between TMCC1 and ribosomal proteins at the rough ER

• Proximity labeling approaches:

  • Use TMCC1 antibodies to validate results from proximity labeling experiments (BioID, APEX)

  • Confirm protein interaction networks identified through proximity labeling

• Correlative light and electron microscopy (CLEM):

  • Use TMCC1 antibodies for immunofluorescence followed by electron microscopy to correlate protein localization with ultrastructural features

  • Investigate TMCC1's precise localization relative to membrane contact sites and other ER subdomains

How can TMCC1 antibodies contribute to understanding the protein's role in neurodegenerative diseases?

TMCC1 antibodies can significantly contribute to understanding the protein's role in neurodegenerative diseases:

• Expression profiling in disease models:

  • Use TMCC1 antibodies for immunohistochemistry and Western blotting to compare expression levels in brain tissues from neurodegenerative disease models versus controls

  • Investigate cell-type specific expression changes in astrocytes versus neurons using co-labeling with cell-type markers

  • Perform quantitative analysis of TMCC1 expression in different brain regions affected by specific neurodegenerative conditions

• Mechanistic studies in astrocyte function:

  • Investigate TMCC1's association with the NF-κB p65-CREB transcriptional complex in astrocytes from disease models

  • Examine how TMCC1 regulates astrocyte transporter EAAT2 expression, which is critical for glutamate clearance and preventing excitotoxicity

  • Study how TMCC1 contributes to astrocytic secretion of proinflammatory mediators in neuroinflammatory conditions

• Therapeutic target validation:

  • Use TMCC1 antibodies to monitor protein levels following experimental therapeutic interventions

  • Validate TMCC1 as a potential biomarker for disease progression or treatment response

  • Investigate if circTmcc1-based interventions improve spatial memory and cognitive functions in models of hepatic encephalopathy

• Interaction with disease-associated proteins:

  • Examine potential interactions between TMCC1 and proteins implicated in specific neurodegenerative diseases

  • Investigate whether TMCC1 co-localizes with protein aggregates characteristic of neurodegenerative conditions

  • Study if TMCC1's role in ER organization is compromised in conditions with ER stress

These approaches can help establish TMCC1 as a promising target for interventions aimed at preventing and treating neuropathophysiological complications in conditions like hepatic encephalopathy and potentially other neurodegenerative disorders .