TMEM101 Antibody

What exactly is TMEM101 and what cellular functions has it been associated with?

TMEM101 (Transmembrane Protein 101) is a multi-pass membrane protein encoded by the TMEM101 gene located on the long arm of human chromosome 17 at locus 17q21.31 . The protein is approximately 28.8 kDa in molecular weight and has been implicated in the activation of NF-κB signaling pathways . Notably, high levels of TMEM101 expression have been linked to breast cancer, suggesting potential roles in cancer biology .

The gene spans approximately 12,758 bp, ranging from position 44,011,188 to 44,023,946 on chromosome 17, and is situated between the NAGS and LSM12 genes . There are five known mRNA transcript variants for TMEM101, including three experimentally confirmed variants (1, 2, and 3) and two computationally predicted variants (X1 and X2) . These variants translate into at least two distinct protein isoforms.

TMEM101 antibodies have been validated for several experimental applications:

• Western Blot (WB): Most commercial TMEM101 antibodies are validated for Western blot applications, typically at dilutions ranging from 1:500 to 1:3000 .

• Enzyme-Linked Immunosorbent Assay (ELISA): Many antibodies are validated for ELISA applications, usually at higher dilutions (approximately 1:10000) .

• Immunofluorescence (IF): Some antibodies, particularly the Prestige Antibodies, are validated for immunofluorescence applications with recommended concentrations of 0.25-2 μg/mL .

• Immunoprecipitation (IP): Certain antibodies have been characterized for immunoprecipitation applications, which can be useful for studying protein-protein interactions involving TMEM101 .

The experimental validation typically involves comparing signals between wild-type samples and knockout controls to confirm specificity, as demonstrated in comprehensive validation studies of transmembrane protein antibodies .

How should researchers validate TMEM101 antibodies before use in critical experiments?

Proper validation of TMEM101 antibodies is essential before use in critical experiments. Based on comprehensive antibody characterization studies, the following validation approach is recommended:

• Knockout/Knockdown Controls: Compare antibody reactivity between wild-type cells and TMEM101 knockout or knockdown cells. This is considered the gold standard for antibody validation. HAP1 cell lines (both parental and TMEM101 KO) have been successfully used for this purpose .

• Multiple Antibody Approach: Use at least two different antibodies targeting distinct epitopes of TMEM101 to confirm findings.

• Western Blot Validation: Run TMEM101 antibodies on Western blots alongside positive controls (e.g., recombinant TMEM101 or cells known to express TMEM101) and negative controls (e.g., knockout cells). The expected molecular weight is approximately 28.8 kDa .

• Peptide Competition Assay: Pre-incubate the antibody with the immunizing peptide to demonstrate that binding is specific to the target epitope.

• Cross-Reactivity Testing: For applications requiring high specificity, test against protein arrays or panels of similar transmembrane proteins to assess potential cross-reactivity .

• Mosaic Cell Strategy: For immunofluorescence validation, co-culture wild-type and knockout cells labeled with different fluorescent trackers to enable side-by-side comparison of antibody performance in a single field of view .

What are the optimal conditions for using TMEM101 antibodies in Western blot applications?

For optimal Western blot results with TMEM101 antibodies, the following conditions are recommended:

Sample Preparation:

• Use gradient polyacrylamide gels (e.g., 4-15%) for optimal separation

• Transfer proteins to nitrocellulose membranes

• Visualize total protein with Ponceau staining as a loading control

Blocking and Antibody Incubation:

• Block membranes with 5% milk in TBST for 1 hour at room temperature

• Dilute primary antibodies in 5% BSA in TBST at the recommended dilution (typically 1:500-1:3000)

• Incubate with primary antibody overnight at 4°C

• Wash three times with TBST

• Incubate with appropriate secondary antibody (typically at ~0.2 μg/mL) in 5% milk in TBST for 1 hour at room temperature

• Wash three times with TBST

Detection:

• Use ECL substrate for detection

• Visualize using autoradiography films or digital imaging systems

Expected Results:

• TMEM101 typically appears at approximately 28.8 kDa

• Multiple bands may be observed due to different isoforms or post-translational modifications

How can researchers troubleshoot non-specific binding with TMEM101 antibodies?

Non-specific binding is a common challenge when working with antibodies against transmembrane proteins like TMEM101. The following troubleshooting approaches are recommended:

• Optimize Antibody Concentration: Titrate the antibody to determine the optimal concentration that maximizes specific signal while minimizing background.

• Modify Blocking Conditions: Test different blocking agents (BSA, milk, commercial blockers) and concentrations to reduce non-specific binding.

• Increase Washing Stringency: Incorporate additional washing steps or increase the concentration of detergent (Tween-20) in wash buffers.

• Pre-adsorption: Pre-incubate the antibody with proteins from non-target species to reduce cross-reactivity.

• Use Knockout Controls: Always include a TMEM101 knockout or knockdown sample as a negative control to distinguish between specific and non-specific signals .

• Change Membrane Type: If using PVDF membranes, consider switching to nitrocellulose, which may provide lower background for some antibodies.

• Optimize Antigen Retrieval: For immunohistochemistry or immunofluorescence applications, test different antigen retrieval methods.

• Fresh Antibody Aliquots: Avoid repeated freeze-thaw cycles of antibodies by preparing single-use aliquots and storing them at -20°C .

How can researchers investigate TMEM101's potential role in NF-κB signaling pathways?

To investigate TMEM101's involvement in NF-κB signaling pathways, researchers can employ several experimental approaches:

• TMEM101 Overexpression and Knockdown Studies:

  • Transfect cells with TMEM101 overexpression constructs to determine if increased TMEM101 levels enhance NF-κB pathway activation

  • Use siRNA or CRISPR-Cas9 to knock down or knock out TMEM101 and assess effects on NF-κB signaling

  • Employ commercially available TMEM101 overexpression lysates as positive controls

• NF-κB Reporter Assays:

  • Utilize luciferase-based NF-κB reporter systems to quantitatively measure pathway activation in the presence or absence of TMEM101

  • Compare NF-κB activity in response to classic activators (e.g., TNF-α, IL-1β) between wild-type and TMEM101-modified cells

• Co-immunoprecipitation Studies:

  • Use validated TMEM101 antibodies for immunoprecipitation to identify protein-protein interactions with NF-κB pathway components

  • Perform reverse co-IP experiments using antibodies against key NF-κB pathway proteins to confirm interactions

• Subcellular Localization Studies:

  • Conduct immunofluorescence experiments to track TMEM101 localization during NF-κB activation

  • Perform subcellular fractionation followed by Western blotting to determine if TMEM101 redistributes within cellular compartments during signaling

• Phosphorylation Analysis:

  • Examine the phosphorylation status of key NF-κB pathway components (e.g., IκB, p65) in the presence or absence of TMEM101

  • Use phospho-specific antibodies in Western blotting to track signaling events

What methods are most effective for studying TMEM101 subcellular localization?

Several complementary techniques can be employed to effectively study TMEM101 subcellular localization:

• Immunofluorescence Microscopy:

  • Use validated TMEM101 antibodies at appropriate dilutions (0.25-2 μg/mL for ICC-IF)

  • Co-stain with established organelle markers (e.g., Golgi, ER, plasma membrane markers)

  • For highest confidence, employ the mosaic cell strategy with labeled wild-type and knockout cells in the same field of view

  • Use fixation with 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100

• Subcellular Fractionation and Western Blotting:

  • Separate cellular compartments (membrane, cytosolic, nuclear, etc.) using fractionation protocols

  • Analyze TMEM101 distribution across fractions using Western blotting with validated antibodies

  • Include compartment-specific marker proteins as controls for fractionation quality

• Live Cell Imaging with Fluorescent Fusion Proteins:

  • Generate TMEM101-GFP (or other fluorescent tag) fusion constructs

  • Validate localization with antibody staining of endogenous protein

  • Perform live cell imaging to track dynamic localization

• Electron Microscopy:

  • Use immunogold labeling with TMEM101 antibodies for high-resolution localization

  • This approach can definitively resolve membrane association and topology

• Protease Protection Assays:

  • Determine the topology of TMEM101 within membranes using selective membrane permeabilization and protease digestion

  • Analyze protected fragments using antibodies targeting different regions of TMEM101

How should researchers approach studying different TMEM101 isoforms?

The study of different TMEM101 isoforms requires careful experimental design:

• Isoform Identification Strategy:

  • The human TMEM101 gene has multiple transcript variants encoding at least two distinct protein isoforms

  • Design PCR primers spanning exon junctions to specifically amplify different transcript variants

  • Use isoform-specific antibodies when available, or antibodies targeting common regions when comparing expression levels

• Western Blot Analysis:

  • Use high-resolution gel systems (e.g., gradient gels) to separate closely sized isoforms

  • The main isoforms have predicted molecular weights around 28.8 kDa

  • Include positive controls expressing specific isoforms to aid in identification

• Expression Vector Construction:

  • Clone individual isoform cDNAs into expression vectors

  • Validate constructs by sequencing before transfection

  • Consider using epitope tags (e.g., FLAG, Myc) to distinguish between isoforms

• Functional Characterization:

  • Compare the functional properties of different isoforms in overexpression studies

  • Assess subcellular localization differences between isoforms

  • Investigate isoform-specific protein-protein interactions

• Tissue Distribution Analysis:

  • Examine the expression pattern of different isoforms across tissues and cell types

  • Use RT-PCR with isoform-specific primers to identify transcript expression

  • Correlate protein expression with transcript presence using validated antibodies

How should researchers interpret multiple bands in Western blots with TMEM101 antibodies?

Multiple bands on Western blots with TMEM101 antibodies require careful interpretation:

• Expected Banding Pattern:

  • The primary TMEM101 band should appear at approximately 28.8 kDa

  • Multiple bands may represent:

    • Different isoforms (human TMEM101 has at least two confirmed isoforms)

    • Post-translational modifications (glycosylation, phosphorylation)

    • Proteolytic processing

    • Oligomeric forms (if sample preparation does not fully denature the protein)

• Validation Approach:

  • Compare with knockout or knockdown samples to identify which bands are specific to TMEM101

  • Use peptide competition assays to determine which bands represent specific binding

  • Compare banding patterns across multiple antibodies targeting different epitopes

  • Examine the molecular weight of observed bands in relation to predicted weights of known isoforms

• Technical Considerations:

  • Ensure complete denaturation of samples (increase SDS concentration or boiling time)

  • Test reducing vs. non-reducing conditions

  • Evaluate fresh vs. frozen samples to rule out degradation artifacts

  • Consider sample preparation modifications if membrane proteins are incompletely solubilized

• Potential Physiological Significance:

  • Consistent additional bands may represent biologically relevant forms

  • Document changes in band patterns across experimental conditions

  • Consider post-translational modification-specific antibodies to confirm the nature of higher molecular weight bands

What approaches can be used to quantify TMEM101 expression levels across different experimental conditions?

Several approaches can be used to quantify TMEM101 expression levels:

• Western Blot Quantification:

  • Use appropriate loading controls (housekeeping proteins or total protein staining)

  • Employ digital imaging systems with a linear detection range

  • Normalize TMEM101 band intensity to loading controls

  • Include a standard curve of recombinant TMEM101 or cell lysates with known expression for absolute quantification

  • For multiple bands, consider quantifying individual bands separately and as a sum

• ELISA-Based Quantification:

  • Utilize validated TMEM101 antibodies in sandwich ELISA format

  • Develop standard curves using recombinant TMEM101

  • This approach is particularly useful for serum or other fluid samples

• qRT-PCR for Transcript Quantification:

  • Design primers specific to TMEM101 transcripts

  • Use reference genes for normalization

  • Correlate mRNA levels with protein expression where possible

• Flow Cytometry:

  • For cell surface-exposed epitopes of TMEM101

  • Use fluorophore-conjugated antibodies or primary-secondary antibody combinations

  • Generate quantitative data on a per-cell basis

• Immunofluorescence Quantification:

  • Use consistent image acquisition settings

  • Measure fluorescence intensity in defined cellular regions

  • Include wild-type and knockout controls in the same field using the mosaic approach

  • Apply appropriate background subtraction

• Mass Spectrometry-Based Approaches:

  • Targeted MS methods can provide absolute quantification of TMEM101

  • Use stable isotope-labeled peptide standards

  • This approach is particularly valuable for distinguishing isoforms

How can researchers determine the specificity of observed TMEM101 antibody signals in tissue samples?

Determining antibody specificity in tissue samples is critical for reliable data interpretation:

• Essential Controls:

  • Positive Control: Include tissues known to express TMEM101

  • Negative Control: Use TMEM101-knockout tissues when available or tissues known not to express TMEM101

  • Secondary Antibody Control: Omit primary antibody to assess secondary antibody background

  • Peptide Competition: Pre-incubate antibody with immunizing peptide to demonstrate specific binding

• Validation Strategies:

  • Compare staining patterns across multiple antibodies targeting different TMEM101 epitopes

  • Correlate protein detection with mRNA expression data from the same tissues

  • For human samples, reference the Human Protein Atlas data for expected TMEM101 distribution patterns

• Technical Considerations:

  • Optimize fixation and antigen retrieval methods for tissue type

  • Test a range of antibody dilutions to identify optimal signal-to-noise ratio

  • Use automated staining platforms when available to ensure consistency

  • Include internal control tissues in the same section when possible

• Advanced Validation Approaches:

  • In situ hybridization for TMEM101 mRNA to correlate with antibody staining

  • Proximity ligation assays to confirm interaction with known binding partners

  • Single-cell analysis to correlate with expected cell-type specific expression patterns

• Multi-modal Confirmation:

  • Validate key findings using orthogonal methods (e.g., Western blot of tissue lysates)

  • Consider laser capture microdissection followed by Western blotting or PCR

  • For translational research, correlate findings with public database expression data

What emerging roles for TMEM101 have been identified in recent research?

While TMEM101 remains relatively understudied, recent findings have begun to illuminate its potential biological roles:

• NF-κB Signaling: TMEM101 has been identified as a putative NF-κB-activating protein , suggesting roles in inflammatory responses and immune regulation.

• Cancer Biology: High expression levels of TMEM101 have been linked to breast cancer , indicating potential involvement in cancer development or progression.

• Membrane Protein Biology: As a multi-pass transmembrane protein, TMEM101 may participate in membrane organization, trafficking, or signal transduction processes.

Future research directions will likely focus on characterizing the precise mechanisms by which TMEM101 influences these biological processes, potentially identifying it as a novel therapeutic target.

What technological advances are improving TMEM101 antibody development and validation?

Recent technological advances are enhancing TMEM101 antibody development and validation:

• Enhanced Validation Approaches:

  • Systematic use of knockout cell lines as gold-standard controls

  • Implementation of the mosaic cell strategy for immunofluorescence validation

  • Comprehensive antibody characterization across multiple applications (WB, IP, IF)

• Standardized Testing Protocols:

  • Development of standardized validation protocols that compare antibody performance in isogenic cell lines

  • Multi-parameter testing using identical experimental conditions for direct comparison

• Advanced Production Methods:

  • Improved recombinant antibody production with consistent quality

  • Development of antibodies against specific post-translational modifications

  • Generation of isoform-specific antibodies

• Bioinformatic Approaches:

  • Enhanced epitope selection using structural prediction algorithms

  • Cross-reactivity prediction to minimize off-target binding

  • Proteome-wide validation using protein arrays

These advances are expected to continue improving the specificity, sensitivity, and reproducibility of TMEM101 antibodies for research applications.