TMEM185A Antibody

What is TMEM185A and what are the available antibody options for its detection?

TMEM185A (Transmembrane protein 185A) is a human protein also known by alternative names including CXorf13, FAM11A, and FRAXF. It is encoded by a gene located on the X chromosome. Currently, several polyclonal antibodies against TMEM185A are commercially available, primarily raised in rabbits . These antibodies target different epitopes of the protein, with some targeting C-terminal regions (amino acids 290-339) and others targeting N-terminal regions . The antibodies are typically available in liquid form in PBS containing glycerol, BSA, and sodium azide .

What applications are validated for TMEM185A antibodies?

TMEM185A antibodies have been validated for multiple research applications with specific recommended dilutions:

These applications have been validated using specific positive controls such as HT-29 cells, K562 cells, and MCF7 cells . For optimal results, researchers should optimize dilutions based on their specific experimental conditions.

What is the molecular weight of TMEM185A and why might it differ in experimental results?

• Post-translational modifications (PTMs) such as glycosylation, phosphorylation, or ubiquitination

• Formation of protein complexes that resist complete denaturation

• Anomalous migration due to the hydrophobic nature of transmembrane proteins

• Splice variants of the protein

When troubleshooting unexpected band sizes, researchers should consider using appropriate positive controls and peptide blocking experiments to confirm antibody specificity .

How should TMEM185A antibodies be stored and handled to maintain optimal activity?

For optimal storage and handling of TMEM185A antibodies:

• Store at -20°C for long-term preservation (up to one year)

• For frequent use and short-term storage (up to one month), store at 4°C

• Avoid repeated freeze-thaw cycles as they can degrade antibody performance

• When aliquoting, use sterile tubes and minimize exposure to room temperature

• For working solutions, dilute in appropriate buffer immediately before use

• Most commercial TMEM185A antibodies are provided in a stabilizing solution containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide , which helps maintain activity

Proper storage and handling are critical as degraded antibodies can lead to weak signals, high background, or non-specific binding.

What controls should be included when validating TMEM185A antibody specificity?

For rigorous validation of TMEM185A antibody specificity, include the following controls:

• Positive control tissues/cells: Use samples known to express TMEM185A, such as K562 cells, HT-29 cells, or MCF7 cells

• Peptide blocking control: Pre-incubate the antibody with the immunizing peptide before application to demonstrate that binding is specific to the target epitope . Images showing antibody reactivity with and without blocking peptide are essential for validation

• Negative control tissues/cells: Use samples with low or no expression of TMEM185A

• Secondary antibody-only control: Omit primary antibody to assess non-specific binding of the secondary antibody

• Isotype control: Use matching concentration of non-specific IgG from the same host species (rabbit IgG for TMEM185A antibodies)

These controls should be run in parallel with experimental samples under identical conditions to accurately assess antibody specificity.

What are the recommended sample preparation protocols for detecting TMEM185A in different applications?

Sample preparation varies by application:

For Western Blot:

• Lyse cells in RIPA buffer containing protease inhibitors

• Determine protein concentration (Bradford or BCA assay)

• Load 20-50 μg of total protein per lane

• Run SDS-PAGE (10-12% gel recommended)

• Transfer to PVDF or nitrocellulose membrane

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

• Incubate with primary antibody (1:500-1:2000 dilution)

• Visualize with appropriate secondary antibody and detection system

For Immunohistochemistry:

• Fix tissues in 10% neutral buffered formalin

• Embed in paraffin and section (4-6 μm thickness)

• Deparaffinize and rehydrate sections

• Perform antigen retrieval using sodium citrate buffer (pH 6.0) at >98°C for 20 minutes

• Block endogenous peroxidase and non-specific binding

• Incubate with primary antibody (1:100-1:300 dilution)

• Apply appropriate detection system (e.g., HRP-conjugated secondary antibody)

For Immunofluorescence:

• Culture cells on coverslips or chamber slides

• Fix with 4% paraformaldehyde (10-15 minutes)

• Permeabilize with 0.1-0.5% Triton X-100 (10 minutes)

• Block with 5% normal serum

• Incubate with primary antibody (1:200-1:1000 dilution)

• Apply fluorophore-conjugated secondary antibody

• Counterstain nuclei (e.g., DAPI) and mount

How can researchers troubleshoot discrepancies between expected and observed TMEM185A expression patterns?

When troubleshooting unexpected TMEM185A expression patterns:

• Verify antibody specificity:

  • Perform peptide blocking experiments

  • Test multiple antibodies targeting different epitopes of TMEM185A

  • Compare results with mRNA expression data (RT-PCR or public databases)

• Optimize experimental conditions:

  • Adjust antibody concentration and incubation time

  • Modify antigen retrieval methods (for IHC/ICC)

  • Try different blocking reagents to reduce background

  • Adjust lysis conditions for more complete protein extraction

• Consider biological variables:

  • Cell/tissue type differences in expression

  • Effects of cell cycle or differentiation state

  • Impact of treatment conditions on protein expression or localization

  • Possibility of splice variants or post-translational modifications

• Technical considerations:

  • For Western blot, use gradient gels to better resolve proteins

  • For IHC/IF, optimize fixation time and conditions

  • Consider detection method sensitivity limitations

Documenting all troubleshooting steps systematically will help identify the source of discrepancies.

What approaches can be used to detect low-abundance TMEM185A in samples?

For detecting low-abundance TMEM185A:

• Enrichment strategies:

  • Immunoprecipitation using TMEM185A antibodies prior to Western blot

  • Subcellular fractionation to concentrate membrane proteins

  • Cell sorting to isolate specific cell populations with higher expression

• Signal amplification methods:

  • For IHC: Use tyramide signal amplification (TSA) system

  • For Western blot: Use high-sensitivity ECL substrates

  • For IF: Use quantum dots or multi-layer detection systems

• Optimization of experimental parameters:

  • Increase primary antibody concentration (1:100-1:200 for WB)

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

  • Increase protein loading (up to 80-100 μg for Western blot)

  • Use PVDF membranes with higher protein binding capacity

• Alternative detection methods:

  • Consider using ELISA with a sensitivity range of 0.156-10 ng/ml

  • Explore proximity ligation assay (PLA) for increased sensitivity

  • Use mass spectrometry-based approaches for targeted protein detection

When implementing these approaches, always include appropriate controls to confirm specificity of the detected signal.

How can researchers evaluate potential cross-reactivity of TMEM185A antibodies with other TMEM family proteins?

To evaluate potential cross-reactivity:

• In silico analysis:

  • Perform sequence alignment of the immunizing peptide against other TMEM family proteins

  • Identify regions of high homology that might lead to cross-reactivity

  • Check epitope uniqueness using tools like BLAST or protein domain databases

• Experimental validation:

  • Test antibody reactivity in cell lines with known expression profiles of different TMEM proteins

  • Perform siRNA knockdown of TMEM185A to confirm signal reduction

  • Overexpress TMEM185A and related family members to assess specificity

  • Use knockout/knockdown validation for definitive confirmation

• Competition assays:

  • Pre-absorb antibody with recombinant proteins of related TMEM family members

  • Compare signal patterns before and after pre-absorption

• Orthogonal method confirmation:

  • Compare protein detection results with mRNA expression data

  • Use multiple antibodies targeting different epitopes of TMEM185A

  • Confirm findings with mass spectrometry-based proteomic approaches

Documented cross-reactivity analysis should be included in research publications to support antibody validation.

What are the considerations for using TMEM185A antibodies in multi-color immunofluorescence experiments?

For successful multi-color immunofluorescence with TMEM185A antibodies:

• Antibody compatibility planning:

  • Select primary antibodies from different host species to avoid cross-reactivity

  • If using multiple rabbit antibodies (common for TMEM185A), consider sequential staining with direct conjugation or Fab fragment blocking between rounds

  • Test each antibody individually before combining to establish optimal dilutions

• Spectral considerations:

  • Choose fluorophores with minimal spectral overlap

  • Include single-color controls for spectral unmixing

  • Consider photobleaching properties when planning imaging sequence

• Technical optimizations:

  • Adjust fixation protocols to preserve epitopes for all target proteins

  • Optimize blocking to minimize background across all channels

  • Determine the optimal order of antibody application (typically start with the weakest signal)

• Imaging parameters:

  • Use appropriate filter sets to minimize bleed-through

  • Acquire control images to set threshold levels

  • Consider confocal microscopy for better resolution of co-localization

• Controls specific to multi-color experiments:

  • Include fluorescence-minus-one (FMO) controls

  • Test secondary antibody cross-reactivity

  • Use co-localization standards to validate analysis methods

These considerations help ensure reliable co-localization analysis with TMEM185A and other proteins of interest.

How can researchers address issues with high background when using TMEM185A antibodies?

To reduce high background with TMEM185A antibodies:

• Antibody optimization:

  • Further dilute primary antibody (try 2-5× more dilute than recommended)

  • Reduce secondary antibody concentration

  • Shorten incubation times for both primary and secondary antibodies

  • Ensure antibodies are properly stored to prevent degradation

• Blocking improvements:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Extend blocking time (1-2 hours at room temperature or overnight at 4°C)

  • Add 0.1-0.3% Triton X-100 to blocking buffer for better penetration

  • Include blocking steps for endogenous biotin or peroxidase activity

• Washing modifications:

  • Increase number and duration of washes

  • Use higher salt concentration in wash buffers (up to 500 mM NaCl)

  • Add 0.05-0.1% Tween-20 to wash buffers

  • Consider using automated washers for consistent results

• Sample-specific considerations:

  • For tissues with high autofluorescence, treat with Sudan Black B or use spectral unmixing

  • For Western blot, ensure complete transfer and use fresh blocking reagents

  • For IHC, optimize antigen retrieval and consider using amplification-free detection systems

Systematic optimization of these parameters can significantly improve signal-to-noise ratio.

What are the critical factors affecting reproducibility when using TMEM185A antibodies across different experiments?

Key factors for ensuring reproducibility include:

• Antibody variables:

  • Use antibodies from the same lot when possible

  • Document complete antibody information (catalog number, lot, dilution)

  • Prepare and store working dilutions consistently

  • Validate new lots against previous results before full implementation

• Sample preparation consistency:

  • Standardize collection, fixation, and processing protocols

  • Control for biological variables (cell density, passage number, treatments)

  • Document and maintain consistent lysis buffers and extraction protocols

  • Use the same protein quantification method across experiments

• Technical parameters:

  • Maintain consistent incubation times and temperatures

  • Use the same detection systems and imaging parameters

  • Control for day-to-day environmental variations

  • Calibrate equipment regularly

• Controls for normalization:

  • Include loading controls for Western blots

  • Use internal reference standards appropriate for each application

  • Apply quantitative analysis methods consistently

  • Perform replicate experiments (both technical and biological)

• Documentation and reporting:

  • Follow comprehensive reporting guidelines (e.g., ARRIVE for animal studies)

  • Document detailed experimental protocols with all parameters

  • Report statistical methods used for data analysis

  • Share raw data when possible to enable independent verification

Implementing these practices significantly enhances the reliability and reproducibility of TMEM185A research findings.

How should researchers interpret discrepancies between TMEM185A protein and mRNA expression levels?

When facing discrepancies between protein and mRNA levels:

• Biological explanations:

  • Post-transcriptional regulation (miRNA targeting, RNA stability)

  • Translational efficiency differences

  • Protein half-life and degradation rates

  • Post-translational modifications affecting antibody recognition

  • Alternative splicing resulting in protein isoforms not detected by the antibody

• Methodological considerations:

  • Different sensitivities of protein (Western blot/IHC) versus mRNA (qRT-PCR/RNA-seq) detection methods

  • Antibody specificity issues including potential cross-reactivity

  • Primer specificity for qRT-PCR

  • Sample preparation differences between protein and RNA extraction

• Technical approaches to resolve discrepancies:

  • Use multiple antibodies targeting different TMEM185A epitopes

  • Employ orthogonal protein detection methods (mass spectrometry)

  • Design primers to detect specific splice variants

  • Perform time-course experiments to account for temporal differences

  • Use polysome profiling to assess translational status

• Integrated analysis:

  • Correlate findings with functional assays

  • Consider cellular localization of protein versus total expression

  • Analyze protein-protein interactions that might mask epitopes

  • Apply statistical methods appropriate for integrating multi-omic data

These discrepancies often reveal important biological regulatory mechanisms rather than technical errors.

What are the considerations for quantitative analysis of TMEM185A expression in tissue microarrays or high-throughput screening?

For quantitative analysis in high-throughput contexts:

• Standardization requirements:

  • Use automated staining platforms for consistent antibody application

  • Include calibration standards on each array/plate

  • Process all samples in parallel under identical conditions

  • Apply consistent image acquisition parameters

• Controls for quantification:

  • Include positive and negative control tissues on each array

  • Use tissue cores with known TMEM185A expression levels as references

  • Include gradient standards for calibrating intensity measurements

  • Employ cell lines with defined TMEM185A expression as controls

• Image analysis considerations:

  • Apply automated segmentation algorithms to identify cellular compartments

  • Develop robust thresholding methods for positive signal detection

  • Implement batch correction for multi-slide experiments

  • Validate quantification algorithms against manual scoring by pathologists

• Statistical analysis approaches:

  • Account for tissue heterogeneity in sampling design

  • Apply appropriate normalization for cross-sample comparisons

  • Use statistical methods that address multiple hypothesis testing

  • Implement machine learning methods for pattern recognition in complex datasets

• Reporting standards:

  • Document complete methodological details including antibody information

  • Report quantification metrics with appropriate statistical measures

  • Include representative images showing the range of expression patterns

  • Share analysis code and raw data when possible

These considerations ensure reliable quantitative assessment of TMEM185A expression across large sample sets.

How can researchers appropriately interpret subcellular localization patterns of TMEM185A using immunofluorescence?

For accurate interpretation of TMEM185A subcellular localization:

• Co-localization studies:

  • Use established markers for cellular compartments (ER, Golgi, plasma membrane)

  • Apply quantitative co-localization analysis (Pearson's coefficient, Manders' overlap)

  • Perform super-resolution microscopy for precise localization

  • Consider 3D reconstruction for complete spatial understanding

• Technical considerations:

  • Optimize fixation methods to preserve membrane structures

  • Use membrane permeabilization protocols appropriate for transmembrane proteins

  • Apply confocal microscopy to minimize out-of-focus signal

  • Consider live-cell imaging with fluorescently tagged TMEM185A to avoid fixation artifacts

• Validation approaches:

  • Confirm patterns with multiple antibodies targeting different epitopes

  • Compare results with subcellular fractionation and Western blotting

  • Use CRISPR-tagged endogenous TMEM185A as a reference standard

  • Employ electron microscopy for ultrastructural localization

• Functional correlation:

  • Relate localization patterns to functional studies

  • Assess changes in localization under different experimental conditions

  • Consider dynamic trafficking between compartments

  • Investigate binding partners that might influence localization

• Common patterns and their interpretation:

  • Perinuclear staining may indicate ER or Golgi localization

  • Punctate cytoplasmic pattern might represent vesicular structures

  • Membrane localization should show distinct cell periphery staining

  • Changes in pattern with treatments may indicate regulated trafficking