TMEM240 Antibody, FITC conjugated

What is TMEM240 and why is it significant for research?

TMEM240 is a small transmembrane protein (19.9 kDa, 173 amino acids) encoded by the TMEM240 gene located on chromosome 1p36.33. It is highly conserved across species and primarily expressed in the brain, cerebellum, small intestine, duodenum, and colon . TMEM240's significance stems from:

• Its role in neurological function, with mutations linked to spinocerebellar ataxia 21 (SCA21)

• Emerging evidence of its potential tumor suppressor activity in colorectal and breast cancers

• Its localization to the cell membrane and synaptic membranes, suggesting involvement in cellular communication

Research involving TMEM240 is providing insights into both neurological disorders and cancer biology, making it an important target for investigation.

What are the key specifications of commercially available TMEM240 antibody with FITC conjugation?

TMEM240 antibody with FITC conjugation typically has the following specifications:

The antibody recognizes human TMEM240 and can be used for various applications including ELISA and immunofluorescence assays .

What are the optimal methods for using TMEM240 antibody (FITC) in flow cytometry experiments?

When using FITC-conjugated TMEM240 antibody in flow cytometry:

• Sample preparation:

  • Harvest cells (1×10^6 cells/100 μL) and wash twice with PBS containing 1% BSA

  • Fix cells if necessary using 4% paraformaldehyde (10 min at room temperature)

  • Permeabilize for intracellular staining if needed (0.1% Triton X-100 for 15 minutes)

• Antibody staining:

  • Determine optimal antibody concentration through titration experiments (typically starting at 1:100-1:500 dilution)

  • Incubate cells with antibody for 30-60 minutes at 4°C in the dark

  • Wash twice with PBS + 1% BSA to remove unbound antibody

• Multicolor panel design:

  • Position TMEM240-FITC appropriately in your panel to minimize spillover with other fluorophores

  • Be cautious about spectral overlap with PE-based dyes, as FITC emission can spill into the PE detector

  • Calculate compensation properly if multiplexing with other fluorophores

• Instrument settings:

  • Use 488 nm laser for excitation

  • Detect emission at ~515-545 nm

  • Include appropriate controls (unstained, isotype control, single-stained controls for compensation)

• Data analysis:

  • Gate properly to exclude debris and doublets

  • Define positive populations using appropriate controls

Remember that FITC is relatively susceptible to photobleaching, so minimize light exposure throughout the procedure .

How can TMEM240 antibody (FITC) be optimized for immunofluorescence microscopy of cancer tissues?

For optimal immunofluorescence microscopy with FITC-conjugated TMEM240 antibody in cancer tissues:

• Tissue preparation:

  • Use fresh frozen or formalin-fixed paraffin-embedded (FFPE) sections (4-6 μm thickness)

  • For FFPE: perform antigen retrieval (citrate buffer pH 6.0 at 95-98°C for 15-20 minutes)

  • Block with 5-10% normal serum from the same species as the secondary antibody for 1 hour

• Antibody incubation:

  • Determine optimal dilution (typically starting at 1:50-1:200) through titration

  • Incubate sections overnight at 4°C in a humidified chamber

  • Wash thoroughly with PBS containing 0.05% Tween-20

• Signal enhancement and counterstaining:

  • For weak signals, consider using a tyramide signal amplification system

  • Counterstain nuclei with DAPI (1 μg/mL for 5 minutes)

  • Mount using anti-fade mounting medium to preserve FITC fluorescence

• Controls and validation:

  • Include positive controls (tissues known to express TMEM240, such as brain or colon tissues)

  • Use negative controls (omitting primary antibody)

  • Consider dual staining with cell membrane markers to confirm localization

• Microscopy settings:

  • Excite FITC using ~488 nm wavelength

  • Collect emission at ~515-530 nm

  • Minimize exposure time to prevent photobleaching

  • Consider confocal microscopy for more precise localization studies

Based on published research, TMEM240 protein expression is typically detected in normal colon tissues but shows reduced expression in colorectal cancer tissues (detected in only 8.3% of tumors) , making it important to include appropriate controls.

How can TMEM240 antibody be used to study its potential role as a tumor suppressor in cancer research?

TMEM240 has demonstrated tumor suppressor properties in both colorectal and breast cancers. To study this role using TMEM240 antibody (FITC):

In published research, TMEM240 protein was detected in 75% of normal colon tissues but in only 8.3% of colorectal tumors and 25% of metastatic tumors, supporting its potential role as a tumor suppressor .

What methodological approaches can be used to study TMEM240 hypermethylation in relation to antibody detection?

TMEM240 hypermethylation has been identified as a potential biomarker in both colorectal and breast cancers. To study the relationship between DNA methylation and protein expression:

• Integrated epigenetic and protein analysis:

  • Perform quantitative methylation-specific PCR (QMSP) to determine TMEM240 promoter methylation status

  • Use TMEM240-FITC antibody on the same samples to correlate methylation with protein expression

  • Create scatter plots showing the inverse relationship between methylation levels and protein expression

• Demethylation experiments:

  • Treat cancer cell lines with demethylating agents like decitabine (DAC)

  • Monitor changes in TMEM240 expression using the FITC-conjugated antibody

  • Quantify fluorescence intensity before and after treatment to measure expression restoration

• Clinical sample analysis:

  • Collect paired samples (tumor and adjacent normal tissue) from patients

  • Analyze both methylation status (by QMSP) and protein expression (by immunofluorescence)

  • Calculate the percentage of cases showing hypermethylation with concurrent protein loss

  • Compare results with published data: 87.8% of colorectal cancers and 54.5% of breast cancers show TMEM240 hypermethylation

• Circulating biomarker development:

  • Isolate cell-free DNA from patient plasma

  • Detect circulating methylated TMEM240 using liquid biopsy approaches

  • Compare with tissue antibody staining results

  • Evaluate sensitivity and specificity (reported as 87.5% and 93.1% respectively for breast cancer progression)

This integrated approach can provide insights into how epigenetic silencing affects TMEM240 protein expression and its potential utility as a biomarker.

How should multicolor flow cytometry panels be designed when including TMEM240 antibody (FITC)?

When designing multicolor flow cytometry panels that include TMEM240-FITC:

• Spectral considerations:

  • FITC (excitation/emission: 499/515 nm) has significant spectral overlap with PE and other green-yellow fluorophores

  • Place FITC in a different detection channel than PE, PE-Cy5, and PE-Cy7 to minimize compensation requirements

  • Be aware that bright FITC signals can spread into the PE detector, potentially affecting detection of dim PE signals

• Marker pairing strategy:

  • Pair TMEM240-FITC with brighter fluorochromes (like PE, APC) for markers that are expressed at lower levels

  • Reserve APC or PE for antibodies detecting antigens with lower expression if TMEM240 is highly expressed in your samples

  • Follow the rule: "Reserve the brightest fluorochromes for dim antibodies, and vice versa"

• Panel design examples:

  Marker	Fluorochrome	Consideration
  TMEM240	FITC	Primary target, 488 nm excitation
  CD45	Pacific Blue	Leukocyte marker, minimal overlap with FITC
  Cell death marker	7-AAD	Minimal overlap with FITC
  CD8	APC	No spectral overlap with FITC
  CD4	PE-Cy7	Some compensation required

• Tandem dye considerations:

  • If using tandem dyes like PE-Cy7 alongside FITC, be aware that tandem dye degradation can occur

  • PE-Cy7 can degrade in the presence of light and fixation, emitting in the PE detector

  • This can complicate compensation when used with FITC

• Controls and validation:

  • Include FMO (fluorescence minus one) controls to set proper gates

  • Use single-stained controls for accurate compensation

  • Validate panel design with preliminary experiments before full-scale implementation

Proper panel design is essential for accurate detection of TMEM240 expression, especially in complex samples like tumor tissues or clinical specimens.

What are the best practices for preserving FITC signal in long-term storage of stained samples?

Preserving FITC signal in stained samples requires careful attention to several factors:

• Fixation protocol optimization:

  • Use fresh 2-4% paraformaldehyde for 15-20 minutes at room temperature

  • Avoid overfixation, which can quench fluorescence

  • After fixation, wash thoroughly to remove any residual fixative

• Storage medium selection:

  • Store fixed cells/tissues in PBS supplemented with 1% BSA or FBS

  • Add sodium azide (0.05-0.1%) as a preservative to prevent microbial growth

  • Consider commercial anti-fade mounting media containing anti-photobleaching agents for slides

• Physical storage conditions:

  • Store at 4°C for short-term (1-7 days) or -20°C for longer periods

  • Keep samples in lightproof containers (wrapped in aluminum foil)

  • Minimize freeze-thaw cycles, as they significantly reduce signal intensity

  • For TMEM240 antibody specifically, storage at -20°C is recommended

• Alternative approaches:

  • Consider capturing images immediately after staining for optimal signal

  • For flow cytometry samples that need re-analysis, consider using a fluorescence stabilizing reagent

  • For tissue sections, use coverslips sealed with nail polish to prevent drying and oxidation

• Quality control measures:

  • Include a fluorescence standard in each batch to track signal decay over time

  • Document initial signal intensity for comparison

  • Reanalyze control samples periodically to assess degradation rate

FITC is particularly susceptible to photobleaching compared to other fluorophores, so minimizing light exposure during storage and handling is critical for maintaining signal integrity.

What are the common issues encountered when using TMEM240 antibody (FITC) and how can they be resolved?

Researchers commonly encounter several issues when working with FITC-conjugated TMEM240 antibody:

• Weak or no signal:

  • Cause: Insufficient antibody concentration, low target expression, or excessive photobleaching

  • Solution: Titrate antibody to determine optimal concentration; use fresh samples; minimize light exposure; try signal amplification methods; verify sample preparation protocol

• High background fluorescence:

  • Cause: Inadequate blocking, insufficient washing, or autofluorescence

  • Solution: Increase blocking time/concentration; add more wash steps; include 0.1% Tween-20 in wash buffer; use tissue-specific autofluorescence quenchers

• Non-specific binding:

  • Cause: Cross-reactivity with other proteins or inadequate blocking

  • Solution: Pre-adsorb antibody with tissue lysates; increase blocking time with 5-10% normal serum; include 0.1-0.3% Triton X-100 for better penetration

• Inconsistent staining patterns:

  • Cause: Variable fixation, sample degradation, or antibody batch variation

  • Solution: Standardize fixation protocol; use fresh samples; include known positive controls with each experiment

• Poor signal-to-noise ratio in tissues with low TMEM240 expression:

  • Cause: Normal low expression or pathological downregulation (as seen in many cancer tissues)

  • Solution: Use signal amplification systems; increase exposure time (while monitoring photobleaching); consider alternative detection methods like immunohistochemistry with HRP

• Discrepancy between methylation and protein expression data:

  • Cause: Post-transcriptional regulation or technical limitations

  • Solution: Perform parallel RNA expression analysis; verify antibody specificity; use multiple antibody clones or epitopes

For optimal results with TMEM240 antibody, include appropriate positive controls such as normal colon tissue or brain tissue, which have been shown to express TMEM240 at detectable levels .

How can researchers validate the specificity of TMEM240 antibody (FITC) for their particular application?

Validating antibody specificity is crucial for generating reliable research data. For FITC-conjugated TMEM240 antibody:

• Positive and negative control tissues:

  • Positive controls: Use tissues known to express TMEM240 (cerebellum, small intestine, duodenum, colon)

  • Negative controls: Include tissues with minimal expression or use siRNA knockdown cell models

  • Compare staining patterns with published literature showing TMEM240 expression primarily in normal colon tissues (75% positive) but low expression in colorectal tumors (8.3% positive)

• Genetic manipulation validation:

  • Perform TMEM240 gene knockdown using siRNA (si-TMEM240) in cell lines

  • Confirm reduced antibody staining following knockdown

  • Conversely, overexpress TMEM240 using plasmid transfection and verify increased signal

  • This approach has been validated in DLD-1 and HCT116 colorectal cancer cell lines

• Cross-validation with alternative detection methods:

  • Compare FITC-conjugated antibody results with other detection methods:

    • Western blot using non-conjugated TMEM240 antibody

    • RT-PCR for mRNA expression

    • Immunohistochemistry using HRP-conjugated antibody

  • Concordance between methods strengthens specificity confirmation

• Epitope blocking experiments:

  • Pre-incubate the antibody with recombinant TMEM240 protein (particularly the immunogen fragment, aa 42-87)

  • Apply the blocked antibody to tissues/cells

  • Specific binding should be significantly reduced or eliminated

• Multiple antibody comparison:

  • Test multiple TMEM240 antibodies targeting different epitopes

  • Compare staining patterns for consistency

  • When using FITC-conjugated antibody, compare with unconjugated primary plus FITC-secondary approach

• Subcellular localization assessment:

  • Verify that staining patterns match the expected subcellular localization (cell membrane/cytoplasm)

  • Use immunofluorescence microscopy with high resolution to confirm membrane localization

  • Co-stain with established membrane markers for co-localization analysis

Thorough validation ensures that experimental findings truly reflect TMEM240 biology rather than technical artifacts.

How can TMEM240 antibody (FITC) be utilized in circulating tumor cell (CTC) detection research?

TMEM240 antibody (FITC) offers potential in developing novel CTC detection methods, particularly given the emerging role of TMEM240 as a cancer biomarker:

• CTC enrichment and identification workflow:

  • Isolate CTCs from patient blood samples using standard methods (immunomagnetic separation, filtration, microfluidics)

  • Stain with TMEM240-FITC antibody along with other markers:

    • Epithelial markers (EpCAM-PE)

    • Leukocyte exclusion marker (CD45-APC)

    • Nuclear stain (DAPI)

  • Analyze using flow cytometry or fluorescence microscopy

• Differential expression approach:

  • Leverage the differential expression of TMEM240 between normal and cancer cells

  • In normal tissue samples, TMEM240 protein expression is relatively high

  • In cancer tissues, TMEM240 protein expression is significantly reduced:

    • 91.7% of colorectal tumors show low expression

    • 88.2% of breast tumors show low expression

  • This pattern may help identify cancer cells of origin in circulation

• Integration with methylation analysis:

  • Combine TMEM240-FITC antibody staining with DNA methylation analysis

  • Sort CTCs based on TMEM240 expression levels

  • Analyze sorted populations for TMEM240 promoter methylation

  • This could help establish correlation between circulating methylated DNA and CTCs

  • Studies show circulating methylated TMEM240 has 87.5% sensitivity and 93.1% specificity for breast cancer progression prediction

• Treatment response monitoring:

  • Monitor TMEM240 expression in CTCs during treatment

  • Track changes in expression patterns as potential indicators of treatment efficacy

  • Correlate with circulating methylated TMEM240 levels in plasma

  • This approach is supported by findings that circulating methylated TMEM240 dramatically decreased in breast cancer patients without disease progression

This novel application could enhance liquid biopsy approaches and provide new insights into metastatic processes.

What are the considerations for using TMEM240 antibody (FITC) in studying neurodegenerative disorders?

TMEM240 is implicated in neurodegenerative disorders, particularly spinocerebellar ataxia type 21 (SCA21). When using FITC-conjugated TMEM240 antibody for such studies:

• Tissue-specific optimization:

  • Brain tissue requires specialized fixation and permeabilization:

    • Use 4% PFA for 24 hours at 4°C for optimal preservation

    • Consider using 0.3% Triton X-100 for sufficient permeabilization

  • Autofluorescence is common in brain tissue:

    • Use Sudan Black B (0.1-0.3%) or commercial autofluorescence quenchers

    • Consider confocal microscopy with narrow bandwidth detection

• Mutation impact studies:

  • TMEM240 mutations cause spinocerebellar ataxia 21

  • Compare antibody staining patterns between:

    • Wild-type TMEM240

    • Common SCA21-associated TMEM240 mutants

  • Assess differences in:

    • Protein localization

    • Expression levels

    • Co-localization with synaptic markers

• Cellular models for neurodegeneration:

  • Develop cellular models expressing wild-type or mutant TMEM240:

    • Primary neuronal cultures

    • iPSC-derived cerebellar neurons

  • Use TMEM240-FITC antibody to:

    • Track protein trafficking

    • Assess membrane insertion

    • Monitor degradation rates

  • Compare findings to those from colorectal cancer research showing TMEM240's role in cell cycle regulation

• Brain region-specific expression:

  • TMEM240 is notably expressed in the cerebellum and caudate

  • Compare expression patterns across brain regions using immunofluorescence

  • Quantify region-specific differences in:

    • Expression levels

    • Subcellular localization

    • Co-localization with neuronal/glial markers

• Integration with functional studies:

  • Correlate TMEM240 expression patterns with:

    • Electrophysiological measurements

    • Behavioral assessments in animal models

    • Neuropathological findings

  • This approach can help establish the functional relevance of TMEM240 alterations in neurodegeneration

The high conservation of TMEM240 across species suggests fundamental neurological functions, making it an important target for neurodegenerative disease research.

How should researchers quantify and analyze TMEM240 expression data from flow cytometry and immunofluorescence experiments?

Proper quantification and analysis of TMEM240 expression data requires rigorous methodological approaches:

Following these guidelines ensures robust and reproducible analysis of TMEM240 expression data across experimental platforms.

How can researchers reconcile contradictory findings between TMEM240 methylation status and protein expression in clinical samples?

Discrepancies between TMEM240 methylation and protein expression data are not uncommon in clinical research. To address and understand these contradictions:

• Systematic evaluation of potential causes:

  • Technical factors:

    • Antibody specificity and sensitivity limitations

    • Methylation assay resolution and coverage

    • Sample processing differences (fixation effects on antigen retrieval)

  • Biological factors:

    • Post-transcriptional regulation (miRNAs, RNA stability)

    • Post-translational modifications affecting protein stability

    • Alternative promoter usage bypassing methylated regions

    • Heterogeneity within tumor samples

• Integrated multi-omics approach:

  • Analyze the same samples using complementary methods:

    • DNA methylation (QMSP or bisulfite sequencing)

    • mRNA expression (qRT-PCR or RNA-seq)

    • Protein expression (FITC-antibody based detection)

  • Plot correlation matrices to identify patterns and outliers

  • Apply machine learning algorithms to identify factors influencing concordance

• Single-cell resolution studies:

  • Use flow cytometry with TMEM240-FITC antibody to isolate cell populations

  • Perform methylation analysis on sorted populations

  • This can reveal whether bulk tissue discrepancies reflect cellular heterogeneity

  • Consider laser capture microdissection for spatial methylation-expression correlation

• Time-course and intervention studies:

  • Track methylation and expression changes over time or treatment course

  • Treat cells with demethylating agents and monitor protein expression recovery

  • Determine lag time between demethylation and protein re-expression

  • Published research shows demethylation treatment can restore TMEM240 expression

• Interpretation framework:

  • Establish decision trees for interpreting discordant cases:

    • High methylation/High expression: Possible compensatory mechanisms or antibody cross-reactivity

    • Low methylation/Low expression: Potential alternative silencing mechanisms

    • Partial methylation with variable expression: Possible allelic-specific expression

  • Document frequency of discordant cases for publication and further investigation

  • Consider additional epigenetic markers (histone modifications) that may provide explanatory power