TM6SF2 Antibody, FITC conjugated

What is TM6SF2 and why is it important in metabolic research?

TM6SF2 (Transmembrane 6 superfamily member 2) is a 377 amino acid multi-pass membrane protein belonging to the TM6SF family that plays a crucial role in liver fat metabolism . The protein comprises 7-10 predicted transmembrane domains and is predominantly expressed in liver and intestine tissues, showing an expression pattern similar to apolipoprotein B (APOB) . The significance of TM6SF2 in metabolic research stems from its regulatory role in triglyceride (TG) secretion and hepatic lipid droplet content . Genome-wide association studies have identified TM6SF2 as a putative causal gene associated with plasma triglyceride concentration, which is a risk factor for coronary heart disease . Functional studies in human liver cells have demonstrated that inhibition of TM6SF2 leads to reduced secretion of TG-rich lipoproteins (TRLs) and increased cellular TG concentration, while TM6SF2 overexpression reduces cellular TG concentration . This makes TM6SF2 a significant target for research into metabolic disorders and liver diseases.

What is the subcellular localization of TM6SF2 protein?

TM6SF2 protein is primarily localized in the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment (ERGIC) of human liver cells . Confocal microscopy studies using TM6SF2 with a C-terminal GFP tag in human hepatoma Huh7 and HepG2 cells revealed a perinuclear lattice-like staining pattern characteristic of ER localization . Colocalization studies quantified using Pearson correlation analysis demonstrated substantial overlap between TM6SF2 and the ER marker calreticulin (CALR) with a Pearson correlation value of 0.83 ± 0.04 . Further immunofluorescence studies showed considerable overlap with both the ER marker protein disulphide isomerase (PDI) and the ERGIC marker protein ERGIC53, with Rcoloc values of 0.78 ± 0.05 and 0.80 ± 0.03, respectively . In contrast, limited overlap was observed with the Golgi marker protein GIANTIN (Rcoloc value of 0.30 ± 0.08), confirming that TM6SF2 predominantly resides in the ER and ERGIC compartments rather than the Golgi apparatus .

What are the key specifications of FITC-conjugated TM6SF2 antibody?

The FITC-conjugated TM6SF2 antibody is a rabbit polyclonal antibody specifically designed for research applications. The key specifications of this reagent are summarized in the following table:

This antibody is specifically designed to recognize the TM6SF2 protein, which is an integral component of cellular membranes and contains transmembrane helices . The FITC conjugation enables direct fluorescence detection without the need for secondary antibodies, making it particularly valuable for multicolor imaging studies and flow cytometry applications.

How can FITC-conjugated TM6SF2 antibody be used to investigate liver fat metabolism?

The FITC-conjugated TM6SF2 antibody serves as a powerful tool for investigating liver fat metabolism through various methodological approaches. To effectively study TM6SF2's role in liver fat metabolism, researchers should employ a multi-faceted experimental design:

First, colocalization studies utilizing confocal microscopy can be performed to examine the spatial relationship between TM6SF2 and other proteins involved in lipid metabolism . The FITC-conjugated antibody can be used alongside markers for lipid droplets (such as BODIPY 493/503 or LipidTOX dyes) to visualize potential associations between TM6SF2 localization and lipid storage compartments. This approach requires careful optimization of fixation protocols to preserve both the fluorophore activity and the cellular structures.

Second, researchers can employ siRNA knockdown or CRISPR-Cas9 gene editing of TM6SF2 followed by immunofluorescence analysis using the FITC-conjugated antibody to confirm protein depletion and examine consequent changes in cellular triglyceride content . This method requires appropriate controls, including non-targeting siRNA or non-edited cells, to validate the specificity of the observed effects.

Third, the antibody can be used in immunoprecipitation experiments followed by mass spectrometry to identify TM6SF2 interaction partners in the triglyceride secretion pathway . For such applications, researchers should optimize lysis conditions to effectively solubilize membrane proteins while maintaining protein-protein interactions.

Finally, the FITC-conjugated TM6SF2 antibody can be utilized in combination with electron microscopy techniques to examine the ultrastructural localization of TM6SF2 in relation to the ER, ERGIC, and lipid droplets during different metabolic states . This approach provides high-resolution insights into the spatial organization of TM6SF2 within the cellular machinery responsible for lipid metabolism.

What methodological considerations are important when using FITC-conjugated TM6SF2 antibody in co-localization studies?

When conducting co-localization studies with FITC-conjugated TM6SF2 antibody, several methodological considerations must be addressed to ensure reliable results:

First, spectral overlap management is critical. FITC emits in the green spectrum (peak emission ~520 nm), which can overlap with other commonly used fluorophores . When designing multi-color imaging experiments, researchers should select fluorophores with minimal spectral overlap with FITC, such as Cy5 or Alexa Fluor 647 for far-red emission. If spectral overlap cannot be avoided, proper compensation controls and sequential scanning should be implemented during confocal microscopy.

Second, fixation protocol optimization is essential. Different fixation methods (e.g., paraformaldehyde, methanol, acetone) can affect both the fluorescence intensity of FITC and the native localization of TM6SF2 within membrane structures . Researchers should systematically test different fixation protocols to determine which best preserves both fluorophore activity and protein localization patterns.

Third, appropriate controls must be included. These should comprise: (1) single-labeled samples to assess bleed-through; (2) secondary antibody-only controls to evaluate non-specific binding; (3) peptide competition assays using the immunizing peptide (P-TM6SF2) to confirm antibody specificity; and (4) samples from TM6SF2-knockdown cells to validate staining patterns .

Fourth, quantitative colocalization analysis should be performed using established methods such as Pearson's correlation coefficient, Manders' overlap coefficient, or object-based colocalization analysis . The selection of appropriate analysis method depends on the specific biological question and the nature of the protein distribution.

Finally, photobleaching considerations are important as FITC is moderately susceptible to photobleaching. Using anti-fade mounting media, minimizing exposure times, and considering sequential rather than simultaneous scanning can help mitigate photobleaching effects during image acquisition.

How can researchers optimize TM6SF2 antibody use for studying genetic variants and their impact on protein expression?

To effectively investigate the relationship between TM6SF2 genetic variants and protein expression using the FITC-conjugated antibody, researchers should implement a comprehensive methodological approach:

First, establish a genotype-phenotype correlation system. Researchers should obtain liver tissue or cell samples with known TM6SF2 genotypes, particularly focusing on the rs10401969 SNP that has been associated with reduced TM6SF2 expression . The FITC-conjugated antibody can then be used to quantify TM6SF2 protein levels via flow cytometry or quantitative immunofluorescence microscopy.

Second, develop appropriate calibration standards. To enable accurate quantification of TM6SF2 protein levels across different genotypes, researchers should use recombinant TM6SF2 protein standards of known concentrations to create calibration curves. This approach allows for the conversion of fluorescence intensity values to absolute protein quantities.

Third, implement multiparametric analysis. The FITC-conjugated antibody can be used alongside markers for lipid droplets, ER stress, and other relevant parameters to comprehensively assess how genetic variants affect not only TM6SF2 expression but also downstream metabolic processes . Flow cytometry is particularly suitable for this application, allowing simultaneous assessment of multiple parameters at the single-cell level.

Fourth, validate findings with complementary methods. While the FITC-conjugated antibody provides spatial information about TM6SF2 expression, researchers should validate their findings using orthogonal approaches such as Western blotting with the unconjugated TM6SF2 antibody (dilution 1:500) and qRT-PCR for mRNA quantification .

Fifth, account for confounding factors. Expression of TM6SF2 can be influenced by various factors beyond genetics, including metabolic state, medication, and liver disease progression. Researchers should carefully document and control for these variables in their experimental design to isolate the effects of genetic variants on TM6SF2 expression.

What is the recommended protocol for immunofluorescence staining using FITC-conjugated TM6SF2 antibody?

For optimal immunofluorescence staining using FITC-conjugated TM6SF2 antibody, the following detailed protocol is recommended:

Materials Required:

• FITC-conjugated TM6SF2 antibody (0.68-0.70 μg/μl concentration)

• Phosphate-buffered saline (PBS)

• 4% paraformaldehyde in PBS

• Permeabilization solution (0.1% Triton X-100 in PBS)

• Blocking solution (3% BSA in PBS)

• Mounting medium with anti-fade properties and DAPI

• Coverslips and slides

Procedure:

• Grow cells on appropriate coverslips or prepare tissue sections on slides.

• Fix samples with 4% paraformaldehyde for 15 minutes at room temperature.

• Wash three times with PBS, 5 minutes per wash.

• Permeabilize cells with 0.1% Triton X-100 for 10 minutes at room temperature.

• Wash three times with PBS, 5 minutes per wash.

• Block non-specific binding with 3% BSA in PBS for 1 hour at room temperature.

• Dilute FITC-conjugated TM6SF2 antibody to a working concentration of 1:50-1:150 in blocking solution .

• Apply diluted antibody to samples and incubate overnight at 4°C in a humidified chamber protected from light.

• Wash five times with PBS, 5 minutes per wash.

• Mount coverslips using mounting medium containing anti-fade agent and DAPI.

• Seal edges with nail polish and store at 4°C protected from light.

• Visualize using appropriate filter sets (excitation ~490 nm, emission ~520 nm) on a fluorescence or confocal microscope.

Critical Considerations:

• Optimize the antibody dilution within the recommended range (1:50-1:150) for your specific sample type .

• Include appropriate controls: (a) untreated cells, (b) secondary antibody-only control, and (c) peptide competition control using P-TM6SF2 .

• For co-localization studies with ER markers, consider a sequential staining approach to avoid potential cross-reactivity .

• Document acquisition parameters carefully to ensure reproducibility across experiments.

What protocol should be followed for Western blot analysis using FITC-conjugated TM6SF2 antibody?

For Western blot analysis using FITC-conjugated TM6SF2 antibody, the following protocol is recommended:

Sample Preparation:

• Harvest cells or tissue and lyse in an appropriate lysis buffer containing protease inhibitors.

• For membrane proteins like TM6SF2, use a lysis buffer containing 1% NP-40 or Triton X-100 to effectively solubilize the protein.

• Centrifuge the lysate at 14,000 × g for 15 minutes at 4°C to remove insoluble debris.

• Quantify protein concentration using a BCA or Bradford assay.

SDS-PAGE and Transfer:

• Prepare protein samples (20-50 μg per lane) in Laemmli buffer with reducing agent.

• Heat samples at 70°C for 10 minutes (avoid boiling as it may cause aggregation of membrane proteins).

• Separate proteins on an 8-12% SDS-PAGE gel.

• Transfer proteins to a PVDF membrane (preferred over nitrocellulose for fluorescence detection).

Antibody Incubation and Detection:

• Block the membrane in 5% non-fat milk or 3% BSA in TBST for 1 hour at room temperature.

• Dilute the FITC-conjugated TM6SF2 antibody 1:500 in blocking buffer .

• Incubate the membrane with diluted antibody overnight at 4°C with gentle agitation, protected from light.

• Wash the membrane 4 times with TBST, 10 minutes per wash.

• For direct fluorescence detection:
  a. Rinse the membrane with PBS to remove Tween-20.
  b. Scan the membrane using a fluorescence imaging system with appropriate filter settings for FITC (excitation ~490 nm, emission ~520 nm).

• For enhanced sensitivity (optional):
  a. Incubate the membrane with an anti-FITC HRP-conjugated antibody (1:2000) for 1 hour at room temperature.
  b. Wash 4 times with TBST, 10 minutes per wash.
  c. Develop using chemiluminescent substrate and image.

Controls and Validation:

• Include a positive control (PC-TM6SF2) to confirm antibody specificity .

• Run a peptide competition assay using P-TM6SF2 to validate specificity .

• Include molecular weight markers to confirm the expected size of TM6SF2 (approximately 40 kDa).

What are the recommended protocols for ELISA and immunoprecipitation using FITC-conjugated TM6SF2 antibody?

ELISA Protocol:

ELISA applications using FITC-conjugated TM6SF2 antibody require careful optimization due to the fluorescent conjugation:

• Coating: Coat 96-well high-binding plates with capture antibody (anti-TM6SF2, unconjugated) at 1-2 μg/ml in coating buffer (50 mM carbonate-bicarbonate, pH 9.6) overnight at 4°C.

• Blocking: Block plates with 3% BSA in PBS for 2 hours at room temperature.

• Sample addition: Add samples and standards diluted in sample buffer (1% BSA in PBS) for 2 hours at room temperature.

• Detection: Dilute FITC-conjugated TM6SF2 antibody 1:4,000 in sample buffer and add to wells for 1 hour at room temperature, protected from light .

• Washing: Wash plates 5 times with PBST (PBS with 0.05% Tween-20).

• Readout: Measure fluorescence using a plate reader with excitation at 490 nm and emission at 520 nm.

• Alternatively, for a more conventional approach, an anti-FITC HRP-conjugated antibody can be used, followed by TMB substrate addition and absorbance measurement at 450 nm.

Immunoprecipitation Protocol:

For immunoprecipitation studies using FITC-conjugated TM6SF2 antibody:

• Sample preparation:

  • Lyse cells or tissue in IP lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1 mM EDTA, protease inhibitors).

  • Clarify lysate by centrifugation at 14,000 × g for 15 minutes at 4°C.

  • Pre-clear lysate by incubating with Protein A/G beads for 1 hour at 4°C, then remove beads by centrifugation.

• Antibody binding:

  • Dilute FITC-conjugated TM6SF2 antibody 1:150 in IP lysis buffer .

  • Add diluted antibody to pre-cleared lysate (typically 1-5 μg antibody per 1 mg of total protein).

  • Incubate overnight at 4°C with gentle rotation, protected from light.

• Immunoprecipitation:

  • Add 30-50 μl of Protein A/G beads to the lysate-antibody mixture.

  • Incubate for 3 hours at 4°C with gentle rotation.

  • Collect beads by centrifugation at 1,000 × g for 2 minutes at 4°C.

  • Wash beads 4 times with IP wash buffer (IP lysis buffer with 0.1% NP-40).

• Elution and analysis:

  • Elute bound proteins by boiling beads in 2X Laemmli buffer at 70°C for 10 minutes.

  • Analyze eluted proteins by SDS-PAGE followed by Western blotting or mass spectrometry.

• Controls:

  • Include an IgG isotype control to assess non-specific binding.

  • Perform a peptide competition assay with P-TM6SF2 to confirm specificity .

  • Include an input control (5-10% of the lysate used for IP).

What are common issues with FITC-conjugated antibodies and how can they be resolved?

When working with FITC-conjugated TM6SF2 antibody, researchers may encounter several common issues. This table outlines these challenges and provides methodological solutions:

How can researchers validate the specificity of FITC-conjugated TM6SF2 antibody?

Validating antibody specificity is crucial for ensuring reliable results. For FITC-conjugated TM6SF2 antibody, a comprehensive validation approach should include:

First, peptide competition assays should be performed using the specific blocking peptide P-TM6SF2 . This involves pre-incubating the antibody with excess immunizing peptide prior to staining. The disappearance or significant reduction of signal in these samples compared to controls confirms specificity for the target epitope.

Second, genetic knockdown or knockout validation is essential. Researchers should use siRNA, shRNA, or CRISPR-Cas9 to reduce or eliminate TM6SF2 expression in cell lines . The FITC-conjugated antibody should show corresponding reduction or absence of signal in these cells compared to control cells.

Third, comparative analysis with alternative antibodies targeting different epitopes of TM6SF2 (such as TM6SF2-212AP which targets amino acids 325-375 versus TM6SF2-201AP which targets amino acids 220-270) should be performed . Consistent localization patterns with antibodies targeting different epitopes provide strong evidence of specificity.

Fourth, overexpression systems can provide additional validation. Cells transfected with TM6SF2 expression vectors should demonstrate increased signal intensity compared to non-transfected controls when stained with the FITC-conjugated antibody .

Fifth, Western blot analysis should be conducted to confirm that the antibody detects a protein of the expected molecular weight (~40 kDa for TM6SF2) . A single band at the correct molecular weight supports antibody specificity.

Finally, mass spectrometry verification can provide definitive validation. Immunoprecipitation using the TM6SF2 antibody followed by mass spectrometry analysis should confirm the presence of TM6SF2 peptides in the immunoprecipitated sample.

What are the best practices for storing and handling FITC-conjugated TM6SF2 antibody?

To maintain optimal activity of FITC-conjugated TM6SF2 antibody, researchers should adhere to the following best practices for storage and handling:

Long-term storage should be at -20°C, as indicated in the product specifications . Upon receipt, the antibody should be immediately aliquoted into small volumes (5-10 μl) to minimize freeze-thaw cycles, as each cycle can reduce antibody activity and fluorescence intensity.

For working solutions, store at 4°C and use within 1-2 weeks. The antibody is supplied in a stabilization buffer (0.68-0.70 μg/μl), which helps maintain activity during storage .

Light protection is crucial at all stages of handling, as FITC is susceptible to photobleaching. Use amber tubes for storage and wrap containers in aluminum foil. Minimize exposure to light during experimental procedures and microscopy sessions.

When diluting the antibody, use high-quality, filtered buffers to prevent contamination and protein degradation. For most applications, dilute in a buffer containing BSA as a carrier protein to maintain stability.

Temperature transitions should be gradual. When removing from -20°C storage, thaw antibody aliquots on ice rather than at room temperature to minimize potential damage to the antibody-fluorophore conjugate.

Quality control testing should be performed periodically using positive controls (such as PC-TM6SF2) to ensure the antibody remains active over time . This is particularly important for older antibody stocks or those that have undergone multiple freeze-thaw cycles.

Contamination prevention is essential. Always use clean pipettes, preferably with filter tips, and work in a clean environment to prevent microbial growth or cross-contamination that could degrade the antibody or introduce experimental artifacts.

How can FITC-conjugated TM6SF2 antibody contribute to research on metabolic disorders?

The FITC-conjugated TM6SF2 antibody offers significant potential for advancing research on metabolic disorders through several methodological approaches:

In clinical biomarker development, the antibody can be used to assess TM6SF2 protein expression levels in patient liver biopsies via quantitative immunofluorescence microscopy. This approach could help stratify patients with non-alcoholic fatty liver disease (NAFLD) based on their TM6SF2 expression profiles, potentially identifying those at higher risk for disease progression .

For drug discovery applications, high-content screening assays utilizing the FITC-conjugated antibody can be developed to identify compounds that modulate TM6SF2 expression or localization. Such screens could reveal therapeutic candidates for treating NAFLD or dyslipidemia by targeting TM6SF2-mediated pathways .

In metabolic pathway elucidation, the antibody enables detailed investigation of TM6SF2's interaction network through proximity ligation assays or FRET (Fluorescence Resonance Energy Transfer) with other fluorophore-labeled proteins involved in lipid metabolism. This approach can reveal novel molecular connections and regulatory mechanisms in lipid homeostasis .

For genotype-phenotype correlation studies, the antibody facilitates examination of how TM6SF2 genetic variants (particularly rs10401969) affect protein expression, localization, and function in patient-derived cells or tissues . Such studies could explain the mechanistic basis for the association between TM6SF2 variants and metabolic traits.

In liver disease progression monitoring, longitudinal studies using the antibody to track changes in TM6SF2 expression patterns during disease progression could reveal critical transition points in NAFLD pathogenesis. This approach may identify optimal intervention windows for therapeutic strategies targeting TM6SF2-related pathways.

What emerging technologies could enhance the utility of FITC-conjugated TM6SF2 antibody?

Several cutting-edge technologies hold promise for expanding the research applications of FITC-conjugated TM6SF2 antibody:

Super-resolution microscopy techniques such as STED (Stimulated Emission Depletion), STORM (Stochastic Optical Reconstruction Microscopy), or PALM (Photoactivated Localization Microscopy) can overcome the diffraction limit of conventional microscopy, enabling nanoscale visualization of TM6SF2 distribution within subcellular compartments . This would provide unprecedented insights into the spatial relationship between TM6SF2 and other components of lipid metabolism machinery.

Live-cell imaging approaches using FITC-conjugated TM6SF2 antibody fragments (Fab or scFv) could enable real-time monitoring of TM6SF2 dynamics in response to metabolic challenges or drug treatments. This approach requires optimization of antibody fragment production and cell permeabilization techniques that maintain cell viability.

Mass cytometry (CyTOF) integration would allow multiplexed analysis of TM6SF2 alongside dozens of other proteins of interest in single cells. By conjugating TM6SF2 antibody to metal isotopes instead of FITC, researchers could perform highly multiplexed analysis without the spectral overlap limitations of fluorescence-based approaches.

Spatial transcriptomics combined with TM6SF2 immunofluorescence would enable correlation of TM6SF2 protein expression with the transcriptional landscape of individual cells within tissue context. This approach could reveal microenvironmental factors that influence TM6SF2 expression and function in different liver zones.

Organ-on-chip technologies incorporating TM6SF2 immunostaining could provide dynamic, physiologically relevant models for studying TM6SF2 function in controlled microenvironments that mimic liver architecture and flow conditions. This would allow assessment of how mechanical forces and intercellular communications affect TM6SF2-mediated lipid metabolism.