TM6SF2 Antibody, HRP conjugated

What is TM6SF2 and why is it significant in lipid metabolism research?

TM6SF2 is a transmembrane protein localized predominantly to the endoplasmic reticulum (ER) and pre-Golgi compartments where lipidation of nascent VLDL occurs in hepatocytes. Its significance in lipid metabolism stems from its role in regulating both the degree of lipidation and the number of secreted VLDL particles. The p.E167K variant of TM6SF2 has been associated with increased hepatic fat content and reduced levels of plasma triglycerides (TG) and LDL cholesterol . This protein has become a focal point in research exploring non-alcoholic fatty liver disease (NAFLD) mechanisms and cardiovascular disease risk, as it appears to represent a divergence point where genetic factors can simultaneously increase liver fat while potentially reducing cardiovascular risk through lower circulating lipids.

TM6SF2 has been demonstrated to interact directly with apolipoprotein B (APOB) and form complexes with ERLIN1 and ERLIN2, all critical for proper VLDL assembly and secretion . The protein's involvement in these essential metabolic pathways makes it a valuable target for antibody-based detection methods in basic and translational research.

What are the primary applications of HRP-conjugated TM6SF2 antibodies in experimental research?

HRP-conjugated TM6SF2 antibodies serve multiple critical functions in experimental research:

• Western Blotting: Direct detection of TM6SF2 protein expression levels without requiring secondary antibody incubation, streamlining workflows and reducing background noise.

• Immunocytochemistry/Immunohistochemistry: Visualization of TM6SF2 localization within cellular compartments, particularly in ER and pre-Golgi regions where TM6SF2 functions in lipid metabolism.

• Protein Complex Detection: Identification of TM6SF2-interacting proteins in pull-down assays and co-immunoprecipitation studies, particularly its interactions with APOB, ERLIN1, and ERLIN2 .

• ELISA Development: Quantification of TM6SF2 levels in cell or tissue lysates.

• Flow Cytometry: Analysis of TM6SF2 expression at the single-cell level when studying cellular heterogeneity in liver cell populations.

The HRP conjugation provides enhanced sensitivity through enzymatic signal amplification, making these antibodies particularly valuable when studying TM6SF2, which may be expressed at relatively low levels in some experimental systems.

How can I validate TM6SF2 antibody specificity for experimental applications?

Validating TM6SF2 antibody specificity is essential for generating reliable research data. A comprehensive validation approach includes:

• Positive and Negative Control Samples:

  • Use cell lines known to express TM6SF2 (HepG2, McA cells) as positive controls

  • Compare with TM6SF2 knockdown cells (using validated shRNA) or knockout cells (CRISPR-edited) as negative controls

• Antibody Validation Experiments:

  • Western blot analysis to confirm single band at expected molecular weight (~40 kDa)

  • Peptide competition assay to demonstrate binding specificity

  • Immunoprecipitation followed by mass spectrometry to confirm target identity

• Correlation with mRNA Expression:

  • Parallel qRT-PCR analysis to verify correspondence between protein and mRNA levels

• Cross-validation with Multiple Antibodies:

  • Use antibodies targeting different epitopes of TM6SF2 to cross-validate findings

• Genetic Models:

  • Verification in cell models with modulated TM6SF2 expression (as described in the research literature using shRNA knockdown or CRISPR knockout approaches)

How can I optimize western blotting protocols for detection of endogenous TM6SF2 using HRP-conjugated antibodies?

Optimizing western blotting for endogenous TM6SF2 detection requires addressing several technical considerations:

• Sample Preparation:

  • Enrich for membrane proteins using specialized lysis buffers containing 1% Triton X-100 or other appropriate detergents

  • Avoid boiling samples (use 37°C incubation instead) to prevent aggregation of transmembrane proteins

  • Add protease inhibitors immediately during cell lysis to prevent degradation

• Gel Electrophoresis and Transfer:

  • Use gradient gels (4-12%) for optimal resolution

  • Consider wet transfer methods with low SDS concentration in transfer buffer

  • Add 0.05% SDS to transfer buffer to facilitate movement of hydrophobic proteins

  • Extended transfer times (overnight at lower voltage) may improve results

• Blocking and Antibody Incubation:

  • Test multiple blocking reagents (BSA vs. milk)

  • For HRP-conjugated antibodies, optimize primary antibody concentration (typically 1:1000 to 1:5000)

  • Extend incubation time to overnight at 4°C to increase signal

• Detection Optimization:

  • Use enhanced chemiluminescent (ECL) substrate with appropriate sensitivity

  • Consider signal enhancers specifically designed for HRP detection

  • Test multiple exposure times to capture optimal signal-to-noise ratio

Research has demonstrated that TM6SF2 protein levels can be reliably detected in human hepatoma cell lines and rodent liver tissues using optimized western blotting protocols, which has been crucial for confirming findings from knockdown and knockout experiments .

What experimental approaches are most effective for studying TM6SF2-protein interactions using antibody-based methods?

For investigating TM6SF2-protein interactions, particularly with APOB, ERLIN1 and ERLIN2, several antibody-based approaches have proven effective:

• Co-immunoprecipitation (Co-IP):

  • Use HRP-conjugated TM6SF2 antibodies for direct detection in western blots after IP with antibodies against interaction partners

  • Crosslinking prior to lysis can stabilize transient interactions

  • Validate interactions bidirectionally by performing reciprocal Co-IPs

• Proximity Ligation Assay (PLA):

  • Allows visualization of protein-protein interactions in situ

  • Requires antibodies recognizing different proteins (TM6SF2 and its interaction partners)

  • Provides spatial information about interaction locations within cells

• Tandem Affinity Purification (TAP):

  • As demonstrated in research, TAP coupled with mass spectrometry effectively identified TM6SF2-interacting proteins

  • This approach revealed that "TM6SF2, ERLIN1 and ERLIN2 formed a protein complex with APOB"

  • Follow with antibody detection to confirm specific interactions

• Mammalian Two-Hybrid System:

  • Can be used to map specific interaction domains

  • Verify results with deletion mutants and co-IP

• Bimolecular Fluorescence Complementation (BiFC):

  • Allows direct visualization of protein interactions in living cells

  • Particularly useful for membrane protein interactions

Research has shown that TM6SF2 forms complexes with ERLIN1, ERLIN2, and APOB, and these interactions are critical for VLDL assembly and secretion . A comprehensive analysis might include domain mapping to identify which regions of TM6SF2 are essential for these protein-protein interactions.

How can TM6SF2 antibodies be used to investigate differences between wild-type and E167K variant protein?

The E167K variant of TM6SF2 (p.E167K) is associated with altered lipid metabolism and NAFLD risk. Investigating the differences between wild-type and variant protein using antibodies requires specific approaches:

• Expression Level Analysis:

  • Western blotting with HRP-conjugated TM6SF2 antibodies can quantify relative expression levels between wild-type and E167K variant

  • Research has demonstrated that the E167K variant typically shows lower expression levels, which may be due to reduced protein stability

• Subcellular Localization Studies:

  • Immunofluorescence microscopy to determine if the E167K variant shows altered localization

  • Co-localization with ER and Golgi markers to assess potential trafficking defects

• Protein-Protein Interaction Analysis:

  • Co-IP experiments to compare interaction efficiency between wild-type and E167K variant with known partners (APOB, ERLIN1, ERLIN2)

  • The E167K mutation may impact these interactions, affecting APOB stabilization

• Protein Stability Assessment:

  • Cycloheximide chase experiments with antibody detection to compare degradation rates

  • Pulse-chase experiments similar to those described in the research literature

• Functional Assays with Antibody Validation:

  • Assess impact on VLDL secretion and TG content

  • Quantify differences in APOB secretion between wild-type and variant

Research has shown that the E167K variant hampers APOB stabilization by reducing the expression of TM6SF2 . This finding helps explain the molecular mechanism behind the association of this variant with altered lipid metabolism and NAFLD.

What are recommended protocols for immunoprecipitation of TM6SF2 and associated proteins?

Optimized immunoprecipitation protocols for TM6SF2 and its associated proteins should address the membrane-bound nature of TM6SF2 and the potentially transient interactions with partners like APOB. Based on research methodologies , the following protocol is recommended:

Reagents and Materials:

• TM6SF2 antibody (HRP-conjugated for direct detection)

• Magnetic protein A/G beads

• NP-40 or Triton X-100 lysis buffer (1%)

• Protease inhibitor cocktail

• Phosphatase inhibitor cocktail

• Hepatic cell lines (HepG2, McA) or liver tissue

Protocol:

• Cell/Tissue Preparation:

  • For adherent cells: Wash twice with ice-cold PBS, scrape in 1 ml PBS, pellet at 500 × g

  • For tissue: Homogenize in cold PBS using Dounce homogenizer

• Lysis:

  • Resuspend in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, protease/phosphatase inhibitors)

  • Incubate with gentle rotation at 4°C for 30 minutes

  • Centrifuge at 16,000 × g for 20 minutes at 4°C

  • Collect supernatant and determine protein concentration

• Pre-clearing:

  • Incubate lysate with protein A/G beads for 1 hour at 4°C

  • Remove beads by centrifugation

• Immunoprecipitation:

  • Add TM6SF2 antibody (2-5 μg) to 500 μg of protein lysate

  • Incubate overnight at 4°C with gentle rotation

  • Add protein A/G beads and incubate for 2-4 hours at 4°C

  • Wash beads 4-5 times with lysis buffer

• Elution:

  • Add 50 μl of 2× Laemmli sample buffer

  • Heat at 37°C for 10 minutes (avoid boiling)

  • Analyze by SDS-PAGE and western blotting

For specifically studying the TM6SF2-APOB-ERLIN complex, modifications to this protocol have been effective:

"We pulled down TM6SF2 and the associated proteins from the CRL1601/TM6SF2-TAP stable cells using the IgG-coupled agarose. After digesting with the TEV protease, supernatants were further immunoprecipitated using the anti-FLAG beads followed by elution using the FLAG peptides" .

This tandem purification approach significantly enhances specificity when identifying true interaction partners.

How can I optimize immunofluorescence protocols to visualize TM6SF2 localization to the ER and pre-Golgi regions?

Visualizing TM6SF2 in its native cellular compartments (ER and pre-Golgi) requires careful optimization of immunofluorescence protocols:

• Cell Preparation:

  • Grow cells on glass coverslips coated with poly-L-lysine or collagen

  • Fix with 4% paraformaldehyde (10 minutes, room temperature)

  • For improved antigen accessibility, test both methanol fixation (-20°C, 10 minutes) and PFA fixation

• Permeabilization and Blocking:

  • Permeabilize with 0.1-0.3% Triton X-100 in PBS (5-10 minutes)

  • Block with 5% normal serum + 0.3% Triton X-100 in PBS (1 hour)

  • Alternatively, use 0.1% saponin for gentler permeabilization that better preserves membrane structures

• Antibody Incubation:

  • For direct detection: Use HRP-conjugated TM6SF2 antibody (1:100-1:500 dilution)

  • For indirect detection: Use unconjugated primary antibody followed by fluorescently-labeled secondary antibody

  • Incubate overnight at 4°C for maximal sensitivity

• Co-localization Markers:

  • ER markers: anti-calnexin, anti-PDI, or ER-Tracker dyes

  • Golgi markers: anti-GM130 (cis-Golgi), anti-TGN46 (trans-Golgi)

  • ERGIC markers: anti-ERGIC-53 for ER-Golgi intermediate compartment

• Signal Amplification:

  • For HRP-conjugated antibodies: Use tyramide signal amplification (TSA) for enhanced sensitivity

  • Optimize concentration and incubation time of TSA reagent

• Imaging Considerations:

  • Use confocal microscopy for accurate co-localization analysis

  • Acquire z-stacks to capture the full cellular volume

  • Employ deconvolution to improve resolution of membrane structures

Research has consistently localized TM6SF2 to the ER and pre-Golgi compartments: "The protein has been localized to the ER and pre-Golgi (where lipidation of nascent VLDL occurs) in human hepatoma lines and in mouse and rat [tissues]" .

How do I interpret conflicting data between TM6SF2 antibody experiments and genetic studies?

Researchers frequently encounter seemingly contradictory results between antibody-based protein detection and genetic manipulation studies of TM6SF2. These discrepancies require careful analytical approaches:

What controls are essential when using TM6SF2 antibodies in studies of the E167K variant?

When investigating the E167K variant of TM6SF2, proper experimental controls are critical for reliable interpretation:

• Essential Genetic Controls:

  • Wild-type TM6SF2 Expression Systems: Cells or tissues expressing confirmed wild-type TM6SF2

  • E167K Variant Expression Systems: Cells expressing the E167K variant (either naturally occurring or engineered)

  • TM6SF2 Knockout Models: Complete absence of TM6SF2 protein as negative control

  • Heterozygous Models: To mimic the common heterozygous state of E167K carriers

• Antibody Controls:

  • Epitope Verification: Confirm that the antibody's epitope is not affected by the E167K mutation

  • Cross-reactivity Testing: Validate that the antibody recognizes both wild-type and variant proteins

  • Quantification Standards: Include concentration gradients of recombinant proteins for quantitative comparisons

• Functional Controls:

  • APOB Secretion Measurements: Monitor APOB secretion as a functional readout

  • TG Secretion and Cellular Content: Measure both secreted and cellular TG levels

  • ER Stress Markers: Monitor UPR pathway components that may be differentially affected

• Technical Controls:

  • Loading Controls: Use appropriate membrane protein controls (Na+/K+ ATPase) rather than cytosolic proteins

  • Signal Linearity Verification: Ensure detection remains within linear range for accurate quantification

  • Subcellular Fractionation Quality Controls: Verify clean separation of membrane compartments

Research has demonstrated that E167K carriers show differences in ER stress response compared to wild-type: "TM6SF2 deficiency resulted in significant downregulation of several genes encoding proteins involved in the unfolded protein response (UPR) to ER stress... There were clear reductions in p-eIF2α, IRE1α, and CHOP protein levels, and a modest decrease in BIP" . These markers should be routinely assessed alongside TM6SF2 protein levels.

How can TM6SF2 antibodies be used in conjunction with lipidomic analyses to advance our understanding of VLDL assembly?

Integrating TM6SF2 antibody-based techniques with lipidomic analyses offers powerful insights into VLDL assembly mechanisms:

• Antibody-Facilitated Subcellular Fractionation:

  • Use TM6SF2 antibodies to immunoisolate ER-derived vesicles and VLDL assembly compartments

  • Perform lipidomic analysis on these isolated fractions to characterize the lipid species present during different stages of VLDL assembly

  • Compare wild-type and E167K variant samples to identify specific lipid alterations

• Proximity-Based Labeling with Lipidomics:

  • Employ TM6SF2 antibodies in conjunction with proximity labeling techniques (BioID, APEX)

  • Identify and characterize lipid transport or modification enzymes in close proximity to TM6SF2

  • Correlate these findings with lipidomic profiles of nascent and mature VLDL particles

• Immunoprecipitation-Mass Spectrometry (IP-MS) Approach:

  • Use TM6SF2 antibodies to pull down TM6SF2-containing complexes

  • Perform both proteomic and lipidomic analyses on the same precipitated material

  • Identify specific lipid species associated with TM6SF2-APOB-ERLIN complexes

• Time-Resolved Analysis:

  • Combine pulse-chase experiments (as described in the literature ) with antibody-based isolation of VLDL assembly intermediates

  • Perform lipidomic analysis at different time points to track lipid composition changes during VLDL maturation

  • Compare wild-type and E167K variant to identify rate-limiting steps

Research has shown that TM6SF2 affects both "the degree of lipidation and the number of secreted VLDL particles" . Advanced lipidomic approaches combined with antibody techniques can help elucidate exactly which lipid species are affected and how this impacts VLDL structure and metabolism.

What emerging technologies might enhance the application of TM6SF2 antibodies in metabolic disease research?

Several cutting-edge technologies show promise for enhancing TM6SF2 antibody applications in metabolic research:

• Single-Cell Protein Analysis:

  • Mass cytometry (CyTOF) using metal-conjugated TM6SF2 antibodies

  • Single-cell western blotting to assess TM6SF2 expression heterogeneity within liver tissue

  • Integration with single-cell transcriptomics for multi-omic analysis

• Advanced Imaging Techniques:

  • Super-resolution microscopy (STORM, PALM) for nanoscale localization of TM6SF2

  • Live-cell imaging with split fluorescent protein systems to monitor TM6SF2-APOB interactions in real-time

  • Correlative light and electron microscopy (CLEM) for ultrastructural context of TM6SF2 localization

• Organoid and 3D Culture Systems:

  • Application of TM6SF2 antibodies in liver organoid systems derived from patient samples

  • 3D hepatocyte spheroid models that better recapitulate physiological VLDL secretion

  • Microfluidic "liver-on-a-chip" systems with integrated antibody-based detection

• CRISPR-Based Protein Tagging:

  • CRISPR knock-in of epitope tags for improved antibody detection of endogenous TM6SF2

  • Combining with split-protein complementation assays for studying protein-protein interactions

• Patient-Derived Models:

  • TM6SF2 antibody applications in hepatocyte spheroids derived from E167K carriers versus controls

  • Induced pluripotent stem cell (iPSC)-derived hepatocyte models from patients with different TM6SF2 genotypes