ABCG8 Antibody

What is ABCG8 and why is it important in sterol transport research?

ABCG8 forms an obligate heterodimer with ABCG5 that mediates Mg(2+)- and ATP-dependent sterol transport across cell membranes. This heterodimer plays an essential role in the selective transport of dietary cholesterol in and out of enterocytes and in the selective sterol excretion by the liver into bile. It is required for normal sterol homeostasis, with the ABCG5/ABCG8 complex having ATPase activity that drives this transport process .

The importance of ABCG8 is highlighted by the fact that defects in this protein cause sitosterolemia (also known as phytosterolemia), a rare autosomal recessive disorder characterized by increased intestinal absorption of all sterols and decreased biliary excretion of dietary sterols into bile . Furthermore, genetic variations in ABCG8 can be associated with susceptibility to gallbladder disease type 4 (GBD4) .

How do I choose between polyclonal and monoclonal ABCG8 antibodies for my research?

The choice depends on your specific research requirements:

Polyclonal Antibodies:

• Recognize multiple epitopes, increasing detection sensitivity

• Often suitable for diverse applications (WB, IHC-P, ICC/IF)

• Examples include ab126493 and ABIN5518720, which recognize regions in the middle section of ABCG8

Monoclonal Antibodies:

• Provide high specificity for a single epitope

• Offer greater consistency between batches

• May have conformation-specific binding properties

• Examples include antibody 1B10A5 and the mAbs 2E10 and 11F4 used in cryo-EM studies

For structural studies or when investigating specific functional domains, monoclonal antibodies like mAbs 2E10 and 11F4 provide high-affinity (around 100 pM) binding to distinctive epitopes on ABCG5/G8, making them valuable for techniques like cryo-EM .

What applications are most commonly supported by commercial ABCG8 antibodies?

Based on the search results, commercial ABCG8 antibodies support various applications:

When selecting an antibody, verify that it has been validated for your specific application and species of interest .

What are the optimal conditions for using ABCG8 antibodies in Western blotting?

For Western blotting with ABCG8 antibodies, follow these methodological considerations:

• Concentration: Use at 0.1-0.5 μg/mL for antibodies like ABIN5518720 or at 1/2000 dilution for antibodies like ab223056

• Sample preparation:

  • The observed molecular weight of ABCG8 is approximately 76 kDa (calculated MW: 75679)

  • Ensure complete protein denaturation in SDS-PAGE

  • Include protease inhibitors during extraction to prevent degradation

• Detection system:

  • Chemiluminescence detection is recommended (e.g., ABIN921124 can be used with ABIN5518720)

  • HRP-conjugated secondary antibodies provide good sensitivity

• Controls:

  • Include positive control (e.g., liver tissue lysate)

  • Consider using ABCG8-knockout samples as negative controls when available

• Buffer conditions:

  • BSA-containing blocking buffers may work better than milk-based ones

  • PVDF membranes may provide better results than nitrocellulose for this transmembrane protein

How can I optimize immunohistochemistry protocols for ABCG8 detection in tissue samples?

For optimal IHC detection of ABCG8 in tissue samples:

• Fixation: Use 10% neutral buffered formalin; overfixation may mask epitopes

• Antigen retrieval:

  • Heat-induced epitope retrieval (HIER) in citrate buffer pH 6.0

  • For antibodies like ab126493, optimize retrieval conditions based on tissue type

• Antibody concentration:

  • Start with manufacturer recommendations and titrate

  • For most ABCG8 antibodies, a 1:100-1:500 dilution range is appropriate

• Detection system:

  • Amplification systems like HRP-polymer or avidin-biotin complex enhance sensitivity

  • Consider fluorescent detection for co-localization studies with ABCG5

• Tissue considerations:

  • Primary sites of interest: liver (hepatocytes) and intestine (enterocytes)

  • Use of positive and negative control tissues is essential

  • Cross-check expression patterns with mRNA expression data

• Validation controls:

  • Blocking peptide controls

  • Comparison with other validated ABCG8 antibodies

  • Correlation with functional assays

How can antibodies be used to study the functional relationship between ABCG5 and ABCG8?

ABCG8 antibodies can provide valuable insights into the functional relationship between ABCG5 and ABCG8 through several methodological approaches:

• Co-immunoprecipitation assays:

  • Use ABCG8 antibodies to pull down the protein complex

  • Analyze the composition to confirm heterodimer formation

  • Investigate associated proteins that may regulate the complex

• Proximity ligation assays:

  • Combine ABCG5 and ABCG8 antibodies for in situ detection of the heterodimer

  • Quantify interaction signals under different experimental conditions

• Modulation of transporter activity:

  • Certain antibodies can directly affect ATPase activity, as demonstrated with mAbs 2E10 and 11F4

  • mAb 2E10 inhibits ATPase activity (IC50 of 49.4 nM), while mAb 11F4 potentiates it (EC50 of 67.2 nM)

  • These functional effects reveal insights into the allosteric regulation of the transporter

• Structural studies:

  • Antibody fragments (Fabs) facilitate cryo-EM structure determination by increasing the size of the protein complex

  • The cryo-EM structure of ABCG5/G8 with Fab fragments was resolved at 3.3Å resolution

  • Such studies reveal the coupling mechanism between nucleotide-binding domains (NBDs) and transmembrane domains (TMDs)

• Trafficking studies:

  • Use fluorescently-labeled ABCG8 antibodies to track the localization and movement of the transporter in live cells

  • Investigate how mutations affect heterodimer formation and cellular localization

What approaches can be used to investigate ABCG8 interactions with sterols using antibody-based methods?

Investigating ABCG8 interactions with sterols requires sophisticated antibody-based approaches:

• Conformational antibodies:

  • Use conformation-specific antibodies that recognize ABCG8 in different states of the transport cycle

  • Changes in antibody binding can indicate conformational shifts upon sterol binding

• ABCG8 immunoprecipitation coupled with lipid analysis:

  • Capture ABCG8 with antibodies and analyze co-precipitated lipids by mass spectrometry

  • Compare lipid profiles under different conditions (e.g., cholesterol loading)

• In vitro reconstitution systems:

  • Purify ABCG5/G8 using immunoaffinity methods

  • Study ATPase activity in reconstituted proteoliposomes with varying sterol compositions

  • Research has shown 1:1 stoichiometry for ATP hydrolysis and cholesterol transport, with bile acids stimulating ATP hydrolysis

• Antibody inhibition studies:

  • Use antibodies that bind to specific domains to inhibit transporter function

  • Analyze the effects on sterol transport in cellular models

• Biolayer interferometry or SPR with antibody capture:

  • Immobilize ABCG8 using antibodies

  • Measure direct binding kinetics of various sterols to the immobilized transporter

How do ABCG8 antibodies contribute to understanding sitosterolemia pathophysiology?

ABCG8 antibodies provide critical tools for understanding sitosterolemia pathophysiology through several methodological approaches:

• Expression analysis in patient samples:

  • Quantify ABCG8 protein levels in intestinal and liver biopsies

  • Compare expression patterns between patients with different mutations

  • Correlate protein expression with clinical phenotypes

• Animal models characterization:

  • ABCG8 antibodies have been used to study knock-out mouse models that recapitulate sitosterolemia

  • G5G8−/− mice show extremely low biliary cholesterol concentrations (0.4 vs. 5.5 μmol/ml in wild-type), increased plasma sitosterol (≈30-fold), and increased absorption of dietary plant sterols (2- to 3-fold)

  • Immunodetection helps validate the model and correlate with physiological findings

• Mechanistic studies:

  • Investigation of how mutations affect protein stability, dimerization, and trafficking

  • Some mutations might allow protein expression but prevent proper function

  • Antibodies targeting different epitopes can help distinguish between trafficking and catalytic defects

• Therapy development:

  • Monitor changes in ABCG8 expression or localization in response to therapeutic interventions

  • Evaluate potential compounds that might rescue specific mutations

• Macrothrombocytopenia and cardiomyopathy investigations:

  • ABCG8 antibodies help study the broader systemic effects of sitosterolemia

  • Research has revealed that Abcg8−/− mice develop macrothrombocytopenia and cardiomyopathy characterized by phytosterol accumulation

How can I address weak or absent ABCG8 signal in Western blotting experiments?

When troubleshooting weak or absent ABCG8 signals in Western blot:

• Sample preparation optimization:

  • Ensure complete protein extraction using detergent mixtures suitable for membrane proteins

  • Avoid repeated freeze-thaw cycles of samples

  • Include protease inhibitors to prevent degradation

• Antibody selection and handling:

  • Verify the antibody is recognizing the correct region of ABCG8 present in your samples

  • For lyophilized antibodies like ABIN5518720, ensure proper reconstitution (add 0.2 mL distilled water for 500 μg/mL)

  • Follow storage recommendations (e.g., store at -20°C, avoid repeated freeze-thaw cycles)

• Protocol adjustments:

  • Increase antibody concentration (e.g., use 0.5 μg/mL instead of 0.1 μg/mL)

  • Extend incubation time (overnight at 4°C)

  • Consider using more sensitive detection methods (enhanced chemiluminescence)

• Transfer efficiency:

  • For this 76 kDa membrane protein, optimize transfer conditions

  • Consider wet transfer at lower voltage for longer periods

• Positive controls:

  • Include tissue samples known to express high levels of ABCG8 (liver, intestine)

  • Consider using recombinant ABCG8 protein as a positive control

What are the best approaches for validating ABCG8 antibody specificity?

To validate ABCG8 antibody specificity:

• Multiple antibody comparison:

  • Use antibodies targeting different epitopes of ABCG8

  • Compare staining patterns across applications

  • Example: Compare antibodies targeting middle region (AA 328-371) like ABIN5518720 with those targeting other regions

• Genetic models:

  • Use ABCG8 knockout tissues or cells as negative controls

  • Abcg8−/− mice provide ideal negative controls for antibody validation

• Peptide blocking:

  • Pre-incubate antibody with the immunizing peptide

  • Specific signal should be eliminated or significantly reduced

  • For antibodies like ABIN5518720, the synthetic peptide corresponding to human ABCG8 (328-371aa) can be used

• Orthogonal methods:

  • Correlate protein detection with mRNA expression (qPCR)

  • Verify subcellular localization matches expected pattern (apical membrane in hepatocytes and enterocytes)

• Cross-reactivity testing:

  • Verify absence of signal in tissues not expressing ABCG8

  • Confirm antibody shows expected species reactivity

  • Many ABCG8 antibodies are reactive to human, mouse, and rat samples

How can immunoprecipitation protocols be optimized for ABCG8 as a membrane protein?

Optimizing immunoprecipitation (IP) for ABCG8 requires specific considerations for membrane proteins:

• Solubilization strategy:

  • Use mild detergents like digitonin, DDM, or CHAPS that preserve protein-protein interactions

  • Optimize detergent concentration to balance solubilization efficiency and preservation of interactions

  • Consider crosslinking before lysis to stabilize transient interactions

• Antibody selection:

  • Choose antibodies with high affinity and specificity

  • Consider using monoclonal antibodies like mAbs 2E10 and 11F4 that have demonstrated high affinity (~100 pM)

  • Ensure antibody epitope is accessible in the native protein

• Immunoprecipitation system:

  • Direct antibody conjugation to beads may reduce background

  • Pre-clear lysates thoroughly to reduce non-specific binding

  • Consider using recombinant protein A/G beads for efficient capture

• Buffer conditions:

  • Include appropriate ions (e.g., Mg2+ for stabilizing the ABC transporter)

  • Adjust salt concentration to reduce non-specific interactions

  • Consider adding cholesterol or bile acids that may stabilize the transporter complex

• Co-immunoprecipitation considerations:

  • For studying ABCG5/G8 heterodimer, determine whether antibody binding affects complex formation

  • IP with anti-ABCG8 followed by ABCG5 detection can confirm heterodimer formation

  • Validate results with reciprocal IP (anti-ABCG5 followed by ABCG8 detection)

How are ABCG8 antibodies being used in cryo-EM structural studies?

ABCG8 antibodies have become indispensable tools in cryo-EM structural studies through several methodological approaches:

• Size enhancement for improved image processing:

  • The ABCG5/G8 heterodimer (~150 kDa) poses challenges for high-resolution cryo-EM

  • Addition of antigen-binding fragments (Fabs) increases protein size by ~47 kDa, improving signal-to-noise ratio

  • This facilitates image alignment and three-dimensional reconstruction

• Fab generation and screening:

  • Researchers generate panels of antibodies and screen for high-affinity, conformation-specific binders

  • Antibodies 2E10 and 11F4 were identified as high-affinity binders (~100 pM) to distinctive epitopes

  • Epitope binning experiments using surface plasmon resonance (SPR) help identify antibodies binding to different regions

• Structural insights from antibody complexes:

  • The cryo-EM structure of human ABCG5/G8 with Fabs was resolved to 3.3 Å resolution

  • Analysis revealed that both Fab 2E10 and Fab 11F4 predominantly bind to the NBD of ABCG8

  • This provided insights into the coupling mechanism between nucleotide-binding and transmembrane domains

• Functional modulation by antibodies:

  • Interestingly, mAb 2E10 inhibits ATPase activity (IC50 of 49.4 nM)

  • In contrast, mAb 11F4 potentiates ATPase activity (EC50 of 67.2 nM)

  • These opposing effects provide insights into allosteric regulation of transporter activity

• Epitope analysis:

  • Fab 2E10 interacts with both RecA and helical domains of the NBD from ABCG8

  • The total buried surface area between Fab 2E10 and ABCG8 is ~1640 Ų

  • Such detailed structural information helps understand protein dynamics and function

What role do ABCG8 antibodies play in investigating sterol homeostasis mechanisms?

ABCG8 antibodies are crucial for investigating various aspects of sterol homeostasis:

• Fractional cholesterol absorption studies:

  • Antibodies help characterize ABCG8 expression in models with altered cholesterol absorption

  • Studies in transgenic mice with both intestine and liver overexpression of human ABCG5/G8 showed decreased fractional cholesterol absorption and increased fecal neutral sterol excretion

• Bile acid-dependent regulation:

  • Immunodetection of ABCG8 helps understand how bile acids affect sterol transport

  • Research has shown that bile acids stimulate ATPase activity, with cholate being most potent, while taurine or glycine conjugates were approximately 50% less effective

• Sterol floppase mechanism:

  • Antibodies aid in investigating the proposed floppase mechanism

  • ABCG5/G8 promotes cholesterol movement to the exofacial leaflet of the canalicular membrane

  • Immunodetection in isolated membranes showed less cholesterol in canalicular membranes of ABCG8-deficient mice

• Regulation of biliary cholesterol secretion:

  • ABCG8 antibodies help quantify expression levels in response to various stimuli

  • Studies in G5G8−/− mice showed extremely low biliary cholesterol concentrations (0.4 vs. 5.5 μmol/ml in wild-type)

  • Immunodetection helps correlate expression levels with functional outcomes

• Whole-body sterol trafficking:

  • Antibodies enable tracking of ABCG8 expression across tissues

  • ABCG5/ABCG8 are key in regulating whole-body sterol trafficking by eliminating sterols via the biliary tree and intestinal tract

• Dietary interventions:

  • Immunodetection helps assess how dietary changes affect ABCG8 expression

  • In G5G8−/− mice, plasma and liver cholesterol levels were reduced by 50% on chow diet but increased dramatically (2.4-fold in plasma, 18-fold in liver) after cholesterol feeding