GPBAR1 Antibody, HRP conjugated

What is GPBAR1 and why is it important in research?

GPBAR1 (G protein-coupled bile acid receptor 1), also known as TGR5, is a 330-amino acid protein belonging to the G-protein coupled receptor 1 family. This membrane-associated receptor is sensitive to bile acids and responds through signaling mechanisms that coordinate energy homeostasis . GPBAR1 is important in research because it represents a potential therapeutic target for common metabolic diseases including obesity, type II diabetes, hyperlipidemia, and atherosclerosis . Furthermore, recent studies have demonstrated GPBAR1's crucial role as a gatekeeper for liver NKT cells, providing protection in models of hepatitis by attenuating inflammation .

What are the primary applications for GPBAR1 antibodies in research?

GPBAR1 antibodies are utilized across multiple experimental applications including:

• Western Blot (WB) for protein expression quantification

• Immunohistochemistry (IHC-P) for tissue localization studies

• Immunofluorescence (IF) for cellular localization

• Flow Cytometry (FCM) for cell population analysis

• ELISA for quantitative protein measurement

The selection of application depends on the specific research question being addressed. HRP-conjugated antibodies are particularly valuable for techniques requiring enzymatic detection systems, such as WB, ELISA, and IHC .

What is the difference between polyclonal and monoclonal GPBAR1 antibodies?

Polyclonal GPBAR1 antibodies, such as the one described as "bs-8874R," recognize multiple epitopes on the GPBAR1 protein, providing enhanced sensitivity but potentially less specificity . These antibodies are typically produced in rabbits using KLH-conjugated synthetic peptides derived from human GPBAR1. In contrast, monoclonal antibodies target a single epitope, offering higher specificity but potentially lower sensitivity. For detecting low-abundance GPBAR1 in certain tissue types, polyclonal antibodies with HRP conjugation provide optimal signal amplification while maintaining acceptable specificity profiles.

How should I optimize Western blot protocols for GPBAR1 detection using HRP-conjugated antibodies?

For optimal Western blot detection of GPBAR1 using HRP-conjugated antibodies:

• Sample preparation: Use RIPA buffer with protease inhibitors; heat samples at 70°C (not 100°C) to prevent GPBAR1 aggregation

• Loading control: Include both membrane (Na⁺/K⁺-ATPase) and cytosolic (GAPDH) controls due to GPBAR1's membrane localization

• Transfer: Use PVDF membranes (not nitrocellulose) with 0.2μm pore size

• Blocking: 5% non-fat milk in TBST for 2 hours at room temperature

• Primary antibody: Use 1:500-1:1000 dilution in 5% BSA/TBST overnight at 4°C

• Secondary antibody: If using non-conjugated primary, select species-appropriate HRP-conjugated secondary

• Detection: Use enhanced chemiluminescence with 1-3 minute exposure

This protocol has been validated in experimental models examining GPBAR1 expression in various tissue types .

What controls should I include when using GPBAR1 antibodies?

Effective experimental design requires appropriate controls:

• Positive controls: DN32.D3 cells and RAW264.7 cells express detectable GPBAR1 levels, as confirmed by both Western blot and IHC analyses

• Negative controls: Omit primary antibody in parallel samples

• Specificity controls: Use GPBAR1 knockout or knockdown samples when available

• Loading controls: For membrane proteins, use Na⁺/K⁺-ATPase rather than traditional cytosolic markers

• Peptide competition: Pre-incubate antibody with the immunizing peptide to validate specificity

The research by Wang et al. demonstrated similar levels of GPBAR1 mRNA in DN32.D3 cells, RAW264.7 cells, and spleen cells isolated from GPBAR1+/+ mice, making these suitable positive control sources .

How do I troubleshoot non-specific binding with HRP-conjugated GPBAR1 antibodies?

When encountering non-specific binding:

• Increase blocking time (3-4 hours) and concentration (5-10% BSA or milk)

• Optimize antibody dilution: Test serial dilutions from 1:250 to 1:2000

• Perform additional washing steps: 5-6 washes of 10 minutes each

• Add 0.1-0.2% Tween-20 to washing buffer

• Use protein-free blocking agents if background persists

• Pre-adsorb antibody with tissue homogenate from negative control samples

• Reduce substrate development time for HRP detection

• If membrane-associated background persists, consider 0.05% SDS in washing buffer

For membrane proteins like GPBAR1, non-specific binding often occurs due to hydrophobic interactions. The storage buffer containing 1% BSA, 0.02% Proclin300, and 50% Glycerol helps minimize this issue .

How can I use GPBAR1 antibodies to study its role in NKT cell regulation?

GPBAR1 functions as a critical regulator of liver NKT cell populations. To investigate this relationship:

• Isolate liver mononuclear cells using Percoll gradient centrifugation

• Perform flow cytometry using HRP-conjugated GPBAR1 antibodies with NKT cell markers (NK1.1, TCRβ)

• Sort NKT cells into different subpopulations (NKT1, NKT2, NKT10)

• Analyze cytokine profiles (IFN-γ, IL-4, IL-10) in response to GPBAR1 activation

• Conduct adoptive transfer experiments with GPBAR1+/+ vs. GPBAR1-/- NKT cells

• Perform ChIP assays to analyze CREB binding to IL-10 promoter following GPBAR1 activation

Recent research demonstrates that GPBAR1 agonism redirects NKT cell polarization toward an IL-10 secreting NKT10 regulatory phenotype. The absence of GPBAR1 biases toward a proinflammatory NKT1 phenotype producing IFN-γ .

What experimental approaches can elucidate GPBAR1's role in hepatic inflammation?

To investigate GPBAR1's role in hepatitis models:

• Compare WT vs. GPBAR1-/- mice in Con A or α-GalCer hepatitis models

• Measure liver enzyme levels (AST, ALT) at defined timepoints post-induction

• Use HRP-conjugated GPBAR1 antibodies for immunohistochemical analysis of liver sections

• Examine inflammatory infiltrates with multi-color immunofluorescence

• Analyze cytokine profiles in liver homogenates and serum

• Test protective effects of GPBAR1 agonists (like BAR501)

• Perform adoptive transfer of NKT cells between genotypes

Research has shown that GPBAR1 genetic ablation worsens liver injury in hepatitis models, while GPBAR1 agonism protects against acute liver damage. The peak of liver injury typically occurs 24 hours after α-GalCer administration, with significantly elevated AST and ALT levels .

How can I investigate GPBAR1 signaling pathways using specific antibodies?

To study GPBAR1 downstream signaling:

• Stimulate cells with bile acids or selective agonists like BAR501

• Use phospho-specific antibodies to detect activated signaling components:

  • Phosphorylated CREB (pCREB) - a direct target of GPBAR1 activation

  • cAMP levels - using ELISA or FRET-based reporters

  • PKA activity assays

• Perform ChIP assays to examine CREB binding to target promoters (particularly IL-10)

• Use immunoprecipitation with GPBAR1 antibodies to identify interacting proteins

• Employ proximity ligation assays to visualize protein interactions in situ

Research has demonstrated that GPBAR1 activation leads to phosphorylation of CREB, which binds to cAMP response elements. CREB binding to the IL-10 promoter is reduced by Con A treatment but restored by BAR501 treatment, highlighting a critical mechanism in the anti-inflammatory effects of GPBAR1 activation .

What are the recommended storage and handling conditions for HRP-conjugated GPBAR1 antibodies?

For optimal stability and performance:

• Storage temperature: Store at -20°C for long-term preservation

• Aliquoting: Divide into single-use aliquots to avoid freeze/thaw cycles

• Buffer composition: Most stable in 0.01M TBS (pH 7.4) with 1% BSA, 0.02% Proclin300, and 50% Glycerol

• Freeze/thaw cycles: Limit to 3-5 cycles maximum

• Working dilutions: Prepare fresh and use within 24 hours

• Light sensitivity: Protect from direct light exposure

• Shipping: Antibodies are typically shipped at 4°C but should be stored at -20°C upon arrival

Following these guidelines ensures antibody integrity and consistent experimental results. Repeated freeze/thaw cycles should be avoided as they can compromise HRP enzymatic activity and antibody binding properties .

How do different fixation methods affect GPBAR1 antibody performance in immunohistochemistry?

Fixation methods significantly impact GPBAR1 detection:

For optimal results with HRP-conjugated GPBAR1 antibodies in IHC-P applications, use 4% paraformaldehyde fixation followed by citrate buffer (pH 6.0) antigen retrieval. GPBAR1's membrane localization requires careful optimization of permeabilization steps to maintain structural integrity while allowing antibody access .

What approaches can validate the specificity of GPBAR1 antibodies in experimental systems?

To ensure antibody specificity:

• Genetic validation: Test antibodies on tissues from GPBAR1 knockout mice

• siRNA/shRNA knockdown: Compare staining in control vs. GPBAR1-silenced cells

• Peptide competition: Pre-incubate antibody with immunizing peptide

• Multiple antibody validation: Use antibodies targeting different GPBAR1 epitopes

• Recombinant expression: Test in overexpression systems with tagged GPBAR1

• Western blot confirmation: Verify single band at expected molecular weight (37-40 kDa)

• Cross-species reactivity: Check consistency across human, mouse, and rat samples

Research has confirmed specificity of certain GPBAR1 antibodies in both Western blot and IHC analyses, with consistent detection in known GPBAR1-expressing cell lines like DN32.D3 and RAW264.7 .

How can GPBAR1 antibodies be used to study metabolic disease mechanisms?

GPBAR1 represents a promising therapeutic target for metabolic disorders. To investigate its role:

• Compare GPBAR1 expression in normal vs. diseased tissue samples using quantitative IHC with HRP-conjugated antibodies

• Analyze hepatic GPBAR1 levels in diet-induced obesity models

• Correlate GPBAR1 expression with metabolic parameters (glucose, insulin, lipids)

• Examine tissue-specific GPBAR1 localization in adipose tissue, muscle, and liver

• Investigate effects of GPBAR1 agonists on energy expenditure and glucose metabolism

• Study co-localization with metabolic regulators using dual immunofluorescence

TGR5-controlled signaling pathways represent potential drug targets for treating common metabolic diseases including obesity, type II diabetes, hyperlipidemia, and atherosclerosis. GPBAR1 antibodies are essential tools for validating target engagement in preclinical studies .

What methods can be used to study GPBAR1 interaction with bile acids?

To investigate the GPBAR1-bile acid interaction:

• Binding assays: Use labeled bile acids and GPBAR1-expressing cells

• Functional assays: Measure cAMP production following bile acid stimulation

• Conformational studies: Analyze receptor changes using conformation-specific antibodies

• Cellular trafficking: Track GPBAR1 internalization after bile acid exposure using immunofluorescence

• Signaling activation: Monitor MAP kinase pathway activation and CREB phosphorylation

Research has demonstrated that bile acids activate GPBAR1, leading to MAP kinase pathway activation, receptor internalization, increased GTP binding in membrane fractions, and rapid intracellular cAMP production. These mechanisms coordinate energy homeostasis and can be effectively studied using GPBAR1-specific antibodies .