GBP1 Antibody

What is GBP1 and why is it significant in immunological research?

GBP1 (Guanylate Binding Protein 1) is a 592 amino acid protein belonging to the GTPase protein family that plays crucial roles in cellular signaling and immune response. It functions by binding guanine nucleotides (GMP, GDP, GTP) and can hydrolyze GTP to GMP through two consecutive cleavage reactions. GBP1 is primarily expressed in endothelial cells of the vascular system and is induced by interferon-gamma during macrophage activation. Its significance in immunological research stems from its involvement in inflammatory cytokine regulation, protection against viral infections (including vesicular stomatitis and encephalomyocarditis viruses), and its role as a cellular activation marker characterizing inflammatory responses in endothelial cells. The protein also plays a key role in inflammasome assembly in response to Gram-negative bacterial infections by forming protein coats around pathogens, facilitating their detection by pattern recognition receptors.

What types of GBP1 antibodies are available for research applications?

Researchers have access to various types of GBP1 antibodies optimized for different experimental applications:

These antibodies are available from multiple suppliers with different validation profiles and applications, allowing researchers to select the most appropriate tool for their specific experimental needs.

How can I determine the optimal conditions for GBP1 antibody use in Western blotting?

Optimizing GBP1 antibody conditions for Western blotting requires systematic titration and protocol adjustments. Begin with a concentration range of 1:500 to 1:2000 dilution (for antibodies supplied at approximately 200 μg/ml) and test with positive control samples expressing GBP1. Interferon-gamma-treated endothelial cells or macrophages provide reliable positive controls, as GBP1 is upregulated following interferon treatment. A critical step is sample preparation: use ice-cold lysis buffer containing protease inhibitors to preserve protein integrity, and consider including phosphatase inhibitors if investigating phosphorylated forms of GBP1. When optimizing blocking conditions, 5% non-fat milk in TBST is generally effective, but for phospho-specific GBP1 antibodies, BSA-based blocking solutions often yield better results. For detection, the standard protein loading amount is approximately 30 μg per well, but this may require adjustment based on GBP1 expression levels in your experimental system. Finally, confirm specificity through GBP1 knockdown or knockout controls to validate that the detected band represents authentic GBP1 protein.

What are the most effective methods for validating GBP1 antibody specificity?

Validating GBP1 antibody specificity requires a multi-faceted approach:

• Genetic controls: Use CRISPR/Cas9 knockout or siRNA knockdown models to confirm signal absence in GBP1-depleted samples. This was effectively demonstrated in THP-1Δ PIM1 cells where phospho-GBP1 signals were absent until reconstituted with functional PIM1.

• Recombinant protein testing: Test antibody against purified recombinant GBP1 protein alongside other GBP family members to assess cross-reactivity. Phospho-specific GBP1 antibodies should be validated against both phosphorylated and non-phosphorylated forms.

• Immunoprecipitation followed by mass spectrometry: This approach confirms that the antibody pulls down authentic GBP1 protein rather than cross-reactive proteins.

• Multiple detection methods: Validate antibody performance across multiple techniques (WB, IF, IHC) to ensure consistent target recognition.

• Peptide competition: Pre-incubate antibody with immunizing peptide to demonstrate signal blockade, confirming epitope specificity.

• Cross-species reactivity: Test antibody against GBP1 from different species to determine conservation of the epitope recognition.

For phospho-specific GBP1 antibodies, additional validation includes phosphatase treatment of samples to confirm loss of signal and use of phospho-mimetic (e.g., S156D) or phospho-dead (e.g., S156A) mutants to verify specificity for the phosphorylated epitope.

How does phosphorylation regulate GBP1 activity and what tools are available to study this process?

GBP1 activity is tightly regulated through phosphorylation, particularly at Ser156, which serves as a molecular switch controlling its function. PIM1 kinase has been identified as the primary kinase responsible for phosphorylating GBP1 at this residue. This phosphorylation creates a binding site for 14-3-3 proteins (especially 14-3-3σ), which lock GBP1 in a GTPase-inactive, monomeric state and restrain its activity in the macrophage cytosol. This regulatory mechanism prevents uncontrolled GBP1 activation that could lead to cell death.

To study this process, researchers can employ several specialized tools:

• Phospho-specific GBP1 antibodies that specifically detect GBP1 phosphorylated at Ser156

• GBP1 mutants (S156A or S156D) that mimic non-phosphorylated or constitutively phosphorylated states

• PIM1 inhibitors or the GBP1:PIM1 interaction inhibitor NSC756093

• Genetic tools for PIM1 or 14-3-3σ depletion

Cryo-electron microscopy has been used to visualize the 14-3-3σ dimer binding to the GBP1 GTPase domain, providing structural insights into this regulatory mechanism. The dynamism of this system is evident during pathogen infection: Toxoplasma gondii infection leads to depletion of PIM1, reducing GBP1 Ser156 phosphorylation and liberating GBP1 from 14-3-3σ sequestration, thereby enabling GBP1 to target pathogen-containing vacuoles.

What mechanisms control GBP1 interaction with 14-3-3 proteins and how can they be experimentally manipulated?

The interaction between GBP1 and 14-3-3 proteins is primarily mediated by phosphorylation at Ser156, which creates a canonical 14-3-3 binding motif. This interaction can be experimentally manipulated through several approaches:

• Site-directed mutagenesis: Creating GBP1 mutants that cannot be phosphorylated (S156A) or mimic constitutive phosphorylation (S156D) directly affects 14-3-3 binding. The S156A mutant cannot be recognized by 14-3-3 proteins, while the S156D mutant may show enhanced binding.

• Modulation of PIM1 activity: Since PIM1 is the kinase responsible for phosphorylating GBP1 at Ser156, manipulating PIM1 expression or activity directly impacts 14-3-3 binding. This can be achieved through:

  • PIM1 overexpression or knockdown

  • PIM1 kinase inhibitors

  • PIM1 kinase-dead mutants (e.g., PIM1 P81S)

  • The specific GBP1:PIM1 interaction inhibitor NSC756093

• 14-3-3 protein modulation: Direct manipulation of 14-3-3 proteins, particularly 14-3-3σ, affects complex formation with GBP1. This can be accomplished through:

  • 14-3-3σ knockdown or knockout

  • 14-3-3 inhibitory peptides or small molecules

  • Expression of dominant-negative 14-3-3 mutants

• Phosphatase treatment: Activating phosphatases that dephosphorylate GBP1 at Ser156 disrupts 14-3-3 binding.

Experimental validation of these interactions can be performed using co-immunoprecipitation assays with phospho-specific GBP1 antibodies, followed by detection of associated 14-3-3 proteins. Single-particle cryo-electron microscopy has been valuable for visualizing the structural basis of this interaction, confirming that a 14-3-3σ dimer binds to the GBP1 GTPase domain, locking it in an inactive conformation.

How does GBP1 contribute to inflammasome assembly during bacterial infection?

GBP1 plays a multifaceted role in inflammasome assembly during bacterial infection through several distinct mechanisms:

• Pathogen vacuole targeting and lysis: GBP1 can target and aid in the lysis of pathogen-containing vacuoles, releasing bacteria into the cytosol where they can be detected by cytosolic pattern recognition receptors.

• Bacterial coating and LPS exposure: Following pathogen release into the cytosol, GBP1 forms a protein coat around Gram-negative bacteria in a GTPase-dependent manner. This coating disrupts the bacterial O-antigen barrier and unmasks lipid A, which is then detected by the non-canonical inflammasome effector CASP4/CASP11.

• Bacterial cytolysis promotion: GBP1 recruits proteins that mediate bacterial cytolysis, leading to the release of double-stranded DNA (dsDNA) that activates the AIM2 inflammasome.

• Facilitation of PAMP detection: By encapsulating pathogens, GBP1 promotes the detection of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors, enhancing inflammasome activation.

The importance of GBP1 phosphorylation state in this process is evident from studies showing that infection with Toxoplasma gondii, which inhibits IFN-γ signaling via its effector protein TgIST, leads to rapid depletion of PIM1. This reduces GBP1 Ser156 phosphorylation, liberates GBP1 from 14-3-3σ sequestration, and enables GBP1 to target pathogen-containing vacuoles more effectively.

What experimental approaches are most effective for studying GBP1's role in pathogen clearance?

To effectively study GBP1's role in pathogen clearance, researchers should employ a combination of cellular, biochemical, and imaging approaches:

• Infection models:

  • Cell-based: Macrophage or epithelial cell infection with bacteria (e.g., Gram-negative bacteria) or protozoa (e.g., Toxoplasma gondii)

  • In vivo: Mouse models with GBP1 knockout or conditional expression

  • Organoid systems: Patient-derived organoids to study GBP1 function in a more physiologically relevant context

• Genetic manipulation techniques:

  • CRISPR/Cas9 knockout of GBP1

  • Expression of GBP1 mutants (e.g., phosphorylation-deficient S156A)

  • Modulation of GBP1 regulators (PIM1, 14-3-3σ)

• High-resolution imaging:

  • Live-cell imaging to track GBP1 recruitment to pathogen vacuoles

  • Super-resolution microscopy to visualize GBP1 coating of pathogens

  • Multicolor immunofluorescence to examine co-localization with inflammasome components

• Biochemical approaches:

  • GTPase activity assays to measure GBP1 function

  • Co-immunoprecipitation to identify GBP1 interacting partners during infection

  • Phospho-specific antibodies to track GBP1 activation state

• Quantitative pathogen clearance assays:

  • CFU (colony-forming unit) assays for bacterial survival

  • Parasite replication assays

  • Flow cytometry-based infection quantification

• Inflammasome activation readouts:

  • IL-1β and IL-18 secretion (ELISA)

  • Caspase-1 activation assays

  • Pyroptosis measurement (LDH release, propidium iodide uptake)

High-throughput imaging has been particularly valuable for studying the kinetics of GBP1 targeting to Toxoplasma-containing vacuoles following PIM1 depletion, providing insights into temporal aspects of GBP1-mediated pathogen clearance.

How is GBP1 involved in cancer biology and what experimental approaches reveal its role?

GBP1's involvement in cancer biology is complex and sometimes contradictory, necessitating careful experimental approaches to elucidate its role:

• Expression analysis: GBP1 expression has been documented in various cancers, including cervical cancer, where bioinformatic analysis of TCGA and GTEx databases reveals widespread expression. This can be validated through:

  • Multicolor immunofluorescence to assess GBP1 expression in tumor tissues

  • Western blot analysis of tumor versus normal tissue samples

  • Single-cell RNA sequencing to identify GBP1-expressing cell populations within tumors

• Functional studies: Knockdown and overexpression experiments in cancer cell lines help determine whether GBP1 acts as a tumor suppressor or promoter. In cervical cancer, both in vitro and in vivo experiments have suggested GBP1 may function as a potential oncogene.

• Mechanism exploration: RNA-seq analysis of GBP1 knockdown and overexpression cell lines has revealed that GBP1 affects numerous RNA alternative splicing events. Although GBP1 itself is not a direct alternative splicing factor (as demonstrated through RNA binding protein immunoprecipitation assays), co-immunoprecipitation coupled with mass spectrometry has identified interactions with alternative splicing factors like Heterogeneous Nuclear Ribonucleoprotein K (HNRNPK).

• Immune microenvironment interaction: Given GBP1's role in immune function, analyzing its relationship with tumor immune infiltration through:

  • Correlation analysis with immune cell markers

  • Spatial transcriptomics to map GBP1-expressing cells relative to immune infiltrates

  • Functional assays examining how GBP1 modulation affects immune cell recruitment and function

• Therapeutic targeting: The GBP1:PIM1 interaction inhibitor NSC756093 has shown promising results in patient-derived tumor organoids, increasing organoid death and preventing organoid reformation, suggesting potential therapeutic applications for disrupting PIM1-driven control of GBP1.

What is the relationship between GBP1 expression and inflammatory skin diseases?

GBP1 expression is markedly elevated in vessels of inflammatory skin diseases such as psoriasis and Kaposi's sarcoma, establishing it as a novel cellular activation marker that characterizes inflammatory responses in endothelial cells. This relationship can be investigated through several experimental approaches:

• Immunohistochemical analysis: Using GBP1 antibodies to compare expression patterns between healthy skin and diseased tissue samples. This approach allows visualization of GBP1 localization and quantification of expression levels.

• Correlation with inflammatory markers: Assessing the relationship between GBP1 expression and other inflammatory cytokines or cellular markers in skin biopsies to understand the inflammatory context.

• Cell-specific expression analysis: Using multicolor immunofluorescence to determine which cell types (endothelial cells, keratinocytes, immune cells) express GBP1 in diseased skin.

• In vitro modeling: Treating skin cell cultures or skin organoids with inflammatory mediators (particularly IFN-γ) to recapitulate disease conditions and monitor GBP1 upregulation.

• Functional consequences: Investigating how GBP1 upregulation affects endothelial cell proliferation and invasiveness through matrix metalloproteinase-1 regulation, which may contribute to the vascular changes observed in these diseases.

The role of GBP1 as a marker of inflammatory responses in endothelial cells suggests it could serve as a biomarker for disease activity or a potential therapeutic target in inflammatory skin conditions. The ability to specifically detect GBP1 using validated antibodies enables researchers to monitor its expression in patient samples and experimental models, providing insights into disease mechanisms and potential intervention strategies.

What are the critical factors for successful GBP1 detection in immunofluorescence and immunohistochemistry?

Successful GBP1 detection in immunofluorescence (IF) and immunohistochemistry (IHC) requires careful consideration of several technical factors:

• Fixation method: GBP1 detection is typically most effective with paraformaldehyde fixation (4% PFA) for 10-15 minutes for cells and 24-48 hours for tissues. Overfixation can mask epitopes, while underfixation may compromise cellular morphology.

• Permeabilization conditions: As a cytoplasmic protein, GBP1 requires effective membrane permeabilization. For IF, 0.1-0.5% Triton X-100 for 5-10 minutes typically provides adequate access while preserving cellular structures. For FFPE tissue sections, antigen retrieval is critical.

• Antigen retrieval: For IHC on paraffin sections, heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is generally effective. The optimal retrieval conditions should be determined empirically for each antibody.

• Antibody selection: Choose antibodies validated specifically for IF/IHC applications. The GBP1 Antibody (1B1) and Anti-GBP1 antibody [OTI1B2] have been validated for these applications. For phospho-specific detection, specialized phospho-GBP1 antibodies are required.

• Signal amplification: For low-abundance expression, consider using fluorophore-conjugated secondary antibodies with higher quantum yields or signal amplification systems like tyramide signal amplification (TSA).

• Controls:

  • Positive control: IFN-γ-treated endothelial cells or macrophages

  • Negative control: GBP1 knockout cells or tissues

  • Absorption control: Pre-incubation of antibody with immunizing peptide

  • Secondary-only control: To assess background fluorescence

• Multiplexing considerations: When performing multi-color IF, carefully select fluorophore combinations to minimize spectral overlap, and perform appropriate compensation controls. The availability of GBP1 antibodies conjugated to various fluorophores (FITC, PE, Alexa Fluor) facilitates multiplexing approaches.

For phospho-specific GBP1 detection, additional precautions include using phosphatase inhibitors during sample preparation and blocking with BSA rather than milk-based blockers to avoid phosphatases in milk.

How can I troubleshoot non-specific binding or weak signals when using GBP1 antibodies?

Troubleshooting non-specific binding or weak signals with GBP1 antibodies requires a systematic approach:

For Non-specific Binding:

• Optimize blocking conditions:

  • Increase blocking time (1-2 hours at room temperature or overnight at 4°C)

  • Try different blocking agents (5% BSA, 5-10% normal serum from the species of secondary antibody, commercial blockers)

  • For Western blots, PVDF membranes often show less background than nitrocellulose

• Antibody dilution optimization:

  • Test serial dilutions of primary antibody (typically starting at 1:200-1:1000)

  • Ensure secondary antibody is appropriately diluted (typically 1:1000-1:5000)

• Washing optimization:

  • Increase number and duration of washes (5-6 washes of 5-10 minutes each)

  • Add low concentrations of detergent to wash buffer (0.05-0.1% Tween-20)

• Pre-absorption of antibody:

  • Pre-incubate with cells/tissues lacking GBP1 expression

  • Commercial pre-absorption kits can reduce cross-reactivity

• Change detection system:

  • Try different secondary antibodies or detection systems

  • Consider monovalent Fab fragments to reduce non-specific binding

For Weak Signals:

• Antigen retrieval optimization:

  • Test different retrieval buffers (citrate pH 6.0, EDTA pH 9.0)

  • Optimize retrieval time and temperature

• Sample preparation:

  • Ensure proteins are not degraded during extraction

  • Include protease and phosphatase inhibitors

  • For phospho-GBP1 detection, treatment with pervanadate can enhance phosphorylation

• Signal amplification:

  • Employ tyramide signal amplification (TSA) systems

  • Use high-sensitivity detection reagents

  • Increase exposure time for WB or imaging time for IF

• Enhance target expression:

  • Use IFN-γ treatment to upregulate GBP1 expression

  • Transfect cells with GBP1 expression vectors as positive controls

• Antibody quality assessment:

  • Check antibody storage conditions and expiration

  • Perform dot blot analysis to confirm antibody activity

  • Consider testing multiple GBP1 antibodies targeting different epitopes

For both issues, refer to published studies using GBP1 antibodies for protocol guidance. Multiple commercial sources provide GBP1 antibodies with different host species, clonality, and epitope targets, allowing flexibility in experimental design and troubleshooting.

How are GBP1 antibodies being utilized to understand the protein's role in novel pathways and diseases?

GBP1 antibodies are increasingly being employed to explore novel roles of this protein beyond its established functions in immunity:

• Cancer immunology: Researchers are using GBP1 antibodies to investigate its expression and function in various cancer types. In cervical cancer, multicolor immunofluorescence combined with RNA-seq analysis has revealed GBP1's relationship with immune invasion and its potential role in controlling RNA splicing through interactions with splicing factors like HNRNPK. This approach is uncovering new connections between GBP1 and cancer progression pathways.

• Phospho-regulation networks: Phospho-specific GBP1 antibodies have been instrumental in identifying a novel regulatory mechanism involving PIM1 kinase and 14-3-3 proteins. This system acts as a molecular switch controlling GBP1's GTPase activity and cellular function, with implications for both normal immunity and disease states.

• Structural biology applications: GBP1 antibodies have facilitated the purification and structural analysis of GBP1 complexes. Single-particle cryo-electron microscopy of the 14-3-3σ/GBP1 complex has provided insights into how protein-protein interactions regulate GBP1 function at the molecular level.

• Systems biology approaches: Integration of GBP1 antibody-based proteomics with transcriptomics and bioinformatics is revealing GBP1's position in broader cellular networks. Correlation analyses between GBP1 expression and miRNAs, lncRNAs, and mutated genes are uncovering novel regulatory relationships.

• Therapeutic development: The GBP1:PIM1 interaction inhibitor NSC756093 demonstrates how understanding GBP1 regulation can lead to therapeutic applications. Antibody-based assays are essential for validating the efficacy of such inhibitors in disrupting protein interactions and modulating GBP1 function.

These emerging research directions highlight how GBP1 antibodies are not only tools for detecting the protein but are enabling comprehensive analysis of its multiple functions and regulatory mechanisms in health and disease.

What advances in GBP1 antibody technology are improving detection sensitivity and specificity?

Recent technological advances are significantly enhancing GBP1 antibody performance:

• Recombinant antibody production: The transition from hybridoma-derived to recombinant antibody technology has improved batch-to-batch consistency. Recombinant monoclonal antibodies like anti-GBP1 [1B1] from Abcam provide more reliable detection with reduced variability compared to traditional production methods.

• Epitope-specific antibody engineering: Advanced epitope mapping and antibody engineering are creating highly specific antibodies targeting distinct regions of GBP1. This includes phospho-specific antibodies that can discriminate between phosphorylated and non-phosphorylated forms at specific residues like Ser156, enabling precise monitoring of GBP1 activation states.

• Enhanced conjugation chemistry: Improved conjugation methods are producing antibodies with optimal fluorophore-to-antibody ratios, enhancing signal-to-noise ratios in fluorescence-based applications. GBP1 antibodies are now available with various conjugates, including agarose, HRP, PE, FITC, and multiple Alexa Fluor options, expanding multiplexing capabilities.

• Fragment-based technologies: Smaller antibody fragments (Fab, scFv) are being developed for applications requiring better tissue penetration or reduced non-specific binding. These formats maintain epitope specificity while offering improved access to densely packed cellular structures.

• Validation across multiple platforms: Comprehensive validation across multiple techniques (WB, IF, IHC, IP, ELISA) ensures antibody performance in diverse experimental contexts. Leading suppliers are implementing stringent validation protocols including genetic knockout controls.

• Application-optimized formulations: Buffer formulations specifically designed for particular applications enhance antibody performance. For instance, specialized formulations for multiplexed immunofluorescence reduce background and cross-reactivity in complex tissue samples.

• Nanobody and alternative scaffold technology: Smaller binding proteins derived from camelid antibodies or synthetic scaffolds offer advantages for certain applications, including improved stability and access to sterically hindered epitopes.

These advances collectively contribute to more sensitive and specific detection of GBP1, enabling researchers to address increasingly sophisticated questions about its expression, localization, interactions, and functional states.