GBP2 Antibody, HRP conjugated

What is GBP2 and why is it significant in immunological research?

GBP2 (Guanylate Binding Protein 2) is a 67.2 kDa member of the dynamin superfamily of large GTPases. It plays critical roles in innate immunity against diverse bacterial, viral, and protozoan pathogens. GBP2 is particularly significant because it serves as a marker of interferon (IFN) responsiveness, being one of the most highly expressed genes after IFN-γ stimulation . The protein hydrolyzes GTP to GMP in consecutive cleavage reactions, with GDP being the major reaction product . GBP2's importance in research stems from its involvement in inflammasome assembly, pathogen restriction, and its association with immune cell infiltration in various cancers .

What are the standard applications for GBP2 antibody (HRP conjugated)?

GBP2 antibody with HRP conjugation is primarily used in the following research applications:

The HRP conjugation eliminates the need for secondary antibody incubation, streamlining experimental workflows while maintaining high specificity and sensitivity in protein detection systems .

For maximum stability and performance of HRP-conjugated GBP2 antibodies, follow these evidence-based storage recommendations:

• Store at -20°C as received in the manufacturer's buffer formulation

• Maintain in aliquots to avoid repeated freeze-thaw cycles that can compromise HRP activity

• The antibody is typically stable for 12 months from the date of receipt when properly stored

• Buffer composition typically includes PBS (pH 7.3) containing 1% BSA and 50% glycerol for stability

• Avoid exposure to light, which can reduce HRP activity over time

How can I optimize antigen retrieval when using GBP2 antibody (HRP conjugated) for IHC applications?

Optimizing antigen retrieval is critical for successful IHC with GBP2 antibodies, particularly when using HRP conjugates. Based on published protocols:

• Buffer selection is critical: Data indicates superior results using TE buffer at pH 9.0 as the primary option for GBP2 detection in tissue samples

• Alternative method: Citrate buffer at pH 6.0 can serve as an alternative, though potentially with reduced epitope accessibility

• Pressure cooking method: For optimal epitope retrieval, use a pressure cooker with the selected buffer, boiling for 2.5 minutes as validated in multiple studies

• Sequential approach: After retrieval, treat sections with 3% hydrogen peroxide to quench endogenous peroxidase activity that could interfere with HRP signal specificity

• Blocking optimization: Following peroxidase quenching, incubate with appropriate blocking buffer to minimize non-specific binding before antibody application

These optimized retrieval conditions have been validated across multiple tissue types including spleen and tonsillitis samples, showing consistent GBP2 detection patterns .

What strategies can address weak signal issues when using GBP2 antibody (HRP conjugated) in Western blot applications?

When encountering weak signal problems with HRP-conjugated GBP2 antibodies in Western blot applications:

• Concentration adjustment: While standard dilution is 1:1000, persistent weak signal may require adjustment to 1:500, particularly for tissues with lower GBP2 expression

• Enhanced protein loading: Consider increasing total protein loading (50-80μg) when detecting GBP2 in samples without interferon stimulation, as baseline expression can be low

• Signal amplification: Implement enhanced chemiluminescence (ECL) substrates specifically designed for HRP with extended signal duration properties

• Membrane optimization: PVDF membranes (as used in published protocols) provide better protein retention and signal-to-noise ratio than nitrocellulose for GBP2 detection

• Transfer conditions: Optimize transfer conditions based on the specific molecular weight of GBP2 (67 kDa):

  • Semi-dry transfer: 15V for 30 minutes

  • Wet transfer: 100V for 60 minutes in 20% methanol buffer

• Stimulation controls: Include positive controls of IFN-γ stimulated cells, as GBP2 expression increases significantly following interferon treatment

How does GBP2 antibody (HRP conjugated) performance compare with other conjugates for multiplex immunofluorescence studies?

When designing multiplex immunofluorescence studies involving GBP2:

• Direct comparison data: HRP-conjugated GBP2 antibodies provide higher sensitivity for chromogenic detection but are generally not optimal for multiplex fluorescence applications unless using tyramide signal amplification (TSA)

• Alternative conjugates for multiplexing: For direct multiplexing, consider:

  • Fluorophore-conjugated GBP2 antibodies (PE, FITC, or Alexa Fluor variants)

  • Unconjugated primary GBP2 antibodies with spectrally distinct secondary fluorescent antibodies

• Sequential multiplexing with HRP: Using HRP-conjugated GBP2 antibodies in sequential TSA methodology provides:

  • Higher sensitivity than direct fluorophore conjugates

  • Ability to use antibodies from the same species with proper stripping between rounds

  • Integration with markers like CD3, CD8, CD68, PD-1 and CTLA4 as demonstrated in published multiplex studies

• Opal multiplex immunohistochemistry: This validated approach allows simultaneous visualization of GBP2 with immune cell markers, enabling spatial relationship analysis between GBP2 expression and immune infiltrates in tissue microenvironments

• Spectral considerations: When designing panels including HRP-TSA approaches, account for spectral overlap and implement appropriate compensation controls

How can GBP2 antibody be used to investigate immune cell infiltration in tumor microenvironments?

GBP2 antibodies have become valuable tools for characterizing tumor immune microenvironments based on multiple published studies:

• Established correlation: High GBP2 expression correlates with increased infiltration of CD3+ T cells, CD8+ T cells, and CD68+ macrophages in tumor tissues

• Multiplex approach: Using HRP-conjugated GBP2 antibody in initial staining followed by immune checkpoint markers (PD-1, CTLA4) allows assessment of both GBP2 expression and immune cell infiltration dynamics

• Tissue microarray application: Systematic analysis using tissue microarrays containing tumor and adjacent normal tissues provides comprehensive evaluation of GBP2 expression patterns and associated immune infiltrates

• Prognostic assessment: GBP2 expression levels can serve as a biomarker for estimating immunological characteristics of tumors and may help identify "immuno-hot" tumors with heightened T-cell infiltration

• JAK-STAT pathway investigation: GBP2 detection can be coupled with JAK-STAT pathway component analysis to investigate the regulatory mechanisms linking interferon signaling, GBP2 expression, and immune cell recruitment

This approach has been successfully implemented in multiple cancer types including clear cell renal cell carcinoma and gastric cancer models .

What protocols exist for investigating GBP2 function in pathogen restriction using knockout/knockdown approaches?

To investigate GBP2 functions in pathogen restriction, researchers can implement these validated approaches:

• Lentiviral shRNA knockdown: Using shRNA lentiviral particles containing 3 target-specific constructs (19-25 nt plus hairpin) designed to knock down GBP2 expression :

  • Plate cells at 50% confluency

  • Replace media with 1 mL containing 5 μg/mL Polybrene

  • Add 20 μL of lentiviral particles containing GBP2-targeting shRNA

  • Culture overnight, then replace with complete medium

  • Select with puromycin (5 μg/mL) and refresh medium every 3-4 days until resistant colonies appear

  • Verify knockdown efficiency by Western blot using anti-GBP2 antibody

• Plasmid-based GBP2 expression: For functional studies or rescue experiments :

  • Clone full-length or truncated GBP2 genes from IFN-γ stimulated cells

  • Insert into expression vectors (e.g., pcDNA3.1-Flag)

  • Create mutations via site-directed mutagenesis for functional domain analysis

  • Transfect into target cells using Lipofectamine 2000

  • Confirm expression using HRP-conjugated anti-GBP2 antibody via Western blot

• Infection model evaluation: Following manipulation of GBP2 expression, challenge cells with relevant pathogens and assess:

  • Pathogen replication rates

  • Inflammasome activation

  • Cytokine production

  • Cell death mechanisms

These approaches have been successfully utilized to demonstrate GBP2's critical role in restricting various pathogens including ectromelia virus (ECTV) .

How do different fixation methods affect GBP2 antibody (HRP conjugated) performance in immunohistochemistry?

Fixation methodology significantly impacts HRP-conjugated GBP2 antibody performance in IHC applications:

• Formalin fixation optimization: Standard 10% neutral-buffered formalin fixation (24-48 hours) provides consistent results when followed by proper antigen retrieval

• Fixation-dependent epitope masking: GBP2 epitopes are particularly susceptible to overfixation, which can cause:

  • Reduced signal intensity

  • False-negative results

  • Necessity for more aggressive antigen retrieval

• Fresh frozen vs. FFPE comparison:

  Fixation Method	Advantages	Limitations	Recommended Antibody Dilution
  FFPE sections	Better morphology, Standard procedure	Requires robust antigen retrieval	1:50 for HRP-conjugated antibodies
  Fresh frozen sections	Better epitope preservation, Less background	Poorer morphological preservation	1:100-1:200 for HRP-conjugated antibodies

• Alcohol-based fixation: Methanol or ethanol fixation may preserve certain GBP2 epitopes better than formalin, particularly for cytoplasmic localization studies

• Post-fixation handling: Regardless of fixation method, tissue processing temperature control is critical—higher temperatures during processing can further compromise GBP2 epitope recognition

What are the most common causes of non-specific background when using GBP2 antibody (HRP conjugated), and how can they be addressed?

Non-specific background is a common challenge with HRP-conjugated antibodies. For GBP2 detection, address these specific causes:

• Endogenous peroxidase activity: Thoroughly quench endogenous peroxidases using:

  • 3% hydrogen peroxide treatment for 10 minutes at room temperature prior to primary antibody application

  • For highly vascular tissues (spleen, lung), extend treatment to 15-20 minutes

• Insufficient blocking: Optimize blocking with:

  • 5% non-fat dry milk for Western blot applications (1 hour at room temperature)

  • 5-10% normal serum (from the same species as secondary antibody would be) for IHC applications

• Non-specific binding to Fc receptors: Particularly problematic in immune tissues rich in Fc receptor-expressing cells:

  • Use commercially available Fc receptor blocking reagents prior to antibody application

  • Consider using F(ab')2 fragments of GBP2 antibodies for tissues with high Fc receptor expression

• Antibody concentration: Titrate antibody concentrations carefully:

  • Start with manufacturer's recommended dilution (typically 1:50-1:500 for IHC)

  • If background persists, increase dilution incrementally while monitoring specific signal retention

• Cross-reactivity validation: Verify specificity using GBP2 knockout/knockdown controls, particularly important when studying tissues with multiple GBP family members

How can I validate the specificity of GBP2 antibody (HRP conjugated) in my experimental system?

Rigorous validation of GBP2 antibody specificity is essential for reliable research outcomes:

• Positive controls: Include samples with confirmed GBP2 expression:

  • IFN-γ stimulated cells (A375, HeLa, RAW264.7) show robust GBP2 upregulation

  • Human/mouse spleen tissue consistently shows detectable GBP2 expression

• Negative controls: Implement appropriate controls:

  • GBP2 knockout/knockdown samples using validated shRNA approaches

  • Isotype controls to distinguish non-specific binding

  • Secondary-only controls (when using non-conjugated primary antibodies)

• Molecular weight verification: Confirm detection at the expected molecular weight:

  • GBP2 consistently appears at approximately 67 kDa in Western blot applications

  • Different sized bands may indicate splice variants, post-translational modifications, or non-specific binding

• Cross-validation with orthogonal methods:

  • Compare protein detection with mRNA expression (qPCR)

  • Use multiple antibodies targeting different GBP2 epitopes

  • Confirm subcellular localization patterns match known GBP2 distribution

• Competitive peptide blocking: Pre-incubate antibody with immunizing peptide to confirm specificity of detected signals

What modifications to standard protocols are needed when using GBP2 antibody (HRP conjugated) for detecting low-abundance expression?

For detecting low-abundance GBP2 expression with HRP-conjugated antibodies:

• Signal amplification systems: Implement:

  • Tyramide signal amplification (TSA) which can increase sensitivity 10-100 fold

  • Enhanced chemiluminescence substrates specifically designed for low-abundance proteins

• Sample enrichment strategies:

  • Immunoprecipitate GBP2 before Western blot analysis to concentrate the target protein

  • Use cellular fractionation to enrich compartments where GBP2 localizes (primarily cytoplasmic)

• Extended antibody incubation:

  • Increase primary antibody incubation to overnight at 4°C instead of standard 1-2 hours

  • Maintain antibody concentration while extending exposure time

• Detection system optimization:

  • For Western blot: Use PVDF membranes with smaller pore size (0.2μm) to prevent protein loss

  • For IHC: Implement polymer-based detection systems with higher sensitivity than standard avidin-biotin complexes

• Interferon pre-treatment: For cell culture systems:

  • Pre-treat cells with IFN-γ (10-100 ng/mL for 12-24 hours) to upregulate GBP2 expression

  • This approach is particularly useful for validating antibody performance in systems with naturally low GBP2 expression

How can GBP2 antibody (HRP conjugated) be used to investigate the relationship between GBP2 expression and cancer prognosis?

GBP2 antibodies have become valuable tools in cancer prognostic research:

• Tissue microarray (TMA) application: Implement systematic analysis using:

  • TMAs containing tumor tissues and adjacent normal controls

  • Standardized IHC protocols with HRP-conjugated GBP2 antibodies

  • Quantitative scoring systems (H-score or percentage of positive cells)

• Multiplex immunophenotyping: Combine GBP2 detection with:

  • Immune cell markers (CD3, CD8, CD68)

  • Immune checkpoint proteins (PD-1, CTLA4)

  • Prognostic markers specific to cancer subtypes

• Correlative analysis workflow:

  • Quantify GBP2 expression levels using digital pathology platforms

  • Correlate with patient survival data and clinical parameters

  • Stratify patients into high/low GBP2 expression groups

  • Perform Kaplan-Meier survival analysis between groups

• Prognostic signature integration: Incorporate GBP2 expression into multi-protein prognostic signatures:

  • A five-protein prognostic signature including GBP2 has been validated in clear cell renal cell carcinoma

  • GBP2 expression serves as a pan-cancer biomarker for estimating tumor immunological characteristics

• JAK-STAT pathway association: Investigate GBP2's role in modulating JAK-STAT signaling in cancer progression through co-detection of pathway components

These approaches have successfully demonstrated that GBP2 expression patterns correlate with immune cell infiltration and can predict patient outcomes in multiple cancer types .

What are the considerations for using GBP2 antibody (HRP conjugated) in studies of GBP2's role in inflammasome activation?

When investigating GBP2's role in inflammasome activation:

• Cell type-specific considerations:

  • Myeloid cells (macrophages, dendritic cells) show different regulation of GBP2 compared to other cell types

  • Baseline expression levels vary significantly between tissue-resident and circulating immune cells

• Stimulation protocols optimization:

  • LPS priming (100 ng/mL, 4 hours) followed by interferon treatment (IFN-γ, 10-100 ng/mL)

  • Sequential stimulation better recapitulates in vivo inflammatory cascades

• Co-detection strategies:

  • Pair GBP2 detection with inflammasome components (NLRP3, AIM2, caspase-1)

  • Assess cleavage products of inflammasome activation (cleaved IL-1β, gasdermin D)

  • Monitor subcellular localization changes using fractionation followed by Western blot

• Pathogen challenge models:

  • GBP2 detection before and after bacterial infections (particularly gram-negative bacteria)

  • Time-course analysis to capture dynamic changes in GBP2 localization

  • Correlate with inflammasome assembly using co-immunoprecipitation approaches

• Genetic manipulation considerations:

  • When using GBP2 knockdown/knockout models, consider compensatory upregulation of other GBP family members

  • Include analysis of IRF8, which regulates GBP2 and other inflammasome components

These approaches have demonstrated GBP2's role in restricting pathogens and promoting inflammasome activation through mechanisms including pathogen vacuole lysis and release of pathogen components into the cytosol .

How should GBP2 antibody (HRP conjugated) protocols be adapted for cross-species studies examining evolutionary conservation of GBP2 functions?

For cross-species comparative studies of GBP2:

• Epitope conservation analysis: Before experimentation:

  • Align protein sequences from target species to identify conserved regions

  • Select antibodies targeting highly conserved epitopes when available

  • Verify cross-reactivity with recombinant proteins from each species if possible

• Demonstrated cross-reactivity: Published evidence shows:

  • Many GBP2 antibodies successfully detect human, mouse, and rat GBP2

  • Clone-specific differences exist in cross-species reactivity

• Species-specific optimization table:

  Species	Recommended Dilution	Tissue Processing Modifications	Positive Control Tissue
  Human	1:50-1:500 for IHC, 1:500-1:2000 for WB	Standard protocols	Spleen, tonsillitis
  Mouse	1:50-1:200 for IHC, 1:500-1:1000 for WB	Shorter antigen retrieval (5-7 min)	Spleen, IFN-γ treated cells
  Rat	Similar to mouse protocols	Similar to mouse protocols	Spleen tissue

• Control considerations:

  • Include species-specific positive and negative controls in each experiment

  • When possible, use tissues from GBP2 knockout models as definitive negative controls

  • For Western blot applications, load protein ladders suitable for cross-species comparative analysis

• Signal interpretation caveats:

  • Species-specific post-translational modifications may affect antibody binding

  • Subcellular localization patterns may differ between species despite protein conservation

  • Expression levels vary significantly between species even under similar stimulation conditions