GLI2 Antibody, HRP conjugated

What is GLI2 and why is it important in research?

GLI2 is a zinc finger transcription factor that functions as a primary mediator of Hedgehog (Hh) signaling pathway. It plays critical roles in embryonic development, stem cell renewal, and has been implicated in various cancers including prostate cancer. Research has demonstrated that GLI2 is significantly overexpressed in primary human prostate tumors and prostate cancer cell lines, making it an important target for cancer research . Studies have shown that GLI2 can activate the expression of other transcription factors like MEF2C, revealing its role in complex transcriptional networks that regulate cell differentiation and proliferation . The importance of GLI2 extends to its ability to mediate the transcriptional activation of target genes including bone morphogenetic proteins, highlighting its role in developmental processes .

What are the key differences between GLI2 antibodies and other GLI family antibodies?

GLI2 antibodies are specifically designed to detect GLI2 protein without cross-reactivity to other GLI family members (GLI1 and GLI3). This specificity is crucial as GLI family proteins share homologous domains but perform distinct functions. For example, research has demonstrated that activation of the Follicle-stimulating hormone (FST) promoter is highly GLI2-specific, with neither GLI1 nor GLI3 being able to significantly increase FST transcription . When selecting a GLI2 antibody, researchers should validate specificity through controls that include GLI1 and GLI3 proteins to ensure accurate experimental results, particularly in systems where multiple GLI family members are expressed.

How do I select the appropriate conjugation for my GLI2 antibody based on my experimental design?

The selection between HRP-conjugated and unconjugated GLI2 antibodies depends on your experimental application:

For detecting GLI2 in Western blot applications where sensitivity is crucial, unconjugated primary antibodies followed by HRP-conjugated secondary antibodies often provide better signal amplification, as demonstrated in studies using GLI2 antibodies at concentrations as low as 0.5 μg/mL .

What are the common applications for GLI2 antibodies in research?

GLI2 antibodies have been validated for multiple research applications:

• Western Blotting (WB): Detecting GLI2 protein (approximately 210KD observed size, though the expected size is 168KD) in cell and tissue lysates .

• Immunohistochemistry (IHC): Visualizing GLI2 expression patterns in tissue sections, particularly in cancer tissues and brain tissues .

• Chromatin Immunoprecipitation (ChIP): Investigating GLI2 binding to DNA regulatory elements and interactions with other transcription factors .

• Immunofluorescence (IF): Examining subcellular localization of GLI2 protein .

• Flow Cytometry (FACS): Quantifying GLI2 expression levels in cell populations .

These applications have been instrumental in elucidating the role of GLI2 in cancer progression, particularly in prostate cancer models where GLI2 knockdown significantly inhibited colony formation and xenograft tumor growth .

How does HRP conjugation impact the sensitivity and specificity of GLI2 antibody detection?

HRP conjugation directly impacts both sensitivity and specificity of GLI2 antibody detection. The conjugation process attaches HRP molecules to the antibody structure, typically at lysine residues. This modification can affect:

For optimal results in critical experiments, antibody validation using GLI2-knockdown samples is highly recommended, as demonstrated in studies where GLI2-specific shRNA was used to confirm antibody specificity .

What validation methods should I use to ensure GLI2 antibody specificity?

To ensure GLI2 antibody specificity, implement these validation methods:

• Knockdown/Knockout Validation: Use GLI2-specific shRNA or CRISPR-Cas9 systems to create GLI2-depleted negative controls. Studies have successfully employed lentiviral-mediated GLI2 shRNA to validate antibody specificity in prostate cancer cell lines .

• Western Blot Analysis: Verify the detection of a single band at the expected molecular weight (though note that GLI2 often shows a discrepancy between expected (168KD) and observed (210KD) molecular weights, likely due to post-translational modifications) .

• Immunoprecipitation Followed by Mass Spectrometry: Confirm that the antibody pulls down GLI2 and not other proteins.

• Cross-Reactivity Testing: Test the antibody against recombinant GLI1 and GLI3 proteins to ensure it doesn't cross-react with these homologous proteins.

• Multiple Antibody Validation: Compare results using antibodies targeting different epitopes of GLI2, such as those targeting the Middle Region versus C-Terminal regions .

How can I optimize experimental conditions for detecting GLI2 in different tissue types?

Optimization strategies for GLI2 detection vary by tissue type:

These protocols are based on validated GLI2 antibody applications in various tissue types . For tissues not listed, start with the brain tissue protocol and adjust as needed based on signal-to-noise ratio in your experiments.

What are the key considerations when using GLI2 antibodies for chromatin immunoprecipitation studies?

When using GLI2 antibodies for ChIP experiments, consider these critical factors:

• Fixation Conditions: Optimize formaldehyde concentration (typically 1-4%) and fixation time (5-15 minutes) to preserve GLI2-DNA interactions without overfixation. Research protocols have successfully used 4% formaldehyde for GLI2 ChIP experiments .

• Chromatin Shearing: GLI2 binding sites, particularly those in the FST promoter region, require efficient chromatin shearing to sizes between 200-500bp for optimal immunoprecipitation .

• Antibody Amount: Use 2-5μg of GLI2-specific antibody per ChIP reaction, as demonstrated in studies examining GLI2-MEF2C interactions .

• Controls: Include IgG negative controls and positive controls (regions known to be bound by GLI2, such as the FST promoter which contains putative GLI binding sites) .

• Binding Site Validation: Confirm GLI2 binding through quantitative PCR analysis of chromatin input, comparing enrichment of specific loci immunoprecipitated with GLI2 antibody versus non-specific IgG. Statistical analysis (ANOVA) should be performed to establish significance .

These considerations are essential for generating reliable ChIP data when studying GLI2's transcriptional regulation activities.

How do I troubleshoot inconsistent results when using GLI2 antibodies in Western blotting?

Inconsistent Western blot results with GLI2 antibodies can be addressed through systematic troubleshooting:

• Protein Size Discrepancy: GLI2 often appears at 210KD rather than the expected 168KD. This is normal and observed across multiple cell lines (U2OS, A549, PC-3, HEK293, Hela) . The size difference is likely due to post-translational modifications.

• Sample Preparation: Use stringent lysis buffers containing protease inhibitors to prevent GLI2 degradation. GLI2 can be unstable in standard RIPA buffers; adding 1% SDS may improve extraction.

• Loading Controls: Due to GLI2's high molecular weight, verify complete transfer of large proteins using Ponceau S staining before blocking.

• Blocking Optimization: If background is high, test different blocking agents:

  Blocking Agent	Advantage	Recommended For
  5% Non-fat Milk/TBS	Good general blocking	Standard GLI2 detection
  5% BSA/TBST	Better for phospho-specific detection	Modified GLI2 detection
  Commercial blockers	Reduced background	Problematic tissues

• Antibody Concentration: Titrate antibody concentrations; optimal concentration for GLI2 detection is typically 0.5-1.0 μg/mL .

• Membrane Washing: Extend wash steps (3 times with TBS-0.1% Tween, 5 minutes each) to reduce background signal .

• Detection System: Enhanced Chemiluminescent detection (ECL) systems provide better sensitivity for detecting GLI2 as demonstrated in validated Western blot protocols .

What are the recommended protocols for immunohistochemical detection of GLI2 in FFPE tissues?

For optimal immunohistochemical detection of GLI2 in formalin-fixed paraffin-embedded (FFPE) tissues, follow this validated protocol:

• Deparaffinization and Rehydration:

  • Xylene: 2 changes, 5 minutes each

  • 100% ethanol: 2 changes, 3 minutes each

  • 95% ethanol: 1 minute

  • 70% ethanol: 1 minute

  • Distilled water: rinse

• Antigen Retrieval:

  • Heat-mediated antigen retrieval in citrate buffer (pH6)

  • 20 minutes at 95-100°C

  • Cool to room temperature (approximately 20 minutes)

• Blocking:

  • 10% goat serum in PBS for 1 hour at room temperature

  • Option: Add 0.3% H₂O₂ in methanol for 10 minutes to block endogenous peroxidase activity

• Primary Antibody Incubation:

  • Dilute GLI2 antibody to 1μg/ml in blocking solution

  • Incubate overnight at 4°C

  • Wash 3x with PBS, 5 minutes each

• Secondary Antibody and Detection:

  • For HRP-conjugated GLI2 antibodies: Proceed directly to step 6

  • For unconjugated GLI2 antibodies: Apply biotinylated goat anti-rabbit IgG for 30 minutes at 37°C

  • Wash 3x with PBS, 5 minutes each

  • Incubate with Strepavidin-Biotin-Complex (SABC) for 30 minutes at 37°C

• Visualization:

  • Develop using DAB chromogen (3-5 minutes monitoring for color development)

  • Counterstain with hematoxylin (30 seconds)

  • Dehydrate, clear, and mount

This protocol has been successfully used to detect GLI2 in various human tissues including mammary cancer and intestinal cancer tissues, as well as mouse and rat brain tissues .

How can I determine the optimal dilution for HRP-conjugated GLI2 antibodies in different applications?

Determining optimal dilutions for HRP-conjugated GLI2 antibodies requires systematic titration:

• Western Blotting Titration:

  • Prepare a dilution series (1:500, 1:1000, 1:2000, 1:5000)

  • Run identical protein samples in multiple lanes

  • Apply different antibody dilutions to different membrane sections

  • Compare signal-to-noise ratio to determine optimal dilution

  • For unconjugated GLI2 antibodies with HRP-secondary antibodies, optimal dilutions of 0.5 μg/mL primary and 1:10000 secondary have been validated

• ELISA Optimization:

  • Perform checkerboard titration with antigen concentration on one axis and antibody dilution on the other

  • Start with manufacturer's recommended range

  • Calculate signal-to-noise ratio for each condition

  • Select dilution with highest signal-to-noise ratio above background

• Immunohistochemistry Dilution Determination:

  • Test serial dilutions on known positive control tissues

  • For GLI2, mammary cancer tissue sections serve as excellent positive controls

  • Compare staining intensity and background

  • Optimal concentration for unconjugated GLI2 antibodies is typically 1μg/ml for IHC applications

• Storage of Diluted Antibody:

  • Prepare working dilutions fresh when possible

  • If storage is necessary, add BSA (0.5-1%) as a stabilizer

  • Store at 4°C for up to 7 days

  • Avoid repeated freeze-thaw cycles of diluted antibody

How is GLI2 antibody being used to investigate cancer pathways and potential therapeutic targets?

GLI2 antibodies have become essential tools in cancer research, particularly for investigating aberrant Hedgehog signaling pathways:

• Cancer Cell Growth and Tumorigenicity: GLI2 antibodies have been instrumental in establishing GLI2's critical role in prostate cancer. Immunoblotting with GLI2 antibodies demonstrated that GLI2 knockdown in human prostate cancer cell lines (DU145, PC3, 22Rν1, and LnCaP) significantly inhibited colony formation and growth, while not affecting non-tumorigenic RWPE1 prostate epithelial cells . This selective effect suggests GLI2 is a potential therapeutic target specific to cancer cells.

• Xenograft Models: GLI2 antibodies have been used to validate GLI2 knockdown in xenograft tumor models, where inhibition of GLI2 expression significantly slowed tumor growth rates (from 20.7 to 3.1 mm³/day) and extended the time needed to reach target tumor volumes .

• Transcriptional Network Analysis: ChIP experiments using GLI2 antibodies have revealed GLI2's role in activating expression of other transcription factors like MEF2C, and its binding to specific promoter regions like those in the FST gene . These findings help map the complex transcriptional networks controlled by GLI2 in cancer progression.

• Therapeutic Response Monitoring: GLI2 antibodies are being used to evaluate tumor response to Hedgehog pathway inhibitors, providing a molecular marker for treatment efficacy.

These applications collectively advance our understanding of GLI2's central role in cancer pathogenesis and help identify potential points for therapeutic intervention.

What are the considerations when using GLI2 antibodies for co-immunoprecipitation studies investigating protein-protein interactions?

When using GLI2 antibodies for co-immunoprecipitation (co-IP) studies investigating protein-protein interactions, consider these critical factors:

• Antibody Selection: Choose GLI2 antibodies validated for immunoprecipitation. Antibodies targeting different regions of GLI2 may have varying efficacy in pulling down intact protein complexes. For instance, antibodies targeting the Middle Region versus C-Terminal regions may preserve different interaction interfaces .

• Lysis Conditions: Use gentle lysis buffers to preserve protein-protein interactions:

  Buffer Type	Composition	Best For
  NP-40 Buffer	50mM Tris-HCl pH 7.4, 150mM NaCl, 1% NP-40, protease inhibitors	Standard GLI2 co-IP
  CHAPS Buffer	30mM Tris-HCl pH 7.5, 150mM NaCl, 1% CHAPS, protease inhibitors	Membrane-associated complexes
  Low Salt Buffer	20mM HEPES pH 7.9, 100mM NaCl, 0.5% NP-40, 10% glycerol, protease inhibitors	Nuclear complexes with GLI2

• Pre-clearing: Pre-clear lysates with protein G sepharose beads to reduce non-specific binding, as demonstrated in successful GLI2 ChIP protocols .

• Controls:

  • IgG control: Use matched isotype IgG (e.g., goat IgG for GLI2 antibodies raised in goat) to identify non-specific binding

  • Input control: Save 5-10% of lysate before immunoprecipitation

  • Reciprocal IP: If studying interaction with a specific protein (e.g., MEF2C), perform reverse IP using antibodies against the interaction partner

• Elution Conditions: For Western blot analysis after co-IP, use reducing SDS sample buffer heated to 70°C rather than 95°C to minimize antibody chain interference.

• Quantification: Calculate enrichment as percent of input chromatin to accurately represent binding efficiency .

These methodological considerations will help ensure reliable results when investigating GLI2 protein interactions with other transcriptional regulators or signaling components.

What are the optimal storage conditions for maintaining GLI2 antibody activity?

Proper storage is critical for maintaining GLI2 antibody activity and extending shelf life:

• Lyophilized Antibody Storage:

  • Store at -20°C for up to one year from date of receipt

  • Protect from moisture by storing with desiccant

  • Always warm to room temperature before opening vial to prevent condensation

• Reconstituted Antibody Storage:

  • Short-term (≤1 month): 4°C

  • Long-term (≤6 months): Aliquot and store at -20°C

  • Very long-term (>6 months): -80°C

  • Avoid repeated freeze-thaw cycles

• HRP-Conjugated Antibody Considerations:

  • More sensitive to temperature fluctuations than unconjugated antibodies

  • Add 50% glycerol (final concentration) for -20°C storage to prevent freeze damage to HRP

  • Sodium azide should NEVER be used with HRP-conjugated antibodies as it inactivates the enzyme

• Reconstitution Recommendations:

  • Use sterile water, PBS, or buffer recommended by manufacturer

  • For 100 μg/vial, reconstitution in 100 μL yields a 1 mg/mL concentration

  • Gentle mixing rather than vortexing prevents antibody denaturation

• Aliquoting Strategy:

  • Prepare single-use aliquots (typically 10-20 μL)

  • Use sterile microcentrifuge tubes

  • Label with antibody name, concentration, and date

Following these storage recommendations will help maintain optimal GLI2 antibody performance in experimental applications over time, ensuring consistent and reliable results.

How do I properly reconstitute lyophilized GLI2 antibodies to ensure maximum activity?

Proper reconstitution of lyophilized GLI2 antibodies is essential for maintaining epitope recognition and signal strength:

• Pre-Reconstitution Preparation:

  • Allow the vial to warm to room temperature (approximately 30 minutes) before opening to prevent condensation that could affect protein stability

  • Briefly centrifuge the vial to collect all material at the bottom

• Reconstitution Process:

  • For a 100 μg vial, add 100 μL of appropriate buffer to achieve 1 mg/mL concentration

  • For buffers, use one of the following:

    • Sterile PBS (pH 7.4)

    • 50mM Tris buffer (pH 7.5) with 150mM NaCl

    • Manufacturer's recommended buffer

  • Add buffer slowly, directing it against the side of the vial rather than directly onto the lyophilized material

  • Replace cap and gently invert several times

  • Allow to stand at room temperature for 10-15 minutes

  • Gently swirl (do not vortex) to ensure complete dissolution

• Post-Reconstitution Processing:

  • For long-term storage, prepare 10-20 μL aliquots in sterile microcentrifuge tubes

  • Store according to recommended conditions: 4°C for one month or -20°C for six months

  • Document reconstitution date, concentration, and buffer composition on each aliquot

• Verification of Activity:

  • After reconstitution, verify antibody activity by performing a quick Western blot using a known positive control cell line such as U2OS or HEK293 whole cell lysates, which have been validated for GLI2 detection

This methodical reconstitution protocol helps preserve the structural integrity and binding capacity of GLI2 antibodies, ensuring optimal performance in experimental applications.

What quality control tests should I perform when receiving a new batch of GLI2 antibodies?

When receiving a new batch of GLI2 antibodies, implement these quality control tests to ensure consistency and reliability:

• Western Blot Validation:

  • Test on positive control lysates (e.g., U2OS, A549, PC-3, HEK293, or Hela cells)

  • Verify correct molecular weight (observed ~210KD, expected 168KD for GLI2)

  • Compare band intensity with previous antibody lot at the same concentration

  • Expected result: Clear single band with minimal background

• Positive Tissue Controls for IHC:

  • Test on known GLI2-positive tissues (mammary cancer, intestinal cancer, or brain tissues)

  • Use established protocol with citrate buffer (pH6) antigen retrieval

  • Compare staining pattern and intensity with previous antibody lot

  • Expected result: Nuclear/cytoplasmic staining pattern consistent with GLI2 localization

• Epitope-Blocking Test:

  • Pre-incubate antibody with 5-10x molar excess of immunizing peptide

  • Perform Western blot or IHC in parallel with unblocked antibody

  • Expected result: Significant reduction in specific signal with blocked antibody

• Cross-Reactivity Assessment:

  • Test against lysates from GLI2-knockout or GLI2-knockdown cells if available

  • Compare with GLI1 and GLI3 overexpression lysates to assess specificity

  • Expected result: Signal in wildtype but not in GLI2-depleted samples; no cross-reactivity with GLI1/GLI3

• Application-Specific Performance Checks:

  Application	Validation Method	Success Criteria
  Western Blot	Titration curve (0.1-1.0 μg/mL)	Clear signal at 0.5 μg/mL
  IHC	Serial dilutions (0.5-5 μg/mL)	Specific staining at 1 μg/mL
  ChIP	% Input analysis	Significant enrichment over IgG control

How are GLI2 antibodies being used to investigate drug resistance mechanisms in cancer therapy?

GLI2 antibodies are providing critical insights into drug resistance mechanisms in cancer therapy:

• Hedgehog Inhibitor Resistance: GLI2 antibodies have revealed that elevated GLI2 expression correlates with resistance to Smoothened (SMO) inhibitors in basal cell carcinoma and medulloblastoma. Immunohistochemical analysis using GLI2-specific antibodies in patient-derived xenograft models has shown that GLI2 can be activated through non-canonical pathways, bypassing SMO inhibition.

• Cross-Talk with Other Signaling Pathways: Western blot analysis with GLI2 antibodies has demonstrated that GLI2 is activated downstream of multiple oncogenic signaling pathways, including:

  • TGF-β/SMAD signaling

  • RAS/RAF/MEK/ERK pathway

  • PI3K/AKT/mTOR signaling

  This explains why single-pathway targeted therapies often develop resistance through GLI2 activation.

• Drug Response Biomarker: GLI2 antibody-based immunohistochemistry is being evaluated as a predictive biomarker for response to various targeted therapies. The nuclear localization of GLI2, detectable by IHC, correlates with transcriptional activity and therapy resistance.

• Combination Therapy Rationale: Research using GLI2 antibodies has provided rationale for combination therapies targeting both upstream activators and GLI2 itself. For example, studies in prostate cancer models have shown that GLI2 knockdown sensitizes tumors to conventional chemotherapeutics and targeted agents .

These applications of GLI2 antibodies are helping researchers develop more effective therapeutic strategies to overcome drug resistance in cancer treatment.

What are the latest developments in using GLI2 antibodies for single-cell protein analysis techniques?

Recent advances in single-cell protein analysis using GLI2 antibodies include:

• Mass Cytometry (CyTOF) Integration: Metal-conjugated GLI2 antibodies are now being incorporated into CyTOF panels for high-dimensional analysis of Hedgehog pathway activation at the single-cell level within heterogeneous tumor samples. This allows researchers to correlate GLI2 expression with dozens of other signaling proteins and cellular markers simultaneously.

• Proximity Ligation Assays (PLA): This technique uses GLI2 antibodies in combination with antibodies against potential interaction partners to visualize protein-protein interactions within intact cells. PLA has been particularly valuable in studying GLI2 interactions with transcriptional cofactors like MEF2C and post-translational modifiers.

• Single-Cell Western Blotting: Microfluidic platforms now allow Western blot analysis of GLI2 expression in individual cells, revealing cell-to-cell variability in GLI2 protein levels and modifications that may explain differential responses to Hedgehog pathway inhibitors.

• Spatial Transcriptomics-Protein Correlation: Combined approaches using GLI2 antibodies for protein detection alongside spatial transcriptomics are revealing the relationship between GLI2 protein localization and target gene expression in tissue contexts.

• CODEX Multiplexed Imaging: This technique allows visualization of GLI2 alongside dozens of other proteins in the same tissue section through iterative antibody staining and imaging cycles, providing unprecedented insights into the spatial relationships between GLI2-expressing cells and their microenvironment.

These emerging techniques are expanding our understanding of GLI2's role in normal development and disease by providing single-cell resolution of protein expression, modification, and interaction patterns.

How can GLI2 antibodies be used to study the relationship between Hedgehog signaling and developmental disorders?

GLI2 antibodies provide valuable tools for investigating the relationship between Hedgehog signaling and developmental disorders:

• Neural Tube Defects: IHC studies using GLI2 antibodies in mouse models and human autopsy specimens have revealed abnormal GLI2 expression patterns in neural tissues associated with holoprosencephaly, anencephaly, and spina bifida. These studies demonstrated that GLI2 is crucial for proper dorsal-ventral patterning of the neural tube, with antibody staining showing graduated expression patterns corresponding to morphogen gradients .

• Craniofacial Abnormalities: GLI2 antibodies have been used to map GLI2 expression in craniofacial development, revealing its role in:

  • Palate formation

  • Midline facial development

  • Tooth morphogenesis

  Altered GLI2 localization has been documented in tissues from patients with cleft palate and other midline defects.

• Skeletal Malformations: Western blot and IHC analyses using GLI2 antibodies have demonstrated GLI2's role in:

  • Endochondral ossification

  • Bone morphogenesis

  • Growth plate organization

  GLI2 antibody staining has revealed aberrant expression in patients with curvature spine disorders and digit abnormalities.

• Genetic Counseling Applications: GLI2 antibody-based diagnostics are being explored for prenatal and postnatal testing to complement genetic screening in families with history of Hedgehog-related developmental disorders.

• Therapeutic Development: GLI2 antibodies are essential tools in developing potential treatments for developmental disorders through:

  • Screening small molecule modulators of GLI2 activity

  • Validating gene therapy approaches aimed at correcting GLI2 function

  • Monitoring response to experimental treatments in animal models

GLI2 antibodies thus serve as critical research tools linking molecular signaling aberrations to developmental phenotypes, advancing both our understanding of disease mechanisms and potential therapeutic interventions.