wnt8b Antibody, HRP conjugated

What is Wnt8b and what biological functions does it serve in cellular systems?

Wnt8b is a secreted glycoprotein belonging to the Wnt family of signaling molecules. It functions as a ligand for members of the frizzled family of seven transmembrane receptors. Biologically, Wnt8b plays a critical role in the development and differentiation of specific forebrain structures, with notable importance in hippocampal development . In canonical Wnt signaling, Wnt8b binding to frizzled receptors leads to stabilization and nuclear translocation of β-catenin, ultimately affecting gene expression patterns and cellular proliferation .

Recent research has revealed that Wnt8b is frequently upregulated in hepatocellular carcinoma (HCC) tissues and significantly associated with poorer patient prognosis. Studies demonstrate that Wnt8b knockdown suppresses HCC cell growth both in vitro and in vivo through inhibition of the canonical Wnt signaling pathway .

What experimental applications are supported by Wnt8b HRP-conjugated antibodies?

Wnt8b HRP-conjugated antibodies support multiple experimental applications with varying recommended dilutions:

The HRP conjugation eliminates the need for secondary antibody incubation in immunodetection methods, streamlining experimental workflows and potentially reducing background signal .

What species reactivity can researchers expect with commercial Wnt8b antibodies?

Commercial Wnt8b antibodies demonstrate variable species reactivity depending on the specific product:

• The Bioss Wnt8b polyclonal antibody (bs-6245R-HRP) shows confirmed reactivity with Human, Mouse, and Rat samples, with predicted reactivity for Pig .

• Some antibodies are species-specific, such as those targeting zebrafish (Danio rerio) Wnt8b (AA 24-358) .

When selecting an antibody, researchers should carefully evaluate the immunogen sequence used for antibody production. For example, the Bioss antibody was raised against a KLH-conjugated synthetic peptide derived from human Wnt8b spanning amino acids 131-230/351 .

What are the optimal storage conditions for maintaining Wnt8b antibody activity?

To maintain optimal activity of Wnt8b HRP-conjugated antibodies, the following storage conditions are recommended:

• Store at -20°C in aliquots to avoid repeated freeze-thaw cycles which can degrade antibody quality .

• The antibody is typically supplied in an aqueous buffered solution containing 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300 and 50% Glycerol .

• For short-term storage (1-2 weeks), antibodies can be kept at 4°C, but long-term storage requires -20°C conditions.

• When working with HRP-conjugated antibodies, avoid repeated exposure to light and oxidizing agents that might compromise the enzyme activity.

How should researchers validate Wnt8b antibody specificity in their experimental systems?

Rigorous validation of Wnt8b antibody specificity should include:

• Positive and negative control samples: Use tissues or cell lines known to express or lack Wnt8b expression. Hepatocellular carcinoma cell lines like Hep3B are appropriate positive controls based on published literature .

• Knockdown/knockout validation: Compare antibody signals between wild-type samples and those with Wnt8b knockdown or knockout. Research shows that siRNA or shRNA targeting Wnt8b in cell lines can effectively reduce Wnt8b expression, providing excellent negative controls .

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide before application to verify signal specificity.

• Western blot analysis: Confirm that the antibody detects a band of the expected molecular weight for Wnt8b (~39-41 kDa).

• Cross-reactivity assessment: Test the antibody on samples from multiple species if working with non-human models to ensure appropriate reactivity as indicated in the manufacturer's specifications .

What are the optimal conditions for using Wnt8b HRP-conjugated antibodies in Western blot applications?

For optimal Western blot results with Wnt8b HRP-conjugated antibodies:

• Sample preparation: Extract proteins using RIPA buffer supplemented with protease inhibitors to prevent degradation of Wnt8b.

• Protein loading: Load 20-50 μg of total protein per lane depending on Wnt8b expression levels.

• Antibody dilution: Begin with a 1:1000 dilution within the recommended range (1:300-5000), optimizing as needed for your specific samples .

• Blocking: Use 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Incubate with diluted Wnt8b HRP-conjugated antibody overnight at 4°C with gentle agitation.

• Washing: Perform 4-5 washes with TBST, 5 minutes each, to reduce background.

• Detection: Since the antibody is HRP-conjugated, proceed directly to chemiluminescence detection without secondary antibody incubation, using ECL substrate with appropriate exposure times.

• Controls: Include positive controls (Hep3B cell lysate) and negative controls (Wnt8b-knockdown samples) to confirm specificity .

How can Wnt8b antibodies be utilized to investigate canonical Wnt signaling pathways?

Wnt8b antibodies can be instrumental in investigating canonical Wnt signaling through several methodological approaches:

• Pathway activation assessment: Monitor β-catenin stabilization and nuclear translocation following Wnt8b treatment. Research has demonstrated that Wnt8b overexpression increases active β-catenin levels, while Wnt8b knockdown leads to decreased β-catenin activation .

• Target gene expression analysis: Use Wnt8b antibodies in combination with antibodies against downstream targets such as Cyclin D1 and c-Myc to correlate Wnt8b expression with pathway output. Studies have shown that Wnt8b knockdown simultaneously reduces expression of these downstream effectors .

• Co-immunoprecipitation studies: Employ Wnt8b antibodies to investigate protein-protein interactions between Wnt8b and its receptors or co-factors.

• Chromatin immunoprecipitation (ChIP): Combine with transcription factor antibodies (e.g., TCF/LEF) to examine regulation of Wnt target genes in response to Wnt8b signaling.

• Dual immunostaining: Perform co-localization studies with Wnt8b and frizzled receptors to investigate spatial relationships in tissues or cultured cells.

Research has confirmed that Wnt8b activates the canonical Wnt pathway in hepatocellular carcinoma, as evidenced by the upregulation of active-β-catenin, total β-catenin, Cyclin D1, and c-Myc following Wnt8b overexpression .

What is the role of Wnt8b in hepatocellular carcinoma progression and how can antibodies help investigate this?

Wnt8b has been identified as a significant factor in hepatocellular carcinoma (HCC) progression through the following mechanisms:

• Upregulation in tumor tissues: qRT-PCR analysis revealed that 53.6% (22/41) of HCC cases showed significant upregulation of Wnt8b mRNA in tumor tissues compared to adjacent non-tumor tissues .

• Correlation with poor prognosis: High Wnt8b expression is significantly associated with poorer patient outcomes, making it a potential prognostic biomarker .

• Promotion of cell proliferation: Functional studies using Wnt8b knockdown demonstrated reduced colony formation, decreased cell proliferation in CCK-8 assays, and suppressed tumor growth in xenograft models .

• Canonical Wnt pathway activation: Wnt8b positively regulates levels of active-β-catenin, total β-catenin, and downstream targets Cyclin D1 and c-Myc .

• Transcriptional regulation: ZNF191 directly binds to the WNT8B promoter at specific binding sites (nt-1491/ATTAATT and nt-1178/ATTCATT) to transactivate WNT8B gene expression .

Researchers can utilize Wnt8b antibodies to:

• Assess Wnt8b protein levels in HCC tissues via IHC or Western blot

• Correlate Wnt8b expression with clinical parameters and patient outcomes

• Monitor changes in Wnt8b expression following experimental interventions

• Evaluate the relationship between Wnt8b and other molecular markers of HCC progression

What controls should researchers include when using Wnt8b antibodies in immunohistochemistry?

For rigorous immunohistochemistry experiments with Wnt8b antibodies, the following controls are essential:

• Positive tissue controls: Include known Wnt8b-positive tissues, such as HCC samples with confirmed Wnt8b expression or appropriate brain regions where Wnt8b is expressed during development .

• Negative tissue controls: Include tissues known to lack Wnt8b expression or use adjacent non-tumor liver tissues which typically express lower levels of Wnt8b .

• Antibody controls:

  • Omission of primary antibody (to assess non-specific binding of detection systems)

  • Isotype control (matched irrelevant antibody of the same isotype)

  • Peptide competition/blocking (pre-incubation with immunizing peptide)

• Technical controls:

  • Consistent tissue processing conditions

  • Standardized antigen retrieval methods (typically heat-induced epitope retrieval in citrate buffer, pH 6.0)

  • Endogenous peroxidase blocking (30 minutes with H₂O₂ solution at room temperature)

  • Hematoxylin counterstaining for nuclei visualization

• Validation controls: Include parallel staining for known Wnt pathway components like β-catenin, which should correlate with Wnt8b expression patterns in functional contexts .

The recommended IHC-P dilution for Wnt8b HRP-conjugated antibodies is 1:200-400, while for IHC-F applications, 1:100-500 is suggested .

What are the most effective transfection approaches for studying Wnt8b function in cell culture models?

Several effective transfection approaches can be employed to study Wnt8b function:

• Overexpression studies:

  • Use of plasmids like Active Wnt8b-V5 (Addgene #43819) which contains the human WNT8B insert under a CMV promoter

  • Transfection using lipid-based reagents for most cell lines or electroporation for hard-to-transfect cells

  • Validation of expression by Western blot using anti-Wnt8b or anti-V5 antibodies

• Knockdown approaches:

  • siRNA transfection for transient knockdown (effective for 48-72 hours)

  • Lentiviral shRNA for stable knockdown, which has been successfully implemented in studies of Wnt8b function in HCC cells

  • Validation of knockdown efficiency by qRT-PCR and Western blot

• CRISPR-Cas9 gene editing:

  • Generation of Wnt8b knockout cell lines for complete elimination of protein expression

  • Targeting of regulatory regions to study transcriptional control mechanisms, such as the ZNF191 binding sites in the WNT8B promoter

• Functional readouts:

  • Colony formation assays to assess proliferative capacity

  • Cell Counting Kit-8 (CCK-8) assays for cell viability assessments

  • TOPFlash/FOPFlash reporter assays to measure canonical Wnt signaling activation

  • Immunoblotting for downstream Wnt target proteins like active-β-catenin, Cyclin D1, and c-Myc

How can researchers accurately quantify Wnt8b expression levels in experimental samples?

Accurate quantification of Wnt8b expression can be achieved through multiple complementary approaches:

• Quantitative RT-PCR (qRT-PCR):

  • Use validated primer pairs specific to Wnt8b

  • Employ the 2^(-ΔΔCt) method for relative quantification, normalizing to housekeeping genes like GAPDH or β-actin

  • Include standard curves to ensure linearity of amplification

• Western blotting with HRP-conjugated Wnt8b antibodies:

  • Load equal amounts of protein (20-50 μg per lane)

  • Use dilutions between 1:300-5000 depending on expression levels

  • Quantify band intensity using densitometry software

  • Normalize to loading controls such as GAPDH or β-actin

• ELISA:

  • Direct ELISA using HRP-conjugated Wnt8b antibodies at 1:500-1000 dilution

  • Sandwich ELISA using capture and detection antibodies for increased specificity

  • Generate standard curves with recombinant Wnt8b protein for absolute quantification

• Immunohistochemistry quantification:

  • Score staining intensity (0-3+) and percentage of positive cells

  • Use digital image analysis software for objective quantification

  • Implement H-score or Allred scoring systems for semi-quantitative assessment

• Flow cytometry:

  • For intracellular Wnt8b detection following permeabilization

  • Allows single-cell quantification and correlation with other markers

What methodological approaches can address potential cross-reactivity with other Wnt family members?

Addressing cross-reactivity with other Wnt family members requires several methodological considerations:

• Epitope selection and antibody design:

  • Use antibodies raised against unique regions of Wnt8b with minimal sequence homology to other Wnt proteins

  • The Bioss antibody (bs-6245R-HRP) targets amino acids 131-230/351 of human Wnt8b, a region with reduced homology to other Wnt family members

• Validation experiments:

  • Perform Western blots to confirm single band detection at the expected molecular weight

  • Test antibody reactivity against recombinant proteins of related Wnt family members

  • Use cell lines with known Wnt expression profiles as positive and negative controls

• Genetic approaches:

  • Employ Wnt8b knockdown or knockout systems to confirm specificity of antibody signals

  • Overexpress Wnt8b using expression vectors like Active Wnt8b-V5 (Addgene #43819) to validate antibody detection

• Peptide competition assays:

  • Pre-incubate antibody with excess immunizing peptide

  • Include related peptides from other Wnt family members to assess cross-reactivity

• Bioinformatic analysis:

  • Perform sequence alignments between the antibody's target epitope and corresponding regions in other Wnt proteins

  • Identify potential cross-reactive epitopes through in silico prediction

How can Wnt8b antibodies be utilized to study neural development and forebrain patterning?

Wnt8b antibodies can be instrumental in investigating neural development through several methodological approaches:

• Temporal expression analysis:

  • Track Wnt8b expression during critical developmental windows using IHC-F (1:100-500 dilution) on embryonic tissue sections

  • Correlate Wnt8b expression with morphological changes in developing forebrain structures

• Spatial expression mapping:

  • Use Wnt8b antibodies in whole-mount immunostaining to visualize expression domains

  • Combine with markers for neural progenitors, differentiating neurons, and regional identity factors

• Functional studies:

  • Manipulate Wnt8b signaling through overexpression or knockdown approaches

  • Assess consequences on hippocampal development, as Wnt8b "may play an important role in the development and differentiation of certain forebrain structures, notably the hippocampus"

• Co-localization studies:

  • Perform dual immunofluorescence with Wnt8b antibodies and frizzled receptor antibodies

  • Investigate potential receptor-ligand interactions in developing neural tissues

• Ex vivo and organoid systems:

  • Apply Wnt8b antibodies to study protein expression in brain organoids

  • Characterize Wnt8b distribution in neural progenitor cultures

• Zebrafish models:

  • Leverage zebrafish-specific Wnt8b antibodies to study neural development in this transparent model organism

  • Combine with transgenic reporter lines for real-time visualization of Wnt pathway activity

What are the methodological considerations for detecting secreted Wnt8b proteins in experimental systems?

Detecting secreted Wnt8b proteins presents unique challenges requiring specialized methodological approaches:

• Medium concentration techniques:

  • Collect conditioned medium from cells expressing Wnt8b

  • Concentrate using ultrafiltration or TCA precipitation

  • Apply to Western blot using HRP-conjugated Wnt8b antibodies (1:300-5000)

• Immunoprecipitation approaches:

  • Use Wnt8b antibodies conjugated to beads to pull down secreted protein from media

  • Elute and analyze by Western blot or mass spectrometry

  • Consider using V5-tagged Wnt8b constructs for more efficient capture

• ELISA optimization:

  • Develop sandwich ELISA using capture and HRP-conjugated detection antibodies (1:500-1000)

  • Include standard curves with recombinant Wnt8b protein

  • Optimize sample collection timing to capture peak secretion

• Subcellular localization studies:

  • Use immunofluorescence to track Wnt8b through the secretory pathway

  • Co-stain for ER, Golgi, and vesicular markers to analyze trafficking

• Consideration of post-translational modifications:

  • Wnt proteins undergo lipid modifications crucial for secretion and activity

  • Account for potential changes in antibody recognition due to these modifications

• Detection of receptor-bound Wnt8b:

  • Optimize fixation protocols to preserve cell surface protein interactions

  • Use non-permeabilizing conditions to specifically detect extracellular Wnt8b