PAFAH1B3 Antibody

What is PAFAH1B3 and why is it significant in research?

PAFAH1B3 is a catalytic subunit of the platelet-activating factor acetylhydrolase IB complex that catalyzes the removal of the acetyl group at the sn-2 position of the glycerol backbone of platelet-activating factor (PAF), producing biologically inactive lyso-PAF . Recent studies have identified PAFAH1B3 as a cancer-relevant metabolic driver with significant upregulation in multiple cancer types, including hepatocellular carcinoma and hypopharyngeal squamous cell carcinoma, making it a promising biomarker and therapeutic target .

What types of PAFAH1B3 antibodies are available for research?

Several types of PAFAH1B3 antibodies are available for research applications:

What are the primary applications for PAFAH1B3 antibodies?

PAFAH1B3 antibodies have been validated for multiple research applications:

• Western blotting for protein expression analysis

• Immunohistochemistry for tissue localization studies

• Immunofluorescence for cellular localization

• ELISA for quantitative analysis

• Immunoprecipitation for protein-protein interaction studies

• Knockdown validation studies in combination with siRNA approaches

What is the recommended storage condition for PAFAH1B3 antibodies?

Most PAFAH1B3 antibodies require storage at -20°C and remain stable for one year after shipment. They are typically provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Aliquoting is generally unnecessary for -20°C storage, though specific products may contain 0.1% BSA for stability .

What are the optimal dilutions for different applications of PAFAH1B3 antibodies?

Based on validated protocols, the following dilutions are recommended:

It is recommended that researchers titrate the antibody in each testing system to obtain optimal results, as sample-dependent variations may occur .

What antigen retrieval methods are recommended for IHC applications?

For immunohistochemistry applications with PAFAH1B3 antibodies, the suggested antigen retrieval method is with TE buffer at pH 9.0. Alternatively, antigen retrieval may be performed with citrate buffer at pH 6.0 . The choice between these methods may depend on the specific tissue type and fixation protocols used.

How can I validate the specificity of a PAFAH1B3 antibody?

Validation of PAFAH1B3 antibody specificity can be accomplished through:

• Western blot analysis using positive control tissues (human, mouse, or rat brain tissues have shown consistent positive results)

• Comparison with siRNA-mediated knockdown of PAFAH1B3 (reduction in signal should be observed)

• Immunofluorescence with appropriate positive control cell lines (e.g., SH-SY5Y cells)

• Cross-validation with multiple antibodies targeting different epitopes of PAFAH1B3

• Inclusion of appropriate negative controls in all experiments

How can PAFAH1B3 antibodies be used to assess cancer prognosis?

PAFAH1B3 expression analysis using antibodies has demonstrated significant value in cancer prognosis studies:

When implementing such studies, researchers should:

• Use standardized scoring systems for immunohistochemical staining

• Correlate expression data with clinicopathological parameters

• Perform both univariate and multivariate analyses to determine independent prognostic value

• Include sufficient sample sizes with appropriate controls

What experimental designs are appropriate for studying PAFAH1B3 function in cancer?

Based on published research, effective experimental designs include:

• Loss-of-function studies:

  • siRNA-mediated knockdown of PAFAH1B3 (validated sequences: 5'-CGACAGGUGAACGAGCUGGUATT-3' and 5'-GGAGAAGAACCGACAGGUGAATT-3')

  • Pharmacological inhibition using selective inhibitors like P11

• Functional assays following PAFAH1B3 manipulation:

  • Cell proliferation assays (e.g., CCK8)

  • Apoptosis detection by flow cytometry (Annexin-V staining)

  • Cell cycle analysis using propidium iodide staining and flow cytometry

  • Migration and invasion assays (e.g., Transwell)

  • Western blot analysis of signaling pathways affected by PAFAH1B3 modulation

• Metabolic pathway analysis:

  • Assessment of glycolysis and lipid synthesis pathways following PAFAH1B3 manipulation

How can I design experiments to investigate the relationship between PAFAH1B3 expression and cancer metabolism?

Research has shown that PAFAH1B3 plays a role in cancer metabolism, particularly in glycolysis and lipid synthesis pathways . To investigate this relationship:

• Employ PAFAH1B3 antibodies for expression analysis in conjunction with metabolic pathway markers

• Use PAFAH1B3 knockdown or inhibition followed by:

  • Western blot analysis of key metabolic enzymes

  • Metabolomic analysis to identify changes in lipid profiles

  • Assessment of glycolytic activity (e.g., glucose uptake, lactate production)

  • Analysis of lipid synthesis pathways

• Correlate PAFAH1B3 expression with metabolic phenotypes in patient samples

What are the considerations for using PAFAH1B3 antibodies in multiplex immunofluorescence studies?

When designing multiplex immunofluorescence studies with PAFAH1B3 antibodies:

• Antibody compatibility:

  • Select antibodies from different host species to avoid cross-reactivity

  • If using multiple rabbit antibodies, sequential staining with appropriate blocking steps may be required

• Fluorophore selection:

  • Choose fluorophores with minimal spectral overlap

  • Consider signal intensity differences between targets and adjust antibody concentrations accordingly

• Controls:

  • Include single-stained controls to assess bleed-through

  • Use appropriate negative controls for each antibody

  • Include positive controls with known PAFAH1B3 expression (e.g., specific cancer cell lines)

• Image acquisition and analysis:

  • Optimize exposure settings for each fluorophore

  • Employ spectral unmixing if necessary

  • Use automated quantification software for colocalization analysis

How can I address weak or absent signal when using PAFAH1B3 antibodies in Western blot?

If experiencing weak or no signal in Western blot:

• Sample preparation:

  • Ensure adequate protein extraction and denaturation

  • Use fresh samples or add protease inhibitors during extraction

  • Verify protein loading with housekeeping controls

• Antibody optimization:

  • Test different antibody concentrations (1:500-1:1000 recommended)

  • Increase incubation time (overnight at 4°C)

  • Try different blocking agents (BSA vs. non-fat milk)

• Detection system:

  • Use more sensitive detection methods (e.g., ECL Plus)

  • Increase exposure time

  • Consider alternative antibodies targeting different epitopes

What could cause non-specific binding when using PAFAH1B3 antibodies in IHC?

Non-specific binding in IHC may result from:

• Antibody factors:

  • Excessive antibody concentration (dilute further, 1:20-1:200 recommended)

  • Non-optimal antigen retrieval (try both TE buffer pH 9.0 and citrate buffer pH 6.0)

  • Insufficient blocking (increase blocking time or concentration)

• Tissue factors:

  • Over-fixation (adjust fixation time)

  • Endogenous peroxidase activity (incorporate H₂O₂ treatment)

  • Non-specific binding to endogenous biotin (use biotin-free detection systems)

• Protocol adjustments:

  • Include additional blocking steps using serum from the secondary antibody host species

  • Optimize incubation temperature and time

  • Add detergents like Tween-20 to reduce non-specific hydrophobic interactions

How should I interpret PAFAH1B3 expression data in cancer research?

When interpreting PAFAH1B3 expression data:

• Consider normal vs. tumor expression:

  • PAFAH1B3 is typically overexpressed in multiple cancer types compared to normal tissues

  • Expression may vary by cancer subtype and stage

• Clinical correlations:

  • Higher expression levels generally correlate with poorer prognosis

  • Expression may be linked to specific clinicopathological features (e.g., stage, grade, metastasis)

• Biological context:

  • PAFAH1B3 functions in metabolic pathways that support cancer progression

  • Expression may correlate with other oncogenic pathways or mutations (e.g., TP53)

• Technical considerations:

  • Use appropriate statistical methods for data analysis

  • Consider sample heterogeneity and potential confounding factors

  • Validate findings across multiple experimental approaches and patient cohorts

How might PAFAH1B3 antibodies be utilized in investigating response to cancer therapeutics?

PAFAH1B3 antibodies can be valuable tools for studying treatment response:

• Predictive biomarker potential:

  • Evaluate PAFAH1B3 expression before and after treatment

  • Correlate expression levels with treatment response

  • Develop IHC-based scoring systems for patient stratification

• Combination therapy studies:

  • Investigate synergistic effects between PAFAH1B3 inhibition and standard therapies

  • Use antibodies to monitor PAFAH1B3 expression changes during treatment

  • Identify resistance mechanisms via changes in PAFAH1B3 expression or localization

• Therapeutic response monitoring:

  • Develop protocols for longitudinal assessment of PAFAH1B3 expression

  • Correlate expression changes with clinical outcomes

  • Identify cutoff values for expression that predict treatment response

What are the methodological considerations for investigating PAFAH1B3 in cancer immunotherapy research?

When exploring PAFAH1B3's role in immunotherapy:

• Tumor microenvironment analysis:

  • Use multiplex immunofluorescence to assess PAFAH1B3 expression in relation to immune cell infiltrates

  • Correlate PAFAH1B3 expression with immune checkpoint molecules

  • Investigate how PAFAH1B3 inhibition affects the immune microenvironment

• Experimental design for immunotherapy studies:

  • Develop protocols combining PAFAH1B3 targeting with immune checkpoint inhibitors

  • Use appropriate in vivo models that retain immune function

  • Employ flow cytometry to assess changes in immune cell populations following PAFAH1B3 manipulation

• Translational considerations:

  • Establish standardized protocols for PAFAH1B3 assessment in clinical samples

  • Develop companion diagnostic approaches using PAFAH1B3 antibodies

  • Consider sampling methodologies that capture tumor heterogeneity

How can single-cell analysis techniques be combined with PAFAH1B3 antibodies for advanced cancer research?

Single-cell approaches with PAFAH1B3 antibodies offer novel research directions:

• Single-cell immunofluorescence:

  • Optimize PAFAH1B3 antibody concentration for single-cell resolution

  • Combine with markers of cell identity and functional states

  • Implement automated image analysis for objective quantification

• Mass cytometry (CyTOF):

  • Develop metal-conjugated PAFAH1B3 antibodies for CyTOF applications

  • Design panels incorporating cancer stem cell markers, proliferation markers, and signaling molecules

  • Implement dimensionality reduction techniques for data analysis

• Spatial transcriptomics integration:

  • Correlate PAFAH1B3 protein expression with spatial gene expression data

  • Identify gene signatures associated with high PAFAH1B3 expression

  • Map PAFAH1B3 expression patterns within the tumor microenvironment