PODXL2 Antibody

What is PODXL2 and what cellular locations should researchers expect to detect it?

PODXL2 (podocalyxin-like 2), also known as Endoglycan, is a type I transmembrane glycoprotein belonging to the CD34/podocalyxin family of sialomucins. It contains an N-terminal acidic domain with polyglutamate tracts, a mucin-like domain, and a membrane proximal globular domain . Unlike other sialomucins, PODXL2 contains chondroitin sulfate modifications .

PODXL2 is primarily expressed in:

• Endothelial cells (including HUVEC cells)

• Hematopoietic precursors

• T-cells, B-cells, and monocytes (with higher expression in memory and germinal center cells than naive B-cells)

• Brain tissue (high expression)

• Pancreas, kidney, and lymphoid node (moderate expression)

• Liver (weak expression)

In immunofluorescence studies, specific staining is typically localized to cell membranes . When designing experiments, researchers should account for this cellular localization pattern.

What are the optimal applications and dilutions for PODXL2 antibodies?

Based on validated data from multiple antibody sources, researchers should consider these applications and dilutions:

Methodological Note: The optimal dilution should be determined empirically by each laboratory for their specific experimental system. Sample-dependent variations may require adjustment of these recommended parameters .

What is the expected molecular weight of PODXL2 in experimental procedures?

When designing Western blot experiments, researchers should expect to observe:

• Calculated molecular weight: 65 kDa (605aa) or 57 kDa (529aa)

• Observed molecular weight: 65 kDa

This information is critical for proper identification of bands in Western blot analysis and for distinguishing specific signals from non-specific binding.

How can researchers validate the specificity of PODXL2 antibodies in their experimental systems?

A multi-step validation approach is recommended:

• Positive and negative control tissues/cells: Use tissues with known expression levels:

  • Positive controls: HEK-293 cells, mouse pancreas tissue, HeLa cells, K-562 cells

  • Tissue panels: Brain (high expression), pancreas, kidney, lymphoid node (moderate expression)

• Knockdown validation: Utilize shRNA silencing of PODXL2, as demonstrated in BT474 breast carcinoma cell line studies . Compare antibody reactivity in wild-type versus knockdown cells.

• Cross-species reactivity assessment: Human PODXL2 shares 73% amino acid sequence identity with mouse PODXL2 within the extracellular domain . This partial homology should be considered when interpreting cross-species experiments.

• Western blot assessment: Validate the observed molecular weight matches expected size (65 kDa) .

• Subcellular localization confirmation: Verify membrane localization pattern in immunofluorescence studies .

What storage conditions are critical for maintaining PODXL2 antibody reactivity?

Proper storage significantly impacts antibody performance. Follow these evidence-based guidelines:

• Store at -20°C (most commercial antibodies)

• Stable for one year after shipment when properly stored

• For reconstituted antibodies, stability varies by formulation:

  • 1 month at 2-8°C under sterile conditions

  • 6 months at -20 to -70°C under sterile conditions

Technical note: Many PODXL2 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3) . Aliquoting is often unnecessary for -20°C storage, but is recommended to avoid freeze-thaw cycles for antibodies in other buffer formulations .

How is PODXL2 implicated in cancer progression and what experimental models are appropriate for its study?

Research has shown PODXL2 plays significant roles in cancer biology:

• Expression correlation with cancer prognosis:

  • PODXL2 overexpression correlates with poor survival of breast cancer patients

  • mRNA analysis from Oncomine showed overexpression in 8 of 20 cancer types compared to normal tissues:

    • Bladder cancer (2/10 studies)

    • Breast cancer (6/43 studies)

    • Cervical cancer (1/10 studies)

    • Colorectal cancer (1/32 studies)

    • Esophageal cancer (2/7 studies)

    • Lung cancer (4/20 studies)

    • Melanomas (1/5 studies)

    • Prostate cancer (1/9 studies)

• Functional impacts in cancer models:

  • Knockdown of PODXL2 in BT474 breast cancer cells:

    • Slightly influenced cell proliferation

    • Suppressed migration

    • Inhibited expressions of Rac1, p-Akt (S473), and p-paxillin (Y31) proteins

    • Reduced expression of cancer stem cell markers (Oct-4, Nanog, ALDH1)

• Recommended experimental models:

  • BT474 invasive ductal breast carcinoma cell line (validated for PODXL2 knockdown studies)

  • Bioinformatics tools: Oncomine database, Human Protein Atlas, METABRIC, TCGA Nature/Cell, and CCLE datasets

What methodological considerations are important when designing experiments to study PODXL2 function?

When investigating PODXL2 function, consider these methodological approaches:

• Gene silencing studies:

  • Short hairpin (sh)-RNA silencing method has been validated for PODXL2 in BT474 cells

  • Western blotting can confirm suppressive efficacy using anti-PODXL2 polyclonal antibodies

• qPCR primer design for PODXL2:

  • Validated primer sets include:

    • PODXL2-sense: 5'-TGC CTT CAG TCA CCC CAA CTA-3'

    • PODXL2-antisense: 5'-AGC CTC GAA CTC TAC CCC AAG-3'

• Co-expression analysis:

  • Utilize datasets like METABRIC, TCGA Nature, and Cell for identifying PODXL2 co-expression patterns

  • Top 10% of co-expressed genes can be analyzed through METACORE database for pathway analysis

• Protein interaction studies:

  • Investigate PODXL2's role as a ligand for vascular selectins

  • Study its involvement in leukocyte rolling over vascular surfaces through interactions with E-, P- and L-selectins

How can researchers address potential discrepancies in PODXL2 antibody performance across different experimental systems?

When confronting inconsistent results with PODXL2 antibodies, researchers should implement this systematic troubleshooting approach:

How does PODXL2 contribute to cellular stemness and what experimental approaches can investigate this function?

Recent research has implicated PODXL2 in maintaining cellular stemness, particularly in cancer contexts:

• Experimental evidence of stemness regulation:

  • Knockdown of PODXL2 in breast cancer cells reduced expression of stem cell markers:

    • Oct-4 and Nanog (embryonic stem cell markers)

    • ALDH1 (breast cancer stem cell marker)

• Recommended experimental approaches:

  • Sphere formation assays to assess self-renewal capacity

  • Flow cytometry analysis of stem cell marker expression

  • In vivo limiting dilution assays to assess tumor-initiating capacity

  • RNA-seq analysis to identify stemness-related pathways affected by PODXL2 modulation

• Key model systems:

  • Breast cancer cell lines (e.g., BT474, validated for PODXL2 knockdown)

  • Primary patient-derived cancer stem cells

  • Embryonic stem cell models to assess developmental roles

What are the technical challenges in detecting PODXL2 in different tissue contexts?

Researchers should be aware of these tissue-specific technical considerations:

• Variable expression levels:

  • High expression in brain, moderate in pancreas, kidney, and lymphoid node, weak in liver

  • Expression higher on memory and germinal center B-cells than naive B-cells

• Co-expression with related proteins:

  • CD34 family members share structural similarities

  • Antibody cross-reactivity must be carefully assessed in tissues expressing multiple family members

• Tissue-specific modifications:

  • Post-translational modifications vary between tissues

  • Glycosylation patterns may affect antibody binding efficiency

• Tissue preparation impact:

  • For immunohistochemistry of mouse testis: Perfusion fixed frozen sections showed optimal results using 1.7 µg/mL Anti-Mouse Endoglycan/PODXL2 antibody

  • For HUVEC cells: Immersion fixation followed by staining with 10 µg/mL Anti-Human Endoglycan/PODXL2 antibody

• Detection system optimization:

  • HRP-DAB staining systems work well for tissue sections

  • For cellular immunofluorescence, NorthernLights™ 557-conjugated secondary antibodies provide strong signal with DAPI counterstaining

How does PODXL2 interact with downstream signaling molecules and what methods can resolve these pathways?

Current research reveals PODXL2 influences several signaling pathways:

• Identified downstream targets:

  • Rac1 (Ras-related C3 botulinum toxin substrate 1)

  • Phosphorylated Akt (S473)

  • Phosphorylated paxillin (Y31)

• Recommended experimental approaches:

  • Proximity ligation assays to detect direct protein interactions

  • Co-immunoprecipitation to identify binding partners

  • Phospho-specific antibody arrays following PODXL2 manipulation

  • Kinase activity assays to assess functional consequences

• Pathway analysis tools:

  • METACORE database for processing networks of co-expressed genes

  • Gene ontology analysis of differentially expressed genes following PODXL2 knockdown

  • Bioinformatic prediction of potential interaction networks

What are the methodological considerations for investigating PODXL2's role in leukocyte-endothelial interactions?

PODXL2 functions as a ligand for vascular selectins, mediating leukocyte rolling. Researchers studying this function should consider:

• In vitro flow chamber assays:

  • Assess leukocyte rolling over PODXL2-expressing endothelial monolayers

  • Measure calcium-dependent interactions with E-, P- and L-selectins

  • Quantify rolling velocity and adhesion strength

• Blocking antibody approaches:

  • Use anti-PODXL2 antibodies to block specific domains

  • Determine which epitopes are critical for selectin binding

  • Compare effects with other selectin ligands

• Domain mapping experiments:

  • Generate domain deletion constructs

  • Assess which regions of PODXL2 are essential for selectin binding

  • Investigate the role of chondroitin sulfate modifications in this process

• Animal models:

  • In vivo imaging of leukocyte trafficking in PODXL2-deficient models

  • Inflammatory challenge models to assess functional significance