ptdss2 Antibody

What is PTDSS2 and why is it important in cellular research?

PTDSS2 catalyzes a base-exchange reaction in which the polar head group of phosphatidylethanolamine (PE) is replaced by L-serine to form phosphatidylserine (PS). Unlike PTDSS1, PTDSS2 is specific for phosphatidylethanolamine and does not act on phosphatidylcholine . This enzyme plays a critical role in membrane phospholipid homeostasis, particularly in regulating PS exposure on cell membranes.

The importance of PTDSS2 extends beyond basic membrane biology. Recent research has demonstrated that PTDSS2 expression levels significantly impact cancer metastasis and B cell receptor signaling , making it a valuable target for both basic and translational research.

How should researchers choose between different PTDSS2 antibody preparations?

Selection criteria for PTDSS2 antibodies should be based on multiple factors:

• Epitope specificity: Most commercial PTDSS2 antibodies target the N-terminal region (AA 1-30) , though antibodies targeting other regions (AA 35-84, AA 381-430) are also available . The epitope location can affect protein detection in different conformational states.

• Species reactivity: Available antibodies show different cross-reactivity profiles:

  • Human-specific: Some antibodies react exclusively with human PTDSS2

  • Multi-species: Others detect PTDSS2 across species including human, dog, guinea pig, mouse, rat, and horse with varying degrees of homology

• Clonality and host: Most PTDSS2 antibodies are rabbit polyclonal antibodies , though rabbit monoclonal options exist for specific applications .

• Purification method: Affinity purified antibodies (through protein A and/or peptide affinity) generally provide higher specificity.

What validation experiments are essential before using a PTDSS2 antibody in critical research?

Thorough validation is crucial for reliable PTDSS2 detection:

• Specificity validation: Test for cross-reactivity with PTDSS1, which shares functional similarity but differs in specificity . PTDSS1 knockout models can help distinguish between these related proteins.

• Knockout validation: Compare antibody signal between wild-type and PTDSS2-knockout samples. Research has shown that PTDSS2 KO does not upregulate PTDSS1 expression and vice versa in several cell lines .

• Peptide competition: Use the immunizing peptide to confirm binding specificity, particularly important for polyclonal antibodies targeting the N-terminal region .

• Cross-species validation: If working with non-human models, verify antibody performance in the target species. Sequence homology data suggests high conservation: Dog (100%), Guinea Pig (100%), Horse (93%), Mouse (100%), Rat (100%) .

How can PTDSS2 antibodies be used to study cancer metastasis mechanisms?

PTDSS2 antibodies enable investigation of a newly discovered metastatic pathway involving phosphatidylserine exposure:

• Detection of ATP11B^lo PTDSS2^hi phenotype: This expression pattern is associated with poor prognosis and enhanced metastasis in breast cancer patients . PTDSS2 antibodies can quantify expression levels across patient samples and cell lines.

• Nonapoptotic PS exposure assessment: Combine PTDSS2 antibody staining with flow cytometry using anti-PS or annexin V antibodies to correlate PTDSS2 expression with PS exposure .

• BRCA1-PTDSS2 regulatory axis: PTDSS2 promoter activity is strongly suppressed by BRCA1. In experimental models, knockdown of BRCA1 increased PTDSS2 mRNA and protein levels, while overexpression of BRCA1 reduced PTDSS2 expression . This relationship can be assessed using PTDSS2 antibodies alongside BRCA1 manipulation.

• Metastasis model development: Research has shown that cells with PTDSS2 overexpression and ATP11B knockdown develop significantly more metastases in mouse models . PTDSS2 antibodies can confirm protein levels in these experimental systems.

What methodological approaches combine PTDSS2 antibodies with functional studies?

Several integrated approaches have proven effective:

• Genetic manipulation with protein validation:

  • CRISPR/Cas9-mediated knockout of PTDSS2

  • Overexpression of wild-type PTDSS2

  • Expression of catalytically dead PTDSS2-R235S mutant (corresponds to human PTDSS2-R313C)

  • Validation of manipulation success using PTDSS2 antibodies

• PS exposure correlation studies:

  • Flow cytometry analysis with anti-PS and anti-annexin V antibodies

  • Correlation with PTDSS2 expression levels detected by antibodies

  • Functional rescue experiments using exogenous PS supplementation

• Phospholipid composition analysis:

  • PTDSS2 knockout typically does not significantly alter phospholipid composition

  • PTDSS1 knockout results in significant phospholipid imbalance

  • Correlation between these findings and PTDSS2 protein levels detected by antibodies

How do researchers utilize PTDSS2 antibodies to study B cell receptor signaling?

PTDSS2 plays a role in B cell receptor (BCR) signaling, particularly in lymphoma cells:

• Expression correlation with BCR dependency: PTDSS2 antibodies can assess expression levels in B cell lymphoma subtypes that show different dependencies on PS synthesis .

• PTDSS1-PTDSS2 interplay: While B cell lymphomas are highly dependent on PS synthesis, this primarily involves PTDSS1 rather than PTDSS2. PTDSS2 knockout does not significantly alter phospholipid composition in lymphoma cells, whereas PTDSS1 inhibition or knockout causes substantial phospholipid imbalance .

• Calcium signaling studies: PTDSS2 antibodies can be used to correlate expression with calcium signaling responses. Research shows that PTDSS1 knockout enhances BCR-induced calcium signaling and apoptosis in B cell lymphoma .

What experimental protocols effectively combine PTDSS2 detection with functional assays in lymphoma research?

Several methodological approaches have proven valuable:

• Combined genetic manipulation:

  • Generate PTDSS1 and PTDSS2 knockout cell lines using CRISPR/Cas9

  • Confirm knockout efficiency using PTDSS2 antibodies

  • Compare effects on phospholipid composition and cellular viability

• Phospholipid profile correlation:

  • Analyze changes in PS, PE, PI, and other phospholipids upon PTDSS2 manipulation

  • Correlate with PTDSS2 protein levels detected by antibodies

  • Particular focus on dominant acyl-chain species (C34:1, C36:1, C36:2)

• BCR signaling assessment:

  • Measure BCR-induced calcium signaling using Fura-2 ratiometric calcium probe

  • Correlate with PTDSS2 expression levels

  • Include rescue experiments with exogenous phospholipids

How can researchers address inconsistent PTDSS2 antibody results between applications?

When facing variable results across experimental systems:

• Application-specific optimization:

  • Western blotting: Optimize protein extraction methods for membrane proteins

  • IHC: Test different antigen retrieval methods for paraffin-embedded tissues

  • Flow cytometry: Adjust cell fixation and permeabilization protocols

• Antibody selection refinement:

  • Test antibodies targeting different PTDSS2 epitopes (N-terminal vs. other regions)

  • Compare polyclonal and monoclonal antibodies

  • Consider antibodies validated specifically for your application of interest

• Validation with orthogonal methods:

  • Complement antibody detection with mRNA analysis

  • Use genetic manipulation (overexpression, knockdown) as controls

  • Include wild-type and PTDSS2-knockout samples when possible

What controls are essential when studying PTDSS2 in complex experimental systems?

Proper controls ensure reliable PTDSS2 antibody-based experiments:

• Expression controls:

  • Positive control: Cell lines with confirmed PTDSS2 expression

  • Negative control: PTDSS2 knockout cells

  • Comparative analysis across multiple cell lines with varying PTDSS2 expression levels

• Specificity controls:

  • Test for cross-reactivity with PTDSS1

  • Include peptide competition assays

  • Use catalytically dead PTDSS2 mutants (e.g., PTDSS2-R235S)

• Functional validation:

  • Correlate antibody detection with PS exposure measured by flow cytometry

  • Include rescue experiments with exogenous phospholipids

  • Assess effects of PTDSS2 manipulation on downstream cellular processes

How can researchers leverage PTDSS2 antibodies to investigate its role in the tumor microenvironment?

Recent findings suggest PTDSS2-regulated PS exposure impacts immune cell recruitment:

• Immunosuppressive microenvironment analysis:

  • Use PTDSS2 antibodies to correlate expression with immune cell infiltration

  • High PTDSS2 expression increases nonapoptotic PS exposure, creating a global immunosuppressive signal

  • This promotes myeloid-derived suppressor cell accumulation and reduces cytotoxic T cell activity

• Therapeutic intervention monitoring:

  • Anti-PS antibody therapy combined with either paclitaxel or docetaxel can effectively overcome metastatic processes associated with ATP11B^lo PTDSS2^hi cancer cells

  • PTDSS2 antibodies can monitor expression changes during treatment response

• Catalytic function assessment:

  • Catalytically dead PTDSS2-R235S mutation completely suppresses lung metastasis induced by PTDSS2 overexpression

  • PTDSS2 antibodies can confirm expression of both wild-type and mutant proteins in experimental models

What are the methodological considerations for studying PTDSS2 in phospholipid transfer research?

PTDSS2 functions within a complex phospholipid regulatory network:

• Membrane contact site analysis:

  • PTDSS2 inhibition affects membrane contact-based lipid transfer machinery

  • Combined immunofluorescence approaches using PTDSS2 antibodies with markers of membrane contact sites

  • Correlation with phospholipid imbalance effects

• Phosphoinositide metabolism integration:

  • PTDSS1 inhibition increases PIP (phosphatidylinositol phosphate) levels

  • PTDSS2 expression may influence this process

  • Combined analysis of PTDSS2 expression with phosphoinositide levels and localization

• Subcellular localization studies:

  • PTDSS2 antibodies can track protein localization

  • Correlation with PS exposure at the plasma membrane

  • Assessment of effects on phospholipid probe localization (e.g., EGFP-2xPH evectin2 for PS, EGFP-2xP4M SidM for PI4P)