ASAH2B Antibody

What is ASAH2B and how does it differ from ASAH2?

ASAH2B is a paralog of ASAH2 (neutral ceramidase) and is formally known as "Putative inactive N-acylsphingosine amidohydrolase 2B." While ASAH2 is an active enzyme involved in sphingolipid metabolism, ASAH2B is considered putatively inactive despite structural similarities. ASAH2 functions as a type II integral membrane protein that can be cleaved to produce a soluble secreted protein involved in the metabolism of dietary sphingolipids and generation of messenger molecules like sphingosine and sphingosine 1-phosphate . ASAH2B, despite its structural similarity, lacks critical residues required for enzymatic activity, suggesting a different physiological role than its active counterpart.

What is the molecular weight of ASAH2B and how does it compare to ASAH2?

The predicted molecular weight of ASAH2B is approximately 19 kDa according to antibody validation data . In contrast, ASAH2 has a calculated molecular weight of 86 kDa based on its 780 amino acid sequence, though it is typically observed at 100-115 kDa in Western blot applications . This size difference is significant and researchers should be aware of this distinction when interpreting Western blot results to avoid misidentification between the two proteins.

What is known about ASAH2B's role in cellular pathways?

Research indicates that lnc-ASAH2B-2 (a long non-coding RNA associated with ASAH2B) is involved in the mTOR signaling pathway, particularly in breast cancer cells. Studies have shown that silencing lnc-ASAH2B-2 inhibits breast cancer cell growth, suggesting a regulatory role in cancer progression . Furthermore, lnc-ASAH2B-2 appears to be upregulated in response to everolimus (an mTOR inhibitor) treatment, potentially contributing to treatment resistance mechanisms in breast cancer .

How can researchers validate the specificity of ASAH2B antibodies?

Validation of ASAH2B antibodies should employ multiple complementary approaches:

• Western blot analysis: Compare bands detected in multiple cell lines (e.g., HeLa, Jurkat, 293T) and tissue lysates (e.g., fetal heart tissue) with the predicted molecular weight (19 kDa) .

• Immunoprecipitation followed by mass spectrometry: This can confirm antibody specificity through protein identification.

• Flow cytometry: Comparing staining in permeabilized cells (e.g., 293 cells) with negative controls helps validate intracellular specificity .

• Knockout or knockdown controls: Using ASAH2B-deficient cells or ASAH2B-silenced cells as negative controls provides strong evidence of specificity.

• Cross-reactivity testing: Test against related proteins, particularly ASAH2, to ensure the antibody doesn't detect homologous proteins.

What epitopes are targeted by commercial ASAH2B antibodies?

Commercial monoclonal antibodies like EPR10539 (ab170949) have been developed against specific epitopes of ASAH2B . This antibody shows reactivity with human samples and has been validated for applications including Western blotting, immunoprecipitation, and flow cytometry. The specific epitope information is typically proprietary but is usually derived from unique regions of ASAH2B that differentiate it from other ASAH family members, particularly ASAH2.

What are the optimal protocols for detecting ASAH2B using Western blotting?

For optimal Western blot detection of ASAH2B:

• Sample preparation: Use freshly prepared total cell lysates from human cell lines (HeLa, Jurkat, 293T) or tissue lysates.

• Protein loading: Load approximately 10-20 μg of total protein per lane .

• Antibody dilution: For primary antibodies like EPR10539, a dilution of 1:1000 is recommended . Secondary antibodies (goat anti-rabbit HRP) can be used at 1:2000 dilution.

• Expected band: Look for a specific band at approximately 19 kDa .

• Controls: Include positive controls (cell lines with known ASAH2B expression) and negative controls (cell lines with low or no ASAH2B expression).

• Optimization note: Due to potential post-translational modifications, the observed molecular weight may vary slightly from the predicted size.

How can ASAH2B be detected in flow cytometry experiments?

For intracellular flow cytometric analysis of ASAH2B:

• Cell preparation: Fix and permeabilize cells (e.g., 293 cells) using standard protocols.

• Antibody concentration: Use anti-ASAH2B antibody [EPR10539] at approximately 1:10 dilution .

• Controls: Include a rabbit IgG negative control at the same concentration .

• Detection: Use appropriate fluorophore-conjugated secondary antibodies.

• Gating strategy: First gate on intact cells based on FSC/SSC, then on single cells, before analyzing ASAH2B expression.

• Data interpretation: Compare the fluorescence intensity shift between the negative control and the ASAH2B antibody-stained sample.

What factors contribute to variability in ASAH2B antibody performance?

Several factors can affect ASAH2B antibody performance:

• Antibody storage conditions: Proper storage at -20°C with minimal freeze-thaw cycles preserves antibody activity .

• Sample preparation: Inefficient cell lysis or protein degradation during sample preparation can significantly impact results.

• Expression levels: ASAH2B expression varies across cell types and tissues, requiring optimization for each experimental system.

• Post-translational modifications: These can alter epitope accessibility or antibody recognition.

• Cross-reactivity: Potential cross-reactivity with ASAH2 or other family members must be carefully controlled for.

How can researchers address weak or absent ASAH2B signal in Western blots?

When encountering weak or absent ASAH2B signals:

• Increase protein loading: Consider loading up to 20-30 μg of total protein.

• Optimize antibody concentration: Titrate antibody concentration; try increasing to 1:500 if 1:1000 yields weak signals .

• Enhance detection sensitivity: Use enhanced chemiluminescence (ECL) substrates with higher sensitivity.

• Adjust exposure time: Increase exposure time for detection of weak signals.

• Verify expression: Confirm ASAH2B expression in your cell type via RT-qPCR or other methods.

• Check for degradation: Use fresh lysates and add protease inhibitors during sample preparation.

• Optimize transfer conditions: Adjust transfer time or buffer composition for proteins in the 19 kDa range.

How is ASAH2B involved in cancer biology, particularly breast cancer?

Research has revealed that lnc-ASAH2B-2 plays a significant role in breast cancer through the mTOR pathway:

• Upregulation in everolimus treatment: lnc-ASAH2B-2 is upregulated after exposure to everolimus (an mTOR inhibitor) in breast cancer cells, both in the presence and absence of serum .

• Growth regulation: Silencing lnc-ASAH2B-2 inhibits proliferation of BT474 and MCF7 breast cancer cells .

• Treatment resistance: The upregulation of lnc-ASAH2B-2 may reduce the efficacy of everolimus treatment, suggesting a potential mechanism of resistance .

• Therapeutic target: lnc-ASAH2B-2 has been proposed as a novel therapeutic target for breast cancer based on these findings .

What approaches can be used to study the interaction between ASAH2B and the mTOR pathway?

To investigate ASAH2B's role in mTOR signaling:

• RNA interference: siRNA or shRNA targeting lnc-ASAH2B-2 can be used to study the effects of its knockdown on:

  • Cell proliferation and colony formation

  • mTOR pathway activation (phosphorylation of S6K, 4E-BP1)

  • Response to mTOR inhibitors like everolimus

• Protein-RNA interactions: RNA immunoprecipitation (RIP) can determine if lnc-ASAH2B-2 directly interacts with proteins in the mTOR pathway.

• Signaling pathway analysis: Western blotting for key mTOR pathway components before and after lnc-ASAH2B-2 manipulation can reveal its position in the signaling cascade.

• Combinatorial treatments: Testing the combination of lnc-ASAH2B-2 silencing with everolimus can assess potential synergistic effects.

• In vivo models: Xenograft models with lnc-ASAH2B-2 knockdown can evaluate its relevance to tumor growth and response to treatment in vivo.

How do ASAH2B antibodies compare in different experimental applications?

How should researchers interpret discrepancies between ASAH2B protein expression and functional data?

When facing discrepancies between ASAH2B expression and functional outcomes:

• Consider protein vs. lncRNA: Distinguish between protein-level ASAH2B expression and lnc-ASAH2B-2 expression, as they may have independent functions.

• Post-translational modifications: Modifications may affect function without changing total protein levels.

• Localization effects: Changes in subcellular localization could alter function without affecting total expression.

• Interaction partners: ASAH2B may require specific interaction partners for function that vary across experimental systems.

• Compensatory mechanisms: Other proteins or pathways might compensate for ASAH2B alterations.

• Technical considerations: Validate results using multiple antibodies and techniques to rule out antibody-specific artifacts.