ASPH Antibody

What are the typical applications for ASPH antibody in research?

ASPH antibodies are commonly used in several research applications:

• Western Blotting (WB): For detection of ASPH protein expression levels in tissue and cell lysates.

• Immunohistochemistry (IHC): For visualization of ASPH expression and localization in tissue sections.

• ELISA: For quantitative measurement of ASPH in biological samples.

• Knockout/Knockdown validation: For confirming the specificity of antibody and evaluating functional changes.

The recommended dilution ranges for different applications are:

It is recommended that researchers titrate the antibody in each testing system to obtain optimal results, as optimal conditions can be sample-dependent .

What is the reactivity profile of commonly used ASPH antibodies?

ASPH antibodies show different reactivity profiles depending on their design and production. For example, the Proteintech antibody 14116-1-AP has been tested and confirmed to react with human and mouse samples in Western blot and immunohistochemistry applications. Literature also cites reactivity with rat samples . When selecting an ASPH antibody for your research, it's important to verify that it has been validated in your species of interest and application.

What are the proper storage conditions for ASPH antibodies?

ASPH antibodies, like the Proteintech 14116-1-AP, are typically stored in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3. The recommended storage temperature is -20°C, where the antibody remains stable for one year after shipment. For 20 μl size preparations, they often contain 0.1% BSA. Aliquoting is generally unnecessary for -20°C storage . Proper storage ensures antibody stability and consistent performance across experiments.

How does the hydroxylase activity of ASPH contribute to cancer cell migration and metastasis?

ASPH hydroxylase activity plays a critical role in promoting cancer cell migration and invasion. Research has shown that specific blockade of ASPH hydroxylase inhibits hepatocellular carcinoma (HCC) cell migration. A polyclonal antibody (FE1) against the Fe-binding His-2 motif at the C-terminal of ASPH, which is a key region for hydroxylase activity, was able to neutralize the hydroxylase activity of ASPH .

In experimental studies, FE1 displayed a dose-dependent inhibitory effect on cell migration in Huh-7 cells that overexpressed wild-type ASPH, with an IC50 of approximately 100 μg/ml resulting in 80% enzymatic inhibition. Importantly, FE1 did not block cell migration in Huh-7 cells overexpressing the enzymatic mutant H679A variant of ASPH, demonstrating the specific dependence on hydroxylase activity .

The relationship between hydroxylase activity and cell migration was further confirmed by the observation that FE1 inhibited cell migration in EHBC-512 cells (which express membrane ASPH) in a dose-dependent manner but was ineffective in MHCC-97L cells (which have low levels of membrane ASPH) .

What is the relationship between ASPH and the Notch signaling pathway in cancer progression?

ASPH has been shown to activate the Notch signaling pathway, which plays a crucial role in tumor development and progression. In pancreatic cancer (PC), ASPH overexpression promotes proliferation, migration, invasion, and malignant transformation of cancer cells through multiple signaling pathways, including Notch .

The relationship between ASPH and Notch signaling was highlighted in a study by Dong et al., which reported that MO-I-1100, a small molecule inhibitor (SMI) of β-hydroxylase, reduced ASPH activity by 80%, inhibited ASPH-induced proliferation, migration, invasion, and colony formation, and suppressed Notch signaling in pancreatic cancer .

Similarly, Sturla et al. demonstrated that SMIs MO-I-1100 and MO-I-1151 significantly reduced viability and directional motility of glioblastoma multiforme (GBM) cells while suppressing Notch activation, confirming the role of ASPH in these processes .

How does ASPH interact with vimentin to promote cancer cell migration?

Mass spectrometry and immunoprecipitation studies have revealed that ASPH interacts directly with vimentin, an intermediate filament protein involved in cell migration. In pull-down assays, vimentin peptides were detected in protein complexes precipitated by anti-Flag antibodies in cells overexpressing FLAG-tagged ASPH. Conversely, ASPH peptides were observed in protein complexes from pull-down assays of HA-tagged vimentin .

The interaction between endogenous ASPH and vimentin was further confirmed by reciprocal immunoprecipitation and immunoblot using vimentin and ASPH antibodies in MHCC-97L cells .

Functional studies demonstrated that:

• Overexpression of ASPH enhanced the expression of vimentin and vice versa

• Downregulation of ASPH slightly decreased vimentin expression

• Overexpression of vimentin greatly promoted HCC cell migration

• Knockdown of vimentin inhibited cell migration

• Overexpression of vimentin effectively reversed the inhibitory effect on cell migration caused by ASPH knockdown

• ASPH overexpression failed to enhance cell migration when vimentin was silenced

These findings suggest that vimentin is a critical mediator of ASPH-induced cell migration in HCC.

What are the current therapeutic approaches targeting ASPH in cancer research?

ASPH has emerged as an important biological target for controlling tumor cell migration and invasion, as its overexpression has been observed in 70-90% of human tumors. Several therapeutic approaches targeting ASPH are being investigated:

• Immunotherapy: ASPH can be used as a tumor-associated antigen (TAA) as it transfers from the endoplasmic reticulum to the plasma membrane in tumor cells, exposing it to the extracellular environment. ASPH-loaded dendritic cells (DCs) have shown substantial anti-tumor effects in HCC models, activating both CD4+ T cells and CD8+ cytotoxic T cells .

• Small Molecule Inhibitors (SMIs): MO-I-1100, an SMI of β-hydroxylase, reduced ASPH activity by 80% and inhibited ASPH-induced proliferation, migration, invasion, and colony formation in pancreatic cancer. Similarly, SMIs MO-I-1100 and MO-I-1151 significantly reduced viability and directional motility of glioblastoma multiforme cells .

• Monoclonal Antibodies: Radiolabeled human monoclonal antibody (mAb) PAN-622 targeting ASPH on the surface of cancer cells has shown promise in imaging and potentially treating metastatic breast cancer. Furthermore, mAb against the ASPH C-terminal (ASPH-C) increased antibody-dependent cellular cytotoxicity of NK cells against HeLa, MCF-7, and HepG2 cells .

• RNA Interference: Antisense oligodeoxynucleotides inhibiting ASPH expression significantly reduced the motility of cholangiocarcinoma cells. Small interfering RNAs (siRNAs) targeting exon 2 of the ASPH gene inhibited ASPH expression and reduced directional motility in HCC cells .

What are the optimal protocols for using ASPH antibody in Western blotting?

For optimal Western blotting results with ASPH antibody:

• Sample Preparation:

  • Total protein extraction from tissues (brain, kidney) or cell lines (A549, HCC cell lines)

  • Include protease inhibitors to prevent degradation

  • Denature samples in loading buffer with reducing agent

• Gel Electrophoresis and Transfer:

  • Use 8-10% SDS-PAGE gels to effectively separate proteins in the 26-141 kDa range

  • Transfer to PVDF or nitrocellulose membrane at 100V for 60-90 minutes

• Blocking and Antibody Incubation:

  • Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • Incubate with primary ASPH antibody (e.g., 14116-1-AP) at 1:200-1:1000 dilution in blocking buffer overnight at 4°C

  • Wash 3 times with TBST, 5 minutes each

  • Incubate with HRP-conjugated secondary antibody at appropriate dilution for 1 hour at room temperature

  • Wash 3 times with TBST, 5 minutes each

• Detection:

  • Apply ECL substrate and image using film or digital imaging system

  • Expected molecular weights: native and phosphorylated forms at 86-141 kDa, and cleavage products at 35-56 kDa and 22-26 kDa

• Controls:

  • Include positive controls such as mouse brain tissue, A549 cells, or human brain tissue

  • Consider using ASPH knockdown/knockout cells as negative controls

How should immunohistochemistry be performed with ASPH antibody?

For optimal immunohistochemistry with ASPH antibody:

• Tissue Preparation:

  • Fix tissues in 10% neutral buffered formalin

  • Embed in paraffin and section at 4-5 μm thickness

• Antigen Retrieval:

  • Use TE buffer pH 9.0 for antigen retrieval (suggested)

  • Alternatively, citrate buffer pH 6.0 can be used

• Blocking and Antibody Incubation:

  • Block endogenous peroxidase with 3% H₂O₂

  • Block non-specific binding with 5-10% normal serum

  • Incubate with ASPH antibody at 1:50-1:500 dilution overnight at 4°C

  • Wash with PBS

  • Incubate with appropriate secondary antibody

  • Develop with DAB substrate and counterstain with hematoxylin

• Controls and Validation:

  • Include positive control tissues (human kidney tissue has been validated)

  • Include negative controls by omitting primary antibody

  • Consider testing different antigen retrieval methods and antibody dilutions for optimization

• Analysis:

  • Evaluate staining pattern, intensity, and localization

  • ASPH is typically localized to the cytoplasm or membrane in cancer cells

How can researchers distinguish between membrane and cytoplasmic ASPH expression?

Distinguishing between membrane and cytoplasmic ASPH expression is important in cancer research since ASPH's translocation to the cell membrane is associated with malignancy. Here are methodological approaches:

• Immunofluorescence without permeabilization:

  • Fix cells but omit the triton X-100 permeabilization step to detect only cell surface ASPH

  • As demonstrated in the research results, EHBC-512 cells showed clear membrane ASPH presence when stained this way, whereas MHCC-97L cells did not

• Flow cytometry:

  • For non-permeabilized cells: detect only surface ASPH

  • For permeabilized cells: detect total ASPH (cytoplasmic and membrane)

  • Research showed that only a subset of EHBC-512 cells expressed ASPH on the cell surface, while nearly all EHBC-512 and MHCC-97L cells expressed ASPH intracellularly

• Cell fractionation and Western blot:

  • Separate membrane and cytoplasmic fractions

  • Perform Western blot with ASPH antibody on each fraction

  • Use appropriate markers for each fraction (e.g., Na+/K+-ATPase for membrane, GAPDH for cytoplasm)

• Confocal microscopy:

  • Use Z-stack imaging to distinguish membrane from cytoplasmic localization

  • Co-stain with membrane markers for colocalization analysis

What are the best approaches to verify ASPH antibody specificity?

To ensure the specificity of ASPH antibodies and validate experimental results:

• ASPH Knockdown/Knockout Controls:

  • Use RNA interference (siRNA or shRNA) to knockdown ASPH

  • The search results indicate that siRNAs targeting exon 2 of the ASPH gene effectively inhibited ASPH expression

  • CRISPR/Cas9-mediated knockout can provide complete absence of the target protein

  • Perform Western blot or immunostaining to confirm absence or reduction of signal

• Peptide Competition Assays:

  • Pre-incubate the antibody with the immunizing peptide

  • As demonstrated in the research results, FE1 antibody signals in EHBC-512 and MHCC-97L were sensitive to antigen peptide competition

• Domain-Specific Antibody Testing:

  • Use different antibodies targeting different domains of ASPH

  • Research showed that FE1 (targeting the Fe-binding His-2 motif at the C-terminal) recognized wild-type ASPH but not the enzymatic mutant, while antibodies targeting the N-terminal recognized both

• Recombinant Protein Expression:

  • Overexpress tagged ASPH in cell lines with low endogenous expression

  • Confirm antibody detection of the overexpressed protein

• Multi-technique Validation:

  • Verify consistent results across different techniques (Western blot, IHC, IF)

  • Confirm expected molecular weight and subcellular localization

How can ASPH hydroxylase activity be measured in experimental settings?

Measuring ASPH hydroxylase activity is crucial for studies investigating its role in cancer progression. Several approaches can be employed:

• α-Ketoglutarate (α-KG) Consumption Assay:

  • ASPH is an α-KG-dependent dioxygenase

  • Measure the consumption of α-KG during Asp β-hydroxylation

  • This method was used in research to demonstrate that the FE1 antibody decreased α-KG consumption by 80% at a concentration of 100 μg/ml

• Site-directed Mutagenesis:

  • Create enzymatic loss-of-function mutants by mutating histidine-679 (an essential residue for ASPH catalytic activities) to alanine

  • Compare the functional effects of wild-type versus mutant ASPH expression

• Inhibitor Studies:

  • Use small molecule inhibitors like MO-I-1100 that reduce ASPH activity

  • Measure the downstream effects on cell migration, invasion, and Notch signaling activation

• Mass Spectrometry:

  • Detect hydroxylated versus non-hydroxylated peptides containing aspartate or asparagine residues

• Functional Readouts:

  • Cell migration assays as indirect measures of hydroxylase activity

  • Notch signaling activation as a downstream consequence

What are the key experimental considerations when studying ASPH in cancer models?

When designing experiments to study ASPH in cancer models, researchers should consider the following:

• Cell Line Selection:

  • Choose cell lines with appropriate endogenous ASPH expression

  • Consider membrane versus cytoplasmic localization

  • The search results indicate that EHBC-512 cells show clear membrane ASPH presence, whereas MHCC-97L cells have low levels of membrane ASPH

• Expression System Design:

  • When overexpressing ASPH, include both wild-type and enzymatic mutant (H679A) versions

  • Use appropriate vectors and tags that don't interfere with protein function

  • The research used FLAG-ASPH and HA-vimentin plasmids constructed by introducing coding sequences into pFLAG-CMV and pCMV-HA vectors

• Functional Assays:

  • Migration assays to assess metastatic potential

  • Proliferation and colony formation assays

  • Invasion assays using Matrigel

  • Signaling pathway analysis (especially Notch pathway components)

• Interaction Studies:

  • Investigate protein-protein interactions (e.g., with vimentin)

  • Use pull-down assays, immunoprecipitation, and mass spectrometry

  • The research successfully identified vimentin interaction with ASPH using these methods

• In Vivo Models:

  • Consider orthotopic models for studying metastasis

  • Evaluate ASPH-targeting therapeutic approaches

  • ASPH-loaded DCs have shown anti-tumor effects in animal models

How does ASPH expression correlate with clinical outcomes in cancer patients?

Understanding the correlation between ASPH expression and clinical outcomes is important for translational research. The available research indicates:

• Hepatocellular Carcinoma (HCC):

  • Wang et al. showed a significant association between ASPH overexpression and higher recurrence and lower survival rates following surgery

  • ASPH overexpression could predict worse surgical outcomes in early-stage HCC patients

• Pancreatic Cancer:

  • ASPH overexpression is observed in pancreatic cancer

  • It plays an important role in promoting proliferation, migration, invasion, and malignant transformation of pancreatic cancer cells through multiple signaling pathways

• Other Cancers:

  • ASPH overexpression has been observed in cholangiocarcinoma, lung cancer, breast cancer, colon cancer, and neoplasms of the nervous system

When designing studies to investigate clinical correlations, researchers should:

• Use tissue microarrays with adequate sample sizes

• Employ multiple antibodies or detection methods

• Correlate expression with established clinical parameters and outcomes

• Consider both expression levels and subcellular localization

• Use multivariate analysis to account for confounding factors

Why might ASPH appear at different molecular weights in Western blots?

ASPH can appear at different molecular weights in Western blots due to several factors:

• Multiple Isoforms and Post-translational Modifications:

  • The calculated molecular weight of ASPH is 86 kDa

  • Native and phosphorylated forms can appear at 86-141 kDa

  • Cleavage products can appear at 35-56 kDa and 22-26 kDa

  • The observed molecular weight reported in the product information is 26 kDa

• Technical Factors:

  • Incomplete denaturation can cause abnormal migration

  • Different gel concentrations can affect apparent molecular weight

  • Insufficient blocking can lead to non-specific bands

• Antibody Specificity:

  • Different antibodies may recognize different domains or isoforms

  • For example, antibodies targeting the N-terminal versus C-terminal of ASPH may detect different forms

• Tissue/Cell Type Variations:

  • Different tissues or cell lines may express different ASPH isoforms

  • Cancer cells often have altered expression patterns compared to normal cells

To address these variations, researchers should:

• Run appropriate molecular weight markers

• Include positive control samples with known ASPH expression

• Consider using multiple antibodies targeting different epitopes

• Validate bands using ASPH knockdown or knockout controls

What should researchers do if they observe discrepancies between different ASPH antibodies?

When faced with discrepancies between different ASPH antibodies:

• Review Antibody Specifications:

  • Check the epitope/immunogen for each antibody

  • Determine if antibodies target different domains (N-terminal vs. C-terminal)

  • The research results show that different antibodies can recognize different forms of ASPH; for example, FE1 (targeting the Fe-binding domain) only recognized wild-type ASPH, while antibodies targeting the N-terminal recognized both wild-type and mutant forms

• Validate with Genetic Approaches:

  • Use ASPH knockdown or knockout controls

  • Overexpress ASPH in null backgrounds

  • Use domain-specific mutants to map epitopes

• Employ Multiple Detection Methods:

  • Compare results from Western blot, IHC, and immunofluorescence

  • Different techniques may provide complementary information

• Consider Post-translational Modifications:

  • Investigate if discrepancies might be due to detection of different modified forms

  • Use phosphatase treatment to remove phosphorylation if relevant

• Consult Literature and Technical Support:

  • Review published validations for each antibody

  • Contact antibody manufacturers for technical assistance

  • Share your experimental conditions for more targeted advice