SASH1 Antibody, HRP conjugated

What is SASH1 and why is it significant in cancer research?

SASH1 is a tumor suppressor protein that belongs to the SLY (SH3 domain containing expressed in lymphocytes) family of signal adapter proteins. It contains sterile α motif (SAM) and Src-homology 3 (SH3) domains that mediate protein-protein interactions. SASH1 has emerged as a critical regulator of tumor progression across multiple cancer types. It has been shown to inhibit proliferation, migration, invasion, and metastasis formation in colorectal carcinoma, hepatocarcinoma, pancreatic cancer, and breast cancer .

Research has demonstrated that SASH1 expression is frequently downregulated in human cancer cell lines compared to non-malignant cells. For example, SASH1 mRNA and protein levels are significantly decreased in pancreatic cancer cell lines compared to the non-malignant hTERT-HPNE cell line . Similar patterns of reduced expression have been observed in hepatocarcinoma, thyroid cancer, and breast cancer cell lines .

The clinical significance of SASH1 varies by cancer subtype. In breast cancer, low SASH1 expression is associated with poor outcomes in HER2+ disease, particularly in HER2+/ER+ cases where low SASH1 is linked to nearly 100% survival (HR 0.06, p = 0.0002) . These findings highlight why SASH1 detection is crucial for understanding cancer biology and potentially developing therapeutic strategies.

What are the advantages of using HRP-conjugated antibodies for SASH1 detection?

HRP-conjugated antibodies offer several significant advantages for detecting SASH1 in research applications:

• Enhanced sensitivity: HRP (40,000 Da) catalyzes reactions with hydrogen peroxide and electron-donating substrates to produce color, fluorescent, or chemiluminescent products, enabling highly sensitive detection of SASH1 even at low expression levels .

• Signal amplification: The enzymatic activity of HRP provides signal amplification through substrate conversion, making it ideal for detecting SASH1 in samples where the protein may be downregulated.

• Versatility across applications: HRP-conjugated SASH1 antibodies are suitable for multiple techniques including Western blotting, ELISA, immunohistochemistry (IHC), and immunocytochemistry (ICC) .

• Stability: HRP remains stable and functional under various conditions including chemical cross-linking, freeze-drying, and prolonged storage at 4°C, making HRP-conjugated SASH1 antibodies reliable research tools .

• Accessibility to antigenic sites: The relatively small size of HRP facilitates better access to antigenic sites or structures in SASH1, particularly important when the protein is part of complex molecular interactions .

What are the optimal protocols for using SASH1 antibody, HRP conjugated in Western blotting?

When using SASH1 antibody, HRP conjugated for Western blotting, the following methodological considerations are recommended:

Sample preparation:

• Extract proteins using RIPA buffer supplemented with protease inhibitors

• For detecting SASH1 in cancer cells, ensure adequate lysis as SASH1 can have varied subcellular distributions

• Include positive controls (cells with known SASH1 expression) and negative controls (SASH1-knockdown cells)

Gel electrophoresis and transfer:

• Use 8-10% SDS-PAGE gels as SASH1 is a relatively large protein (~170 kDa)

• Transfer to PVDF membranes (rather than nitrocellulose) for optimal protein binding

• Use semi-dry transfer at 15V for 60 minutes or wet transfer at 100V for 90 minutes

Antibody incubation and detection:

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

• Dilute SASH1 antibody, HRP conjugated at 1:1000-1:5000 in blocking buffer

• Incubate overnight at 4°C with gentle rocking

• Wash extensively with TBST (4-5 times, 5 minutes each)

• Proceed directly to enhanced chemiluminescent (ECL) detection without secondary antibody since the antibody is already HRP-conjugated

• For optimal sensitivity, use a digital imaging system with various exposure times

The HRP enzyme conjugated to the SASH1 antibody catalyzes the oxidation of luminol in the presence of hydrogen peroxide, producing light that can be detected with appropriate imaging equipment. This direct detection eliminates the need for secondary antibody incubation, reducing background and improving specificity.

How can I effectively detect SASH1 in immunohistochemistry applications?

For immunohistochemical analysis of SASH1 expression in tissue samples, consider the following protocol:

Tissue preparation:

• Use either formalin-fixed paraffin-embedded (FFPE) or frozen tissue sections

• For FFPE sections (4-6 μm thickness), perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• For frozen sections, fix with cold acetone or 4% paraformaldehyde

Staining procedure:

• Quench endogenous peroxidase activity with 3% hydrogen peroxide for 10 minutes

• Block non-specific binding with 5-10% normal serum in PBS for 30-60 minutes

• Apply HRP-conjugated SASH1 antibody (1:100-1:500 dilution) and incubate overnight at 4°C

• Wash thoroughly with PBS (3-4 times, 5 minutes each)

• Develop with DAB (3,3'-diaminobenzidine) substrate

• Counterstain with hematoxylin, dehydrate, and mount

Important considerations:

• SASH1 expression can be heterogeneous within tumor tissue, so examine multiple fields

• Include positive controls (normal tissues with known SASH1 expression) in each batch

• For hypoxic tissues, additional considerations may be necessary as hypoxia has been shown to inhibit SASH1 expression

• When studying EMT processes, co-staining with epithelial markers (E-cadherin) and mesenchymal markers (vimentin) alongside SASH1 may provide valuable insights

What controls should I include when validating SASH1 antibody specificity?

Proper validation of SASH1 antibody specificity is crucial for generating reliable research data. Include the following controls:

Positive controls:

• Cell lines with confirmed high SASH1 expression (e.g., normal epithelial cells)

• Recombinant SASH1 protein for Western blot analysis

• Tissues known to express SASH1 (e.g., normal colon tissue)

Negative controls:

• SASH1 knockdown or knockout cells using siRNA or CRISPR-Cas9

• Cell lines with confirmed low SASH1 expression (e.g., many cancer cell lines including HepG2, HCCLM3, PANC-1)

• Primary antibody omission control

• Isotype control antibody

Validation experiments:

• Peptide competition assay: Pre-incubate the SASH1 antibody with excess purified SASH1 protein or immunizing peptide before use in your application. Signal reduction confirms specificity.

• Molecular weight verification: Confirm that the detected band corresponds to the expected molecular weight of SASH1 (~170 kDa).

• Orthogonal method comparison: Compare results with alternative detection methods such as RT-qPCR using validated SASH1 primers (5′-TCCCGTCACAGGAAGAAACG-3′ and 5′-GATACCCATCACGTCGGTCC-3′) .

• Signal pattern analysis: Verify that the subcellular localization pattern matches known SASH1 distribution (typically cytoplasmic with occasional nuclear localization).

How can I design experiments to study SASH1's role in cancer signaling pathways using HRP-conjugated antibodies?

SASH1 has been implicated in regulating several signaling pathways relevant to cancer progression. Here are experimental approaches using HRP-conjugated SASH1 antibodies to investigate these mechanisms:

For studying SASH1 in the PI3K/Akt/mTOR pathway:

• Co-immunoprecipitation studies:

  • Immunoprecipitate SASH1 from cell lysates and probe for PI3K, Akt, or other pathway components

  • Western blot with HRP-conjugated SASH1 antibody to confirm successful immunoprecipitation

  • Investigate changes in these interactions under various treatments or in different cell lines

• Pathway activation analysis:

  • Compare phosphorylation levels of PI3K and Akt in cells with normal versus overexpressed or knocked-down SASH1

  • Use HRP-conjugated SASH1 antibody alongside phospho-specific antibodies for PI3K/Akt

  • Research has shown that SASH1 overexpression prevents hypoxia-induced activation of the PI3K/Akt pathway

For investigating SASH1's role in the Sonic Hedgehog (Shh) signaling pathway:

Design experiments to demonstrate the relationship between SASH1 and Shh pathway components as observed in hepatocarcinoma :

Use HRP-conjugated SASH1 antibody for Western blotting to confirm expression levels while simultaneously probing for Shh pathway components (Shh, Smo, Ptc, Gli-1) to establish correlation or causation .

What are the considerations for using SASH1 antibody, HRP conjugated when studying hypoxia-induced epithelial-mesenchymal transition?

Hypoxia has been shown to inhibit SASH1 expression and promote epithelial-mesenchymal transition (EMT) in cancer cells. When designing experiments to study this relationship using HRP-conjugated SASH1 antibodies, consider:

Experimental design considerations:

• Hypoxic conditions:

  • Use a controlled hypoxia chamber (typically 1% O2, 5% CO2, 94% N2)

  • Include normoxic controls (21% O2) for comparison

  • Consider time-course experiments (6h, 12h, 24h, 48h) as SASH1 expression changes dynamically under hypoxia

• EMT marker panel:

  • Monitor epithelial markers (E-cadherin) and mesenchymal markers (vimentin) alongside SASH1

  • Research has shown that hypoxia decreases E-cadherin and increases vimentin, while SASH1 overexpression reverses these effects

• HIF-1α assessment:

  • Include HIF-1α detection, as SASH1 has been shown to negatively regulate HIF-1α expression

  • Compare HIF-1α mRNA and protein levels under normoxia versus hypoxia, with and without SASH1 overexpression

Protocol modifications for hypoxic samples:

• Sample collection and processing should be rapid to preserve hypoxic conditions

• Consider using hypoxia-mimetic agents (e.g., CoCl2) as additional controls

• When analyzing protein extracts from hypoxic cells, include reducing agents in sample buffers to prevent oxidation artifacts

Expected results based on published data:

These patterns have been observed in pancreatic cancer cell lines (PANC-1) and may vary in other cancer types .

How can I troubleshoot non-specific binding or weak signal when using SASH1 antibody, HRP conjugated?

When experiencing technical issues with SASH1 antibody, HRP conjugated, consider the following troubleshooting approaches:

For weak signal:

• Antibody concentration:

  • Try a more concentrated antibody dilution (e.g., 1:500 instead of 1:1000)

  • Extend primary antibody incubation time (overnight at 4°C)

• Antigen retrieval enhancement:

  • For FFPE tissue sections, optimize antigen retrieval (test both citrate pH 6.0 and EDTA pH 9.0 buffers)

  • Increase retrieval time or temperature

• Detection system enhancement:

  • Use a more sensitive ECL substrate for Western blotting

  • For IHC, consider using amplification systems like tyramide signal amplification (TSA)

• Sample preparation:

  • Ensure complete lysis of cells/tissues

  • Avoid repeated freeze-thaw cycles of lysates

  • Consider concentration of dilute samples by immunoprecipitation

For non-specific binding/high background:

• Blocking optimization:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Increase blocking time (2 hours or more)

  • Add 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

• Wash protocol intensification:

  • Increase number and duration of washes

  • Use higher salt concentration in wash buffers (up to 500 mM NaCl)

• Antibody specificity verification:

  • Perform peptide competition assay

  • Test antibody on known negative controls

  • Consider antibody purification using antigen affinity chromatography

• Endogenous enzyme inactivation:

  • For tissues with high endogenous peroxidase activity, increase H2O2 quenching time

  • Use additional blocking steps for endogenous biotin if applicable

How should I interpret contradictory findings on SASH1 expression across different cancer subtypes?

SASH1 expression patterns and their clinical implications vary across cancer types and even within subtypes of the same cancer. When interpreting seemingly contradictory findings:

• Consider tissue-specific baseline expression:

  • Different tissues have varied normal SASH1 expression levels

  • Compare cancer samples to appropriate normal tissue controls

• Evaluate cancer subtype heterogeneity:

  • In breast cancer, SASH1's relationship with outcome is subtype-dependent:

    • Low SASH1 expression is associated with better outcomes in ER-negative and triple-negative basal-like cases

    • In contrast, low SASH1 is associated with worse outcomes in HER2+ disease, particularly HER2+/ER+ cases

• Examine contextual signaling pathway activation:

  • SASH1's tumor-suppressive effects may depend on which downstream pathways are active in a given cancer

  • In hepatocarcinoma, SASH1 suppresses the Sonic Hedgehog pathway

  • In pancreatic cancer, SASH1 regulates HIF-1α under hypoxic conditions

  • In breast cancer, SASH1 inhibits the PI3K-Akt-mTOR pathway

• Consider genetic and epigenetic alterations:

  • Some cancers may have mutations in SASH1 that alter protein function without affecting detection by antibodies

  • Epigenetic silencing mechanisms may vary across cancer types

When designing experiments, include controls representing multiple cancer subtypes when possible, and correlate SASH1 expression with other molecular markers to contextualize findings.

How can I use HRP-conjugated SASH1 antibody to investigate connections between SASH1 and drug resistance?

Emerging evidence suggests SASH1 may influence drug sensitivity in cancer. To investigate this using HRP-conjugated SASH1 antibodies:

• Drug response correlation studies:

  • Treat cancer cell panels with various chemotherapeutic agents

  • Use HRP-conjugated SASH1 antibody to quantify expression by Western blotting

  • Correlate SASH1 levels with IC50 values for each drug

  • Research has shown that chloropyramine increases SASH1 protein levels in breast cancer cells and reduces cell confluency

• SASH1 modulation experiments:

  • Generate stable cell lines with SASH1 overexpression or knockdown

  • Confirm expression changes using HRP-conjugated SASH1 antibody

  • Assess drug sensitivity changes using dose-response curves

  • Analyze changes in apoptotic markers in response to treatment

• Signaling pathway analysis:

  • Investigate how SASH1 modulation affects established drug resistance pathways

  • Monitor EMT markers, as EMT is associated with chemoresistance

  • Assess PI3K/Akt/mTOR pathway activation, which is both regulated by SASH1 and implicated in drug resistance

Experimental design recommendation:

For each condition, collect samples at multiple time points (24h, 48h, 72h) to track dynamic changes in SASH1 expression and drug response.

What methods are used to conjugate HRP to SASH1 antibodies and how might they affect performance?

The conjugation process can significantly impact antibody performance. Understanding these methods helps researchers select the appropriate HRP-conjugated SASH1 antibody:

Common conjugation methods:

• Glutaraldehyde method:

  • Uses glutaraldehyde to crosslink amino groups between HRP and antibody

  • Advantages: Simple, cost-effective

  • Limitations: Can cause antibody aggregation, potential loss of antibody activity

• Periodate method:

  • Oxidizes carbohydrate residues on HRP to create aldehydes that react with antibody amino groups

  • Advantages: Milder conditions, less impact on antibody binding

  • Limitations: Less stable linkage, potential for heterogeneous conjugation

• Heterobifunctional linker method:

  • Uses reagents like sulfo-SMCC and SATA to create defined linkages

  • Sulfo-SMCC activates HRP to create reactive maleimide groups for coupling to sulfhydryl groups in antibodies

  • SATA introduces protected sulfhydryl groups into the antibody

  • Advantages: Greater control over conjugation, more homogeneous products, preservation of antibody activity

  • This is a preferred method for creating stable antibody-HRP conjugates with retention of antigen-binding activity

How conjugation affects performance:

The conjugation method can impact:

• Antigen binding affinity

• Sensitivity and specificity

• Stability during storage

• Background signal in assays

When selecting a commercial SASH1 antibody, HRP conjugated, consider requesting information about the conjugation method used and validation data showing retained specificity after conjugation.

What techniques can I use to verify the quality and activity of my SASH1 antibody, HRP conjugated?

Before using HRP-conjugated SASH1 antibody in critical experiments, verify both antibody quality and HRP enzyme activity:

Antibody quality verification:

• Western blot analysis:

  • Run known positive and negative controls for SASH1

  • Verify correct molecular weight band (~170 kDa)

  • Check for absence of non-specific bands

• Dot blot titration:

  • Apply serial dilutions of recombinant SASH1 protein to membrane

  • Probe with HRP-conjugated SASH1 antibody

  • Determine limit of detection and linear range

• Immunocytochemistry:

  • Test on cell lines with known SASH1 expression patterns

  • Verify expected subcellular localization

HRP enzyme activity verification:

• Direct enzyme activity assay:

  • Spot dilutions of antibody-HRP conjugate onto nitrocellulose

  • Develop with TMB (3,3',5,5'-tetramethylbenzidine) or other HRP substrate

  • Compare color development intensity with a standard HRP curve

• Absorbance measurement:

  • Measure absorbance ratio (A403/A280) to determine HRP:antibody ratio

  • Optimal conjugation typically yields 2-4 HRP molecules per antibody

• Storage stability testing:

  • Test activity after various storage conditions (4°C, -20°C, -80°C)

  • Compare fresh vs. stored conjugate performance

Quality control checklist:

• Specific binding to SASH1 antigen

• Low background in negative controls

• Consistent lot-to-lot performance

• Strong and proportional signal with increasing antigen concentration

• Stable signal over the recommended storage period

By performing these quality checks, researchers can ensure reliable results when using SASH1 antibody, HRP conjugated for critical experiments investigating SASH1's role in cancer biology.