EPSTI1 Antibody, HRP conjugated

What is EPSTI1 and why is it a significant research target?

EPSTI1 (Epithelial Stromal Interaction 1) is a protein highly expressed in placenta, small intestine, spleen, salivary gland, and testes, with lower expression in normal breast tissue . It has emerged as an important research target because:

• It plays a critical role in M1 macrophage polarization and regulates gene expression during M1 versus M2 macrophage differentiation

• It is significantly upregulated in breast carcinomas (5.6-72.1 fold increase observed in 14 tested breast carcinomas)

• It functions in RELA/p65 and STAT1 phosphorylation and nuclear localization during macrophage activation

• It has been identified as a direct transcriptional target of KLF8, which is involved in cancer progression

• It promotes cell invasiveness in breast cancer models

For optimal research results, consider tissue-specific expression patterns when designing experiments targeting EPSTI1.

What should researchers consider when selecting an EPSTI1 antibody for HRP conjugation?

When selecting an EPSTI1 antibody for HRP conjugation, researchers should evaluate:

Host and clonality:

• Polyclonal antibodies (like 11627-1-AP) offer broad epitope recognition but may have batch-to-batch variation

• Monoclonal antibodies (like clone 3G7) provide consistent specificity to a single epitope

Target epitope region:

• N-terminal antibodies (AA 1-100) are available for specific applications

• Middle region antibodies may target functional domains

• Consider epitope accessibility in your application

Validated applications:
The antibody should be validated for your intended application. Available EPSTI1 antibodies have been validated for:

• Western blotting (WB): 1:1000-1:6000 dilution range

• Immunohistochemistry (IHC): 1:20-1:200 dilution range

• ELISA applications

• Immunofluorescence (IF)

Species reactivity:
Confirm reactivity with your research model:

• Human reactivity is common to most available antibodies

• Mouse reactivity is confirmed for some antibodies

• Pig reactivity is reported for select antibodies

What are the key differences between commercially available EPSTI1 antibodies?

What is the optimal protocol for conjugating an EPSTI1 antibody with HRP?

For optimal HRP conjugation of EPSTI1 antibodies, follow this methodological approach:

Pre-conjugation considerations:

• Ensure antibody purity: The antibody should be in 10-50mM amine-free buffer (e.g., MES, MOPS, HEPES, PBS) with pH between 6.5-8.5

• Avoid buffers containing primary amines, chelating agents, and sodium azide as they may interfere with conjugation

• Determine optimal antibody:HRP ratio:

  • 1:4 ratio (10μg antibody:HRP) for highest sensitivity

  • 1:1 ratio (40μg antibody:HRP) for strongest specific signal

Conjugation protocol:

• Add modifier reagent (1μl per 10μl of antibody)

• Add antibody+modifier solution directly to lyophilized HRP

• Resuspend gently by pipetting

• Incubate at room temperature (20-25°C) for 3 hours in the dark

• Add quencher reagent (1μl per 10μl of antibody)

• Incubate for 30 minutes before use

This protocol produces ready-to-use conjugates that do not require purification.

How can I determine the success of my EPSTI1 antibody-HRP conjugation?

To verify successful HRP conjugation to your EPSTI1 antibody:

Functional testing:

• Perform a Western blot using both the unconjugated and conjugated antibody on samples known to express EPSTI1 (e.g., HUVEC cells, mouse spleen tissue , or breast cancer cell lines )

• Compare signal intensity and specificity between conjugated and unconjugated antibodies using standard procedures:

  • A successful conjugation should show a band at 35-38 kDa (observed molecular weight for EPSTI1)

  • Evaluate background noise levels

  • Compare sensitivity at different dilutions

Control experiments:

• Include a known HRP-conjugated antibody as a positive control

• Include negative controls (samples without EPSTI1 expression)

• Test for non-specific binding by pre-absorbing the conjugated antibody with recombinant EPSTI1 protein

Spectrophotometric analysis:

• Measure absorbance at 280nm (protein) and 403nm (HRP) to calculate conjugation ratio

• Compare with expected theoretical ratio based on your conjugation protocol

What factors might affect the stability and performance of HRP-conjugated EPSTI1 antibodies?

Several factors can impact the stability and performance of HRP-conjugated EPSTI1 antibodies:

Storage conditions:

• Store at -20°C in suitable buffer (e.g., PBS with 0.02% sodium azide and 50% glycerol, pH 7.3)

• Avoid repeated freeze-thaw cycles

• Consider aliquoting before storage

Buffer composition:

• Sodium azide inactivates HRP and should be avoided in working solutions

• Glycerol improves stability but may affect viscosity at high concentrations

• pH shifts can denature both antibody and HRP

Time-dependent degradation:

• Even at optimal storage conditions, enzymatic activity may decrease over time

• Typically stable for one year after shipment when properly stored

• Regular activity testing is recommended for long-term storage

Contaminants:

• Heavy metals can inhibit HRP activity

• Microbial contamination can degrade proteins

• Oxidizing and reducing agents can affect HRP catalytic site

To maximize performance, regularly validate activity, store according to manufacturer recommendations, and prepare fresh working solutions.

How can I optimize Western blotting using HRP-conjugated EPSTI1 antibodies?

For optimal Western blot results with HRP-conjugated EPSTI1 antibodies:

Sample preparation:

• Use appropriate positive controls:

  • HUVEC cells and mouse spleen tissue have shown positive EPSTI1 expression

  • MDA-MB-231 breast cancer cells express high levels of EPSTI1

• Include proper lysis buffer with protease inhibitors to prevent EPSTI1 degradation

• Load 50μg total protein per lane as recommended for EPSTI1 detection

Electrophoresis and transfer:

• Use 12% SDS-PAGE gels for optimal EPSTI1 separation

• Transfer to PVDF membrane for better protein retention

• Include molecular weight markers spanning 30-50 kDa range to identify EPSTI1 (35-38 kDa observed)

Blocking and antibody incubation:

• Block with 5% non-fat milk or BSA in TBST

• For HRP-conjugated EPSTI1 antibodies, start with 1:1000 dilution and optimize if needed

• Expected working dilution range: 1:1000-1:6000

• Incubate overnight at 4°C for optimal binding

Signal detection:

• Use enhanced chemiluminescence (ECL) substrates like Immobilon Western HRP Substrate

• Expose for multiple time points to capture optimal signal

• If signal is weak, consider using signal enhancers or amplification systems

Troubleshooting:

• If multiple bands appear, validate specificity with knockdown experiments

• If no signal appears, verify EPSTI1 expression in your samples with RT-qPCR

• Note that calculated molecular weight (47 kDa) differs from observed (35-38 kDa)

What are effective strategies for using HRP-conjugated EPSTI1 antibodies in immunohistochemistry?

For effective immunohistochemistry with HRP-conjugated EPSTI1 antibodies:

Tissue preparation:

• Validated tissue samples include:

  • Human breast cancer tissue

  • Human kidney tissue

• Use appropriate fixation (e.g., 10% neutral buffered formalin)

• For paraffin sections, antigen retrieval is critical:

  • Recommended: TE buffer pH 9.0

  • Alternative: citrate buffer pH 6.0

Staining protocol:

• Deparaffinize and rehydrate sections using standard protocols

• Perform antigen retrieval based on recommended conditions

• Block endogenous peroxidase with 3% H₂O₂

• Block non-specific binding with appropriate serum

• Apply HRP-conjugated EPSTI1 antibody:

  • Starting dilution: 1:50 (for directly conjugated antibody)

  • Optimization range: 1:20-1:200

• Incubate in humidified chamber (4°C overnight or room temperature for 1-2 hours)

• Develop with DAB substrate

• Counterstain, dehydrate, and mount

Controls:

• Positive control: Include human breast cancer tissue known to express EPSTI1

• Negative control: Omit primary antibody

• Consider including normal breast tissue for comparison (low EPSTI1 expression)

Interpretation:

• EPSTI1 expression is upregulated in breast carcinomas compared to normal tissue

• Both tumor and stromal cells may express EPSTI1

• Note subcellular localization patterns for biological insights

How can EPSTI1 antibodies be used to study the NF-κB signaling pathway?

EPSTI1 plays a critical role in NF-κB signaling, making HRP-conjugated EPSTI1 antibodies valuable tools to study this pathway:

Methodology for studying EPSTI1-mediated NF-κB activation:

• Cellular models:

  • Use MCF-7 cells with inducible EPSTI1 expression (MCF7-iE1) or similar models

  • Compare cells with normal versus overexpressed/knockdown EPSTI1

• Detection of pathway components:

  • Monitor IκBα degradation by Western blot:

    • EPSTI1 overexpression decreases IκBα protein levels

    • VCP-N187 peptide expression can rescue this effect

  • Measure nuclear translocation of p65 and p50 subunits:

    • EPSTI1 expression increases nuclear levels and decreases cytoplasmic levels of both subunits

• Functional assays:

  • NF-κB transcriptional activity:

    • Use an NF-κB-responsive promoter reporter assay

    • EPSTI1 expression causes 3-4× increase in reporter activity

  • Inflammatory cytokine expression:

    • Measure IL-1β, IL-6, and TNF-α levels by ELISA

    • EPSTI1 knockdown attenuates expression of these proinflammatory factors in LPS-treated cells

• Mechanistic studies:

  • Examine EPSTI1-VCP interaction via co-immunoprecipitation

  • Study the effect of VCP-N187 peptide (aa 1-187) on EPSTI1-mediated processes:

    • VCP-N187 inhibits cell invasiveness induced by EPSTI1

    • VCP-N187 prevents EPSTI1-mediated IκBα degradation

• Pathway analysis:

  • Monitor phosphorylation of pathway components:

    • Measure p-p65/p65 and p-IκBα/IκBα ratios

    • EPSTI1 knockdown decreases these ratios in LPS-stimulated cells

This approach provides a comprehensive framework for studying how EPSTI1 regulates NF-κB signaling in various contexts, including inflammation and cancer.

Why is there a discrepancy between the calculated (47 kDa) and observed (35-38 kDa) molecular weight of EPSTI1?

The discrepancy between calculated (47 kDa) and observed (35-38 kDa) molecular weight of EPSTI1 can be explained by several factors:

Potential biological explanations:

• Alternative splicing: EPSTI1 has multiple isoforms produced by alternative splicing , which may result in proteins with different molecular weights

• Post-translational modifications: Certain modifications can alter protein migration:

  • Proteolytic processing could remove portions of the protein

  • Some modifications increase mobility (e.g., certain phosphorylation patterns)

• Protein structure: Highly structured regions may cause anomalous migration

• Tissue-specific variants: Different cell types might express variant forms

Technical considerations:

• SDS-PAGE conditions: Gel percentage, buffer composition, and running conditions can affect migration

• Protein loading: Overloading can distort band appearance

• Molecular weight markers: Calibration differences between marker sets

• Protein purity: Contaminants or degradation products

Verification approaches:

• Mass spectrometry analysis of the immunoprecipitated protein

• Testing multiple antibodies targeting different epitopes

• Expressing recombinant EPSTI1 with tags and comparing migration

• Confirming specificity with EPSTI1 knockdown experiments

This discrepancy is consistently observed across studies, suggesting it represents a biological characteristic rather than a technical artifact.

How can I validate the specificity of my HRP-conjugated EPSTI1 antibody?

To rigorously validate the specificity of an HRP-conjugated EPSTI1 antibody:

Genetic manipulation approaches:

• Knockdown validation:

  • Perform siRNA or shRNA knockdown of EPSTI1 in appropriate cell lines

  • Compare antibody signal between control and knockdown samples

  • Quantify reduction in signal corresponding to EPSTI1 depletion

  • Example: EPSTI1 knockdown in MDA-MB-231 cells has been effective

• Overexpression validation:

  • Generate cells with inducible EPSTI1 expression (e.g., 10A-iE1, MCF7-iE1)

  • Compare signal before and after induction

  • Verify size of detected protein matches expected EPSTI1 size

Biochemical approaches:

• Peptide competition:

  • Pre-incubate antibody with excess immunizing peptide/recombinant EPSTI1

  • Specific signal should be blocked while non-specific binding remains

  • Use a graduated peptide concentration series for quantitative assessment

• Multiple antibody validation:

  • Test different antibodies targeting distinct EPSTI1 epitopes

  • Compare detection patterns across techniques

  • Consistent results across antibodies increase confidence in specificity

Cross-reactivity assessment:

• Tissue panel testing:

  • Test antibody on tissues with known EPSTI1 expression profiles

  • High expression: placenta, small intestine, spleen, salivary gland, testes

  • Low expression: normal breast tissue

  • Signal intensity should correspond to known expression levels

• Species specificity:

  • Test across species if working with non-human models

  • Confirmed reactivity in human and mouse samples for selected antibodies

What are the emerging applications of EPSTI1 antibodies in cancer and inflammation research?

HRP-conjugated EPSTI1 antibodies are increasingly valuable in several cutting-edge research areas:

Cancer metastasis studies:

• Epithelial-stromal interactions:

  • EPSTI1 can substitute for peritumoral activated fibroblasts in tumor environment assays

  • HRP-conjugated antibodies enable visualization of EPSTI1 in tumor-stroma boundaries

• Metastatic potential assessment:

  • EPSTI1 and KLF8 co-overexpression in metastatic breast cancer correlates with invasiveness

  • Quantitative EPSTI1 detection may serve as a biomarker for metastatic potential

• Therapeutic target validation:

  • Monitoring EPSTI1 expression changes in response to experimental therapies

  • Correlating EPSTI1 levels with treatment efficacy

Inflammatory response research:

• Macrophage polarization:

  • EPSTI1 influences M1 vs. M2 macrophage differentiation

  • HRP-conjugated antibodies can track EPSTI1 dynamics during polarization

• LPS-induced inflammation:

  • EPSTI1 is highly expressed after LPS exposure

  • Knockdown alleviates inflammatory injury through NF-κB pathway inactivation

• Inflammatory biomarkers:

  • EPSTI1 levels correlate with IL-1β, IL-6, and TNF-α expression

  • HRP-conjugated antibodies enable multiplex detection in inflammatory conditions

Methodological advances:

• Multiplex immunohistochemistry:

  • Combining HRP-conjugated EPSTI1 antibodies with other markers

  • Enables simultaneous detection of multiple proteins on a single tissue section

• Flow cytometry applications:

  • Using HRP-conjugated antibodies for intracellular EPSTI1 detection

  • Correlating EPSTI1 with cell surface markers

• High-throughput screening:

  • Adapting EPSTI1 detection to plate-based formats

  • Screening compounds that modulate EPSTI1 expression or function

These emerging applications highlight the growing importance of EPSTI1 in understanding complex disease mechanisms and developing new therapeutic approaches.