SERPINI1 Antibody, HRP conjugated

What is SERPINI1 and why is it significant in research?

SERPINI1, also known as Neuroserpin or PI12, is a member of the serpin superfamily of serine proteinase inhibitors. It is primarily secreted by axons in the brain and preferentially inhibits tissue-type plasminogen activator. SERPINI1 plays crucial roles in the regulation of axonal growth, development of synaptic plasticity, and protection of neurons from cell damage. Research interest in SERPINI1 has increased due to its associations with neurological disorders and various cancers, including hepatocellular carcinoma (HCC) .

What are the common applications for SERPINI1 antibodies in research?

SERPINI1 antibodies are widely used in multiple research applications including:

• Western Blot (WB) for protein detection and quantification

• Immunohistochemistry (IHC) for tissue localization

• Immunofluorescence (IF) for cellular localization

• Enzyme-Linked Immunosorbent Assay (ELISA) for quantitative detection

• Immunoprecipitation (IP) for protein purification

• Co-Immunoprecipitation (Co-IP) for protein-protein interaction studies

What are the advantages of using HRP-conjugated SERPINI1 antibodies?

HRP (Horseradish Peroxidase) conjugated antibodies offer several advantages:

• Direct detection without need for secondary antibodies, reducing protocol time and potential cross-reactivity

• Enhanced sensitivity through enzymatic signal amplification

• Compatibility with colorimetric, chemiluminescent, and chemifluorescent detection systems

• Long shelf life compared to fluorescent conjugates

• Cost-effectiveness for routine laboratory assays

What sample types have been validated with SERPINI1 antibodies?

SERPINI1 antibodies have been validated for detection in multiple human cell lines and tissues including:

• Hepatocellular carcinoma cell lines (HepG2, HuH-7)

• Normal liver cell line (L02)

• Human Umbilical Vein Endothelial Cells (HUVEC)

• Brain tissue samples

• Serum samples from both healthy individuals and HCC patients

How should I optimize western blot protocols for SERPINI1 detection?

For optimal western blot results with SERPINI1 antibodies:

• Use appropriate lysis buffers containing protease inhibitors to prevent degradation

• Load 20-50 μg of total protein per lane

• Use 10-12% SDS-PAGE gels for optimal separation

• For HRP-conjugated antibodies, dilute to 1:500-1:2000 based on signal strength

• When detecting SERPINI1, look for bands at approximately 45 kDa

• Include positive controls such as HepG2 or HuH-7 cell lysates

• Optimize blocking conditions (typically 5% BSA or non-fat milk) to reduce background

• For enhanced sensitivity with HRP-conjugated antibodies, consider extended exposure times with chemiluminescent substrates

What are the critical parameters for immunohistochemistry using SERPINI1 antibodies?

When performing IHC with SERPINI1 antibodies:

• Optimize antigen retrieval methods - recommended to use TE buffer pH 9.0 or citrate buffer pH 6.0

• Use appropriate dilutions (typically 1:50-1:500) based on antibody sensitivity

• Include positive control tissues (such as human brain tissue or liver cancer tissue)

• Include negative controls (omitting primary antibody)

• For HRP-conjugated antibodies, ensure appropriate substrate (DAB or AEC) is used

• Consider counterstaining with hematoxylin for better visualization

• When interpreting results, note that SERPINI1 is primarily localized in the cytoplasm and sometimes secreted extracellularly

How can I validate specificity of SERPINI1 antibodies in my experimental system?

To ensure specificity of SERPINI1 antibody detection:

• Perform western blots with positive control samples (HepG2 cells, brain tissue)

• Include knockout/knockdown controls using SERPINI1 siRNA (validated sequences include: 5'-GCUGUGCUGUAUCCUCAAGUUTT-3'/5'-AACUUGAGGAUACAGCACAGCTT-3')

• Conduct peptide competition assays using the immunizing peptide

• Test multiple antibodies targeting different epitopes of SERPINI1

• Validate antibody specificity across multiple applications (WB, IHC, IF)

• Compare results with mRNA expression data from qPCR

• Consider orthogonal validation using mass spectrometry-based proteomics

How can I use SERPINI1 antibodies to study its role in hepatocellular carcinoma?

For studying SERPINI1 in HCC research:

• Perform comparative expression analysis in HCC vs. normal tissues using IHC or western blot

• Assess serum SERPINI1 levels in HCC patients vs. healthy controls using ELISA

• Correlate SERPINI1 expression with clinicopathological features (tumor size, differentiation degree, vascular invasion)

• Combine AFP and SERPINI1 detection for improved diagnostic accuracy

• Use SERPINI1 knockdown/overexpression in HCC cell lines to study effects on:

  • Cell proliferation (MTT, EdU assays)

  • Migration (wound healing assay)

  • Invasion (transwell invasion assay)

• Investigate downstream molecular pathways by monitoring EMT markers (E-cadherin, vimentin, MMP9) after modulating SERPINI1 expression

What are the best approaches to study SERPINI1 protein-protein interactions?

To investigate SERPINI1 protein interactions:

• Perform co-immunoprecipitation (Co-IP) using anti-SERPINI1 antibodies followed by mass spectrometry

• Conduct reverse Co-IP with antibodies against suspected interaction partners

• Use proximity ligation assays (PLA) to visualize protein interactions in situ

• Consider bimolecular fluorescence complementation (BiFC) for live-cell interaction studies

• For extracellular interactions, use solid-phase binding assays with purified SERPINI1 protein

• Validate interactions through functional assays, particularly focusing on known targets like tissue-type plasminogen activator

• For HRP-conjugated antibodies in Co-IP experiments, use special elution protocols to prevent interference from the HRP moiety

How can SERPINI1 antibodies be used in multiplex immunofluorescence studies?

For multiplex IF studies involving SERPINI1:

• When using HRP-conjugated antibodies, consider tyramide signal amplification (TSA) for sequential multiplex staining

• Select antibodies raised in different host species to avoid cross-reactivity

• Optimize the order of antibody application (typically start with the weakest signal)

• Use appropriate spectral unmixing for fluorophores with overlapping emission spectra

• Include single-stained controls for each antibody to establish specificity

• Consider automated image analysis software for quantification of co-localization

• To study SERPINI1 in relation to EMT markers, design multiplex panels including E-cadherin, vimentin, and MMP9

What are common issues when using SERPINI1 antibodies and how can they be resolved?

How should I design experiments to study SERPINI1 function through antibody-based approaches?

For functional studies of SERPINI1:

• Combine knockdown/overexpression with antibody-based detection to validate phenotypic changes

• For secreted SERPINI1, use antibodies for both cellular and extracellular detection

• Design time-course experiments to track SERPINI1 expression and localization during cellular processes

• Use recombinant neuroserpin as a positive control in functional assays

• Compare results across multiple cell lines with different baseline SERPINI1 expression

• Utilize HRP-conjugated antibodies for higher sensitivity in low-expression systems

• Consider neutralizing antibodies to block SERPINI1 function in live cell experiments

What are the best practices for quantifying SERPINI1 in patient samples?

For quantitative analysis of SERPINI1 in clinical samples:

• Standardize sample collection procedures (for serum: centrifuge at 3000 rpm for 20 minutes, store at -80°C)

• Use validated ELISA kits with established reference ranges

• Include standard curves with each experiment (r² > 0.98)

• Run samples in triplicate to ensure reproducibility

• Include quality control samples across multiple plates

• For serum analysis, consider pre-analytical variables (fasting status, time of collection)

• When analyzing immunohistochemistry, use digital image analysis with validated scoring systems

• For prognostic studies, correlate SERPINI1 levels with long-term patient outcomes

How is SERPINI1 research evolving beyond traditional antibody applications?

Recent advances in SERPINI1 research include:

• Development of highly sensitive proximity extension assays for SERPINI1 detection in limited samples

• Application of CRISPR/Cas9 technology for precise genome editing to study SERPINI1 function

• Integration of SERPINI1 in multi-biomarker panels for improved cancer diagnostics

• Single-cell analysis of SERPINI1 expression in heterogeneous tissues

• Development of aptamer-based detection methods as alternatives to antibodies

• Investigation of SERPINI1 in liquid biopsies for non-invasive cancer monitoring

• Exploration of SERPINI1 as a therapeutic target in cancer and neurological disorders

What are the emerging techniques for studying SERPINI1 protein dynamics?

Cutting-edge approaches for SERPINI1 protein dynamics include:

• Live-cell imaging using SERPINI1 fusion proteins with fluorescent tags

• FRAP (Fluorescence Recovery After Photobleaching) to study SERPINI1 mobility

• FRET (Förster Resonance Energy Transfer) to investigate SERPINI1 protein-protein interactions

• Advanced mass spectrometry techniques for quantitative proteomics

• Super-resolution microscopy for detailed subcellular localization

• Microfluidic platforms for real-time monitoring of SERPINI1 secretion

• Computational modeling of SERPINI1 structure-function relationships

How should researchers interpret contradictory findings regarding SERPINI1 expression in different cancer types?

When facing contradictory findings about SERPINI1 in cancer research:

• Consider tissue-specific effects - SERPINI1 may function differently in different cellular contexts

• Examine methodological differences between studies (antibody clones, detection methods, sample preparation)

• Evaluate the cellular models used (cell lines vs. primary cells vs. tissue samples)

• Consider post-translational modifications that might affect antibody recognition

• Analyze subtypes of cancers separately, as SERPINI1 may have different roles in different subtypes

• Integrate findings with genomic data (mutations, copy number alterations)

• Consider the tumor microenvironment's influence on SERPINI1 expression and function

• Validate findings across multiple patient cohorts to establish reproducibility