ASGR2 Antibody

What is ASGR2 and why is it significant in research?

ASGR2 is the minor subunit of the asialoglycoprotein receptor, a hetero-oligomeric protein expressed predominantly in hepatocytes. It mediates the endocytosis of plasma glycoproteins with terminal galactose or N-acetylgalactosamine residues after sialic acid removal . ASGR2 is significant in research because:

• It serves as a hepatocyte-specific marker for liver studies

• It may facilitate hepatic infection by viruses including hepatitis B

• It represents a potential target for liver-specific drug delivery

• Its alternatively spliced variant (H2) has been proposed as a marker for liver fibrosis

• It has emerging roles in cancer biology, particularly gastric cancer

Which applications are most suitable for ASGR2 antibody detection?

ASGR2 antibodies can be utilized across multiple experimental platforms:

Each application requires optimization based on specific experimental conditions and antibody clones .

How should I select between monoclonal and polyclonal ASGR2 antibodies?

The choice depends on your experimental goals:

Monoclonal antibodies (e.g., clone 2327C, EPR16975, 1D7):

• Provide higher specificity to a single epitope

• Show better reproducibility between batches

• Recommended for quantitative assays and detection of specific isoforms

• Example: The rabbit monoclonal antibody clone EPR16975 is validated for IP, WB, and IHC-P applications

Polyclonal antibodies:

• Recognize multiple epitopes, potentially increasing signal strength

• Better for detecting denatured proteins in applications like Western blotting

• Useful when protein levels are low

• Example: Rabbit polyclonal antibodies (e.g., 11501-2-AP) show reactivity across multiple applications

For critical quantitative experiments requiring high reproducibility, monoclonal antibodies are generally preferred .

What are the optimal sample preparation methods for ASGR2 detection in different tissues?

Sample preparation varies by application and tissue type:

For liver tissues (Western blot):

• Homogenize fresh or frozen tissue in RIPA buffer containing protease inhibitors

• Centrifuge at 14,000 × g for 15 minutes at 4°C to remove debris

• Determine protein concentration (BCA/Bradford assay)

• Denature samples in reducing conditions (with β-mercaptoethanol)

• Load 10-30 μg protein per lane

• Use Immunoblot Buffer Group 1 for optimal results

For paraffin-embedded liver sections (IHC):

• Cut 4-6 μm sections and mount on positively charged slides

• Deparaffinize and rehydrate sections

• Perform antigen retrieval using TE buffer (pH 9.0) or citrate buffer (pH 6.0)

• Block endogenous peroxidase activity with 3% H₂O₂

• Use 1 μg/mL antibody concentration for 1 hour at room temperature

• Develop with DAB and counterstain with hematoxylin

For flow cytometry:

• Prepare single-cell suspensions (≤1×10^6 cells/100 μL)

• Fix with 4% paraformaldehyde if required

• Block with 5% normal serum

• Use 0.25 μg antibody per 10^6 cells

• Set quadrant markers based on appropriate control antibody staining

What controls should be included when validating ASGR2 antibody specificity?

Comprehensive controls are essential for reliable ASGR2 detection:

Positive controls:

• Human liver tissue (primary site of expression)

• HepG2 cells (human liver cancer cell line)

• HEK293 cells transfected with human ASGR2

Negative controls:

• Non-hepatic tissues (kidney, heart, etc.)

• Cell lines that don't express ASGR2 naturally

• HEK293 cells with irrelevant transfectants

Specificity controls:

• Antibody preabsorption with recombinant ASGR2 protein

• ASGR2 knockdown (siRNA) or knockout cells

• Isotype control antibodies (e.g., MAB1050)

• Testing multiple antibody clones against the same sample

Including comprehensive controls helps distinguish specific from non-specific binding and validates experimental findings .

How can I troubleshoot weak or absent ASGR2 signal in Western blot experiments?

When encountering signal issues with ASGR2 detection:

• Sample preparation issues:

  • Confirm protein stability (use fresh samples, add protease inhibitors)

  • Ensure adequate protein loading (20-50 μg total protein)

  • Verify proper reducing conditions (ASGR2 requires reducing agents)

• Technical adjustments:

  • Optimize antibody concentration (try 2-5 μg/mL range)

  • Increase antibody incubation time (overnight at 4°C)

  • Use 5% NFDM/TBST as blocking/dilution buffer

  • Try Immunoblot Buffer Group 1 as recommended

• Detection optimization:

  • Use more sensitive detection systems (ECL Plus/Prime)

  • Increase exposure time

  • Try HRP-conjugated secondary antibody (Catalog # HAF008)

• Membrane considerations:

  • PVDF membranes are recommended for ASGR2 detection

  • Ensure efficient protein transfer (confirm with Ponceau S staining)

• Antibody selection:

  • The observed molecular weight should be ~45 kDa

  • Consider trying different antibody clones if persistent issues occur

How can ASGR2 antibodies be utilized in extracellular vesicle (EV) research?

ASGR2 antibodies have emerged as valuable tools in EV research, particularly for hepatocyte-derived EVs:

• EV isolation and characterization:

  • ASGR2 antibodies can perform immunoaffinity capture of hepatocyte-derived EVs

  • Captured EVs can be confirmed by co-detection of EV markers (ALIX, CD81, Tsg101)

• Nano-plasmonic enhanced scattering (nPES) assay:

  • ASGR2 antibodies serve as capture antibodies on protein A/G functionalized slides

  • Captured EVs are detected using gold nanorods conjugated with anti-CD63 antibodies

  • This assay enables direct quantification of hepatocyte-derived EVs in small plasma volumes (4 μL)

• Clinical applications:

  • ASGR2-positive EV detection correlates with small EVs in NASH patients

  • Optical response with ASGR2 antibody is decreased in post-bariatric surgery patients

  • ASGR2-positive EVs serve as potential biomarkers for NAFLD and NASH

This approach enables quantitative assessment of liver-derived EVs without extensive EV isolation procedures, offering advantages for biomarker studies .

What is the role of ASGR2 in cancer research and how can antibodies facilitate these studies?

ASGR2 has emerging roles in cancer biology that can be investigated using antibodies:

These findings position ASGR2 as a potential biomarker for hematogenous recurrences after curative resection for gastric cancer .

How can fluorophore-conjugated ASGR2 antibodies be optimized for multicolor flow cytometry and imaging?

Several conjugated ASGR2 antibodies are available for multicolor applications:

• Available conjugates:

  • Alexa Fluor 488 (green fluorescence)

  • Alexa Fluor 647 (far-red fluorescence)

  • Alexa Fluor 700 (near-infrared fluorescence)

• Panel design considerations:

  • Combine ASGR2-AF488 with PE-conjugated ASGR1 antibodies for co-expression analysis

  • Use ASGR2-AF647 for multicolor panels including FITC or PE-conjugated markers

  • Consider spectral overlap and employ proper compensation controls

• Optimization protocols:

  • Titrate antibodies to determine optimal concentration (start with 0.25 μg per 10^6 cells)

  • Always protect conjugated antibodies from light during storage and staining

  • Do not freeze conjugated antibodies (store at 2-8°C for up to 12 months)

  • Set quadrant markers based on control antibody staining

• Applications in co-expression studies:

  • Co-staining with ASGR1 and ASGR2 antibodies can assess receptor heterooligomer formation

  • Dual staining with eGFP-transfected cells allows measurement of transfection efficiency

Proper handling and optimization of conjugated antibodies ensures reliable multiparameter analysis of ASGR2 expression .

How does ASGR2 expression vary across different tissues and disease states?

ASGR2 exhibits distinct expression patterns:

Normal tissue distribution:

• Predominantly expressed in hepatocytes (liver parenchymal cells)

• Localized to cytoplasm in hepatocytes as demonstrated by IHC

• Limited or absent expression in non-hepatic tissues

Disease-associated variations:

• Altered levels in liver fibrosis (alternatively spliced H2 variant)

• Increased expression in certain gastric cancer types, associated with hematogenous recurrence

• Detectable on extracellular vesicles in NAFLD/NASH patients

Experimental models:

• Detectable in human liver tissue and fetal liver lysates

• Expressed in HepG2 cells (hepatocellular carcinoma line)

• Successfully expressed in transfected HEK293 cells

• Limited expression in K562 cells (chronic myelogenous leukemia)

Understanding these expression patterns is crucial for experimental design and proper interpretation of ASGR2 antibody staining results .

What is the molecular weight of ASGR2 and how can I confirm specific detection?

ASGR2 protein characteristics must be understood for accurate detection:

Molecular weight considerations:

• The observed molecular weight of ASGR2 is approximately 45 kDa in Western blot under reducing conditions

• This is consistent with literature descriptions (PMID:3040719)

• The calculated molecular weight is 39 kDa

• The discrepancy may be due to post-translational modifications

Confirmation methods:

• Size comparison with recombinant ASGR2 protein (human ASGR2 Gln80-Ala311)

• Detection in human liver tissue (primary expression site)

• Comparison between wild-type and ASGR2-transfected cells:

  • Transfected HEK293 cells should show strong band compared to non-transfected controls

  • The difference between lanes confirms specificity

• Antibody validation with siRNA knockdown

Alternative splicing consideration:

• ASGR2 has alternatively spliced variants

• The H2 variant may appear at a different molecular weight

• Consider using antibodies that can distinguish between variants for specific studies

How can I quantitatively assess ASGR2 expression levels across different experimental conditions?

For rigorous quantitative analysis of ASGR2:

• Western blot quantification:

  • Use housekeeping proteins (β-actin, GAPDH) as loading controls

  • Apply densitometric analysis to normalize ASGR2 signals

  • Include a standard curve using recombinant ASGR2 at known concentrations

  • Use antibody dilutions in the linear response range (1:200-1:1000)

• Flow cytometry quantification:

  • Use antibody capture beads to establish standard curves

  • Express data as molecules of equivalent soluble fluorochrome (MESF)

  • Compare median fluorescence intensity (MFI) after subtracting isotype control values

  • Use consistent staining conditions (0.25 μg per 10^6 cells)

• RT-qPCR for mRNA levels:

  • Use ASGR2-specific primers

  • Normalize to appropriate reference genes

  • Compare with protein levels to assess correlation

• IHC scoring systems:

  • Develop H-score (intensity × percentage positive cells)

  • Use digital image analysis for objective quantification

  • Establish clear scoring criteria for inter-observer reproducibility

• Statistical considerations:

  • Apply appropriate statistical tests based on data distribution

  • Use multiple biological and technical replicates

  • Consider power analysis to determine sample size requirements

How can I use ASGR2 antibodies in co-immunoprecipitation studies to identify binding partners?

Co-immunoprecipitation (Co-IP) with ASGR2 antibodies can uncover protein interactions:

• Antibody selection:

  • Use antibodies validated for IP applications (e.g., EPR16975)

  • Recommended usage: 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate

• Protocol optimization:

  • Extract proteins under non-denaturing conditions to preserve interactions

  • Pre-clear lysates with Protein A/G beads to reduce non-specific binding

  • Perform IP at 4°C overnight for efficient capture

  • Include appropriate negative controls (isotype control antibodies)

• Known interactions to validate method:

  • ASGR1-ASGR2 interaction (heterooligomer formation)

  • Test detection of both subunits in the immunoprecipitated complex

  • Verify with reciprocal IPs using anti-ASGR1 antibodies

• Applications:

  • Identify novel ASGR2 binding partners in hepatocytes

  • Investigate interactions with endocytic machinery proteins

  • Study ASGR2's role in virus binding (e.g., hepatitis B)

  • Examine associations with other lectins or carbohydrate-binding proteins

This approach can provide insights into ASGR2's functional networks and molecular mechanisms .

What considerations are important when using ASGR2 antibodies for immunohistochemistry in liver pathology?

For optimal IHC applications in liver tissues:

• Tissue preparation:

  • Use freshly fixed tissues (10% neutral buffered formalin)

  • Limit fixation time to 24-48 hours to preserve antigenicity

  • Process tissues consistently for comparable results

• Antigen retrieval methods:

  • TE buffer (pH 9.0) is recommended as primary method

  • Citrate buffer (pH 6.0) can be used as an alternative

  • Heat-induced epitope retrieval (pressure cooker or microwave) is effective

• Antibody optimization:

  • Titrate antibody concentration (starting with 1 μg/mL)

  • Optimize incubation time and temperature (typically 1 hour at room temperature)

  • Consider signal amplification systems for low expression levels

• Detection systems:

  • Anti-Rabbit IgG VisUCyte™ HRP Polymer Antibody system works effectively

  • DAB (brown) provides good contrast with hematoxylin (blue) counterstain

  • Consider multiplex IHC for co-localization studies

• Interpretation in liver pathology:

  • Normal ASGR2 staining shows cytoplasmic localization in hepatocytes

  • Evaluate distribution patterns (zonal variations in lobule)

  • Assess changes in cirrhosis, hepatitis, or tumors

  • Compare with normal liver controls

These considerations enhance the reliability of ASGR2 detection in liver specimens for diagnostic and research applications .

How can ASGR2 antibodies be utilized in developing targeted drug delivery systems to the liver?

ASGR2 antibodies enable development of hepatocyte-targeted therapeutics:

• Target validation approaches:

  • Use anti-ASGR2 antibodies to confirm expression levels in target tissues

  • Employ flow cytometry to quantify receptor density on hepatocytes

  • Evaluate internalization kinetics using fluorophore-conjugated antibodies

  • Assess expression in disease models to confirm targeting potential

• Drug delivery system development:

  • Conjugate therapeutic payloads to anti-ASGR2 antibodies or fragments

  • Develop nanoparticles decorated with ASGR2-targeting ligands

  • Create bispecific antibodies targeting both ASGR2 and therapeutic targets

  • Validate binding specificity using competition assays with unconjugated antibodies

• Functional testing:

  • Use in vitro cell models (primary hepatocytes or HepG2 cells)

  • Track internalization and intracellular trafficking using imaging techniques

  • Assess therapeutic efficacy in hepatocyte-specific disease models

  • Evaluate potential for off-target effects in non-hepatic tissues

• Applications:

  • Liver-specific gene delivery systems

  • Targeted therapies for hepatic diseases

  • Treatment of viral hepatitis where ASGR2 may facilitate infection

  • Delivery of siRNA or antisense oligonucleotides to hepatocytes

This research area leverages ASGR2's hepatocyte specificity for precision medicine applications targeting liver diseases .

What is the potential of ASGR2 as a biomarker for liver fibrosis and how can antibodies facilitate its detection?

ASGR2's emerging role in liver fibrosis assessment:

• Biomarker potential:

  • The alternatively spliced H2 variant can be shed and has been proposed as a marker for liver fibrosis

  • Changes in ASGR2 expression correlate with fibrotic progression

  • Non-invasive assessment could supplement or replace liver biopsy

• Detection strategies:

  • Develop ELISA systems using ASGR2 antibodies for serum/plasma quantification

  • Use antibodies that specifically recognize the shed H2 variant

  • Combine with other fibrosis markers for improved diagnostic accuracy

  • Correlate with established fibrosis scoring systems (METAVIR, Ishak)

• Clinical validation approaches:

  • Compare ASGR2 levels across fibrosis stages

  • Assess prognostic value for fibrosis progression

  • Evaluate response to anti-fibrotic therapies

  • Determine sensitivity and specificity compared to existing biomarkers

• Research limitations to address:

  • Need for antibodies specifically recognizing the shed H2 variant

  • Standardization of measurement protocols

  • Understanding of confounding factors affecting serum levels

  • Validation across different etiologies of liver disease

This research direction could establish ASGR2 as a valuable addition to the liver fibrosis biomarker panel .

How does ASGR2 function in extracellular vesicle biology and what techniques can be used to study this relationship?

Exploring ASGR2's role in EV biology requires specialized techniques:

• EV isolation and characterization:

  • Differential ultracentrifugation to separate large and small EVs

  • Immunoaffinity capture using anti-ASGR2 antibodies

  • Validation with immuno-gold electron microscopy

  • Western blotting for EV markers (ALIX, CD81, Tsg101) in ASGR2-positive EVs

• Advanced detection methods:

  • Nano-plasmonic enhanced scattering (nPES) assay using ASGR2 antibodies as capture antibodies

  • Flow cytometry of isolated EVs using fluorophore-conjugated anti-ASGR2 antibodies

  • Single-vesicle analysis using super-resolution microscopy

  • Correlation of ASGR2-positive EVs with clinical parameters

• Functional studies:

  • Tracking ASGR2-positive EVs and their cellular uptake

  • Investigating EV cargo in ASGR2-positive versus negative populations

  • Assessing the effect of ASGR2 knockdown on EV production and content

  • Exploring the role of ASGR2-positive EVs in intercellular communication

• Clinical applications:

  • ASGR2-positive EVs correlate with small EVs in NASH patients

  • Potential biomarker for liver diseases including NAFLD

  • Monitoring changes in ASGR2-positive EVs during disease progression or treatment

These approaches reveal ASGR2's significance in EV-mediated communication, particularly in liver pathology contexts .

What role does ASGR2 play in viral hepatitis infection and how can antibodies help study this interaction?

ASGR2's involvement in viral hepatitis mechanisms:

• Viral entry studies:

  • ASGR2 may facilitate hepatic infection by viruses including hepatitis B

  • Anti-ASGR2 antibodies can block receptor binding to assess functional roles

  • Co-localization studies using fluorophore-conjugated antibodies can visualize virus-receptor interactions

  • Knockdown/knockout studies can confirm the necessity of ASGR2 for viral entry

• Mechanism investigation techniques:

  • Co-immunoprecipitation to identify viral proteins interacting with ASGR2

  • Flow cytometry to quantify ASGR2 expression changes during infection

  • Live-cell imaging using labeled antibodies to track receptor trafficking

  • Competitive binding assays to characterize virus-receptor interactions

• Therapeutic implications:

  • Development of entry inhibitors targeting ASGR2-virus interactions

  • Screening compounds that modulate ASGR2 expression or function

  • Creating decoy receptors based on ASGR2 structure

  • Evaluating ASGR2 expression patterns in responders versus non-responders to antiviral therapy

• Diagnostic applications:

  • Monitoring ASGR2 levels as potential markers of infection status

  • Developing assays to detect virus-ASGR2 complexes in patient samples

  • Correlating ASGR2 polymorphisms with infection susceptibility

This research area has implications for understanding viral hepatitis pathogenesis and developing novel therapeutic strategies .