CD82 Antibody, HRP conjugated

What is CD82/KAI1 and what cellular functions does it regulate?

CD82/KAI1 is a member of the tetraspanin family of proteins that functions as a structural component of specialized membrane microdomains known as tetraspanin-enriched microdomains (TERMs). These microdomains serve as platforms for receptor clustering and signaling . CD82 participates in diverse biological functions including cell signal transduction, adhesion, migration, and protein trafficking.

CD82 acts primarily as an attenuator of EGF signaling by facilitating ligand-induced endocytosis of the receptor and its subsequent desensitization . Mechanistically, it modulates ligand-induced ubiquitination and trafficking of EGFR via E3 ligase CBL phosphorylation by PKC . Additionally, CD82 increases cell-matrix adhesion by regulating membrane organization of various integrins, including alpha4/ITA4, alpha6/ITGA6, and beta1/ITGB1, thereby suppressing cell migration .

Beyond its well-established role in cancer metastasis suppression, CD82 also participates in immune regulation. It associates with CD4 or CD8 and delivers costimulatory signals for the TCR/CD3 pathway, plays a role in TLR9 trafficking, inhibits LPS-induced inflammatory responses, and contributes to macrophage activation into anti-inflammatory phenotypes .

What are the key differences between polyclonal and monoclonal CD82 antibodies for research applications?

The selection between polyclonal and monoclonal CD82 antibodies depends on experimental requirements and desired specificity:

Polyclonal CD82 antibodies, such as ab66400, have demonstrated effectiveness in multiple applications including Western blot and immunohistochemistry on paraffin-embedded tissues . For example, ab66400 at 1/50 dilution successfully stained CD82 in paraffin-embedded mouse salivary gland tissue and human tonsil tissue (1:80 dilution), clearly highlighting follicular dendritic cell networks .

By contrast, monoclonal CD82 antibodies like EPR4112 (ab109529) provide high specificity for a single epitope, making them especially valuable for distinguishing between closely related proteins or specific conformational states of CD82 .

How does HRP conjugation enhance CD82 antibody utility in experimental protocols?

HRP conjugation provides significant methodological advantages in CD82 research applications:

• Direct detection capability: HRP-conjugated CD82 antibodies (such as CSB-PA13269B0Rb) enable direct detection without requiring secondary antibodies, simplifying experimental protocols and reducing potential sources of variability .

• Increased sensitivity: The enzymatic amplification provided by HRP can enhance signal detection, especially beneficial when studying samples with low CD82 expression levels.

• Reduced background: Direct conjugation eliminates potential cross-reactivity associated with secondary antibodies, potentially reducing non-specific background signals.

• Time efficiency: HRP-conjugated antibodies streamline experimental workflows by eliminating secondary antibody incubation and washing steps, particularly valuable in high-throughput studies.

• Versatility: HRP-conjugated CD82 antibodies can be utilized across multiple applications including ELISA, Western blot, and immunohistochemistry with appropriate detection substrates .

The HRP-conjugated CD82 antibody CSB-PA13269B0Rb, for example, is specifically optimized for ELISA applications with human samples, utilizing recombinant human CD82 antigen protein (amino acids 111-228) as an immunogen .

What are the optimal experimental conditions for using CD82 Antibody, HRP conjugated in ELISA applications?

For optimal ELISA results with HRP-conjugated CD82 antibodies, researchers should carefully consider multiple methodological parameters:

Sample Preparation:

• For cell lysates: Extract proteins using RIPA buffer containing protease inhibitors

• For serum/plasma: Use appropriate dilution (typically 1:10-1:100) in blocking buffer

• For tissue extracts: Homogenize in PBS with protease inhibitors, centrifuge, and use supernatant

Protocol Optimization:

• Coating: Use purified CD82 protein or capture antibody (1-10 μg/ml) in carbonate buffer (pH 9.6), incubate overnight at 4°C

• Blocking: 3-5% BSA or non-fat milk in PBS-T (PBS + 0.05% Tween-20) for 1-2 hours at room temperature

• HRP-conjugated CD82 antibody application: For CSB-PA13269B0Rb, use at manufacturer-recommended dilution (typically 1:1000 to 1:5000) in blocking buffer

• Incubation: 1-2 hours at room temperature or overnight at 4°C with gentle agitation

• Washing: 4-6 times with PBS-T to remove unbound antibody

• Detection: Use TMB substrate followed by stop solution (2N H₂SO₄)

• Quantification: Read absorbance at 450 nm with 570 nm reference wavelength

The CSB-PA13269B0Rb antibody has been specifically validated for ELISA applications with human samples, with optimal concentration determined through careful titration experiments .

What are the critical steps for successful Western blot detection of CD82 using HRP-conjugated antibodies?

Successful Western blot detection of CD82 requires attention to several critical methodological details:

Sample Preparation:

• Cell lysis: Use RIPA or NP-40 buffer containing protease inhibitors

• Protein quantification: Bradford or BCA assay to ensure equal loading

• Sample denaturation: Heat at 70°C for 10 minutes (not boiling) to preserve tetraspanin structure

Electrophoresis and Transfer:

• Load 10-30 μg total protein per lane

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

• Transfer to PVDF membrane (preferred over nitrocellulose for tetraspanins)

• Use semi-dry transfer at 15V for 30 minutes or wet transfer at 30V overnight at 4°C

Immunodetection:

• Blocking: 5% non-fat milk in TBS-T for 1 hour at room temperature

• Primary antibody: For non-conjugated antibodies like ab109529, use at 1:1000 dilution

• For HRP-conjugated antibodies: Apply directly at manufacturer-recommended dilution

• Incubation: Overnight at 4°C with gentle agitation

• Washing: 4 × 5 minutes with TBS-T

• Detection: Use enhanced chemiluminescence (ECL) substrate

• Exposure: Start with 30-second exposure, adjust as needed

Band Interpretation:
CD82 typically appears as multiple bands between 40-60 kDa due to glycosylation. In U-87 MG and Jurkat cell lines, specific bands have been validated with the EPR4112 monoclonal antibody .

What controls should be included when validating CD82 detection specificity?

Proper experimental controls are essential for validating CD82 antibody specificity:

Positive Controls:

• Cell lines with confirmed CD82 expression: Jurkat (human T cell leukemia) and U-87 MG (human glioblastoma-astrocytoma) have been validated as positive controls

• Tissue sections: Human tonsil tissue sections have demonstrated strong membrane staining of follicular dendritic cells with CD82 antibodies

Negative Controls:

• Primary antibody omission: Process samples following standard protocol but omit primary antibody

• Isotype controls: Use non-specific IgG from same species at equivalent concentration

• CD82-knockdown samples: Use siRNA or shRNA to generate CD82-depleted samples as demonstrated in 2.5.2A breast cancer cells

Blocking Peptide Controls:

• Pre-incubate antibody with excess immunizing peptide (for CSB-PA13269B0Rb, this would be recombinant Human CD82 antigen protein amino acids 111-228)

• Apply pre-absorbed antibody to duplicate samples

• Specific staining should be eliminated or significantly reduced

Cross-Reactivity Assessment:

• Test antibody against related tetraspanin family members

• Evaluate species cross-reactivity if working with non-human samples

Proper controls are critical for distinguishing specific CD82 signals from background or non-specific binding, particularly in complex samples like tissue sections.

What are common causes of weak or absent signals when using CD82 Antibody, HRP conjugated?

Several factors can contribute to suboptimal signal when using CD82 antibodies:

When working with CD82 antibodies, researchers should be aware that CD82 is often post-translationally modified with high levels of glycosylation, which can affect detection . Additionally, proper antigen retrieval is critical for immunohistochemistry applications. For example, ab66400 has been successfully used with heat-mediated antigen retrieval in CC1 Cell Conditioning Buffer using a Ventana Standard Retrieval program .

How can researchers determine the optimal dilution of CD82 Antibody, HRP conjugated for specific applications?

Determining optimal antibody concentration requires systematic titration:

For Western Blot:

• Prepare a known positive control sample (e.g., Jurkat or U-87 MG cell lysate)

• Run multiple identical lanes of the same sample

• Test a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000)

• Process following standard Western blot protocol

• Compare signal-to-noise ratio across dilutions

• Select dilution that provides best combination of specific signal and minimal background

For ELISA:

• Coat plate with target antigen at constant concentration

• Perform checkerboard titration with antibody dilutions (e.g., 1:1000 to 1:10,000)

• Process following standard ELISA protocol

• Generate standard curve for each antibody dilution

• Calculate signal-to-noise ratio and determine linear range for each dilution

• Select dilution providing optimal sensitivity within linear detection range

For Immunohistochemistry:

• Use known positive control tissue (e.g., human tonsil)

• Prepare multiple sections of identical tissue

• Test antibody dilution series (e.g., 1:50, 1:100, 1:200)

• Process following standard IHC protocol

• Evaluate specific membrane staining versus background

• Select dilution with optimal specific staining and minimal background

For ab66400, the experimentally validated dilutions are 1:50 for mouse salivary gland tissue and 1:80 for human tonsil tissue in immunohistochemistry applications .

What approaches can address non-specific binding with CD82 Antibody, HRP conjugated?

Non-specific binding can significantly impact experimental outcomes, requiring systematic troubleshooting:

Optimization of Blocking Conditions:

• Test different blocking reagents (BSA, non-fat milk, normal serum, commercial blockers)

• Increase blocking time (2-3 hours at room temperature or overnight at 4°C)

• Add 0.1-0.3% Triton X-100 to blocking solution for membrane permeabilization

Washing Optimization:

• Increase washing frequency (6-8 washes instead of standard 3-5)

• Extend washing duration (10 minutes per wash)

• Use higher concentration of detergent (0.1% vs. 0.05% Tween-20) in wash buffer

Antibody Incubation Modifications:

• Dilute antibody in blocking solution containing 1-5% of protein from the same species as the sample

• Add 0.1-0.5 M NaCl to antibody diluent to reduce non-specific ionic interactions

• Pre-adsorb antibody against tissues or cell extracts from species of interest

Background Reduction Methods:

• For tissue sections: Use avidin/biotin blocking kit if endogenous biotin is present

• For cells with high peroxidase activity: Increase H₂O₂ blocking (e.g., 3% H₂O₂ for 10 minutes)

• For high autofluorescence: Use Sudan Black B treatment

For immunohistochemistry applications with CD82 antibodies, specific optimization strategies have been documented, including 3% H₂O₂ for 4 minutes at 37°C for peroxidase blocking prior to antibody application .

How can CD82 Antibody, HRP conjugated be used to investigate CD82's role in EGFR signaling?

CD82 plays a critical role in regulating EGFR signaling through several mechanisms that can be investigated using CD82 antibodies:

Ubiquitylation Analysis:
CD82 specifically suppresses ubiquitylation of EGFR after stimulation with heparin-binding EGF (HB-EGF) or amphiregulin (AR), but not with EGF itself . Researchers can design experiments to examine this selective regulation:

• Stimulate cells with different EGFR ligands (EGF, HB-EGF, AR)

• Immunoprecipitate EGFR

• Perform Western blot analysis for ubiquitin and CD82

• Compare ubiquitylation levels between CD82-expressing and CD82-depleted cells

Studies have demonstrated that ubiquitylation of EGFR in CD82-depleted cells increased up to 3-fold following HB-EGF stimulation compared to parental cells .

Receptor Trafficking Studies:
CD82 affects EGFR trafficking following ligand stimulation, which can be analyzed through:

• Surface biotinylation followed by internalization assays

• Immunofluorescence co-localization with endosomal markers

• EGFR recycling assays comparing CD82-positive and CD82-negative cells

PKC Phosphorylation Analysis:
CD82 increases phosphorylation of threonine 654 (PKC phosphorylation site) in the juxtamembrane domain of EGFR . This can be examined using:

• Phospho-specific antibodies against EGFR-T654

• Kinase inhibitor studies to determine PKC involvement

• Co-immunoprecipitation of CD82 and EGFR following ligand stimulation

These methodologies allow researchers to dissect the complex regulatory role of CD82 in EGFR signaling, particularly its discriminative control of c-Cbl activity toward heparin-binding ligand-EGFR pairs .

What approaches can be used to study the interaction of CD82 with heparan sulfate proteoglycans (HSPGs)?

The interaction between CD82 and HSPGs represents an important regulatory mechanism in EGFR signaling that can be investigated through several approaches:

Heparin-Binding Domain Studies:
Research has shown that the heparin-binding domain of HB-EGF is essential for CD82-induced changes in the ubiquitylation of EGFR . Experimental approaches include:

• Comparing wild-type HB-EGF versus HB-EGF with deleted heparin-binding domain (sΔHB-EGF)

• Analyzing EGFR ubiquitylation following stimulation with modified ligands

• Using heparinase treatment to degrade cell surface HSPGs before stimulation

Studies have demonstrated that ubiquitylation of EGFR in both control and CD82-expressing cells was robust and comparable following stimulation with sΔHB-EGF, confirming the critical role of the heparin-binding domain .

Co-immunoprecipitation Studies:

• Immunoprecipitate CD82 and probe for HSPGs

• Perform reverse co-IP with antibodies against specific HSPGs

• Use crosslinking approaches to stabilize transient interactions

Surface Plasmon Resonance (SPR):

• Immobilize purified CD82 on SPR chip

• Flow over different HSPG variants

• Measure binding kinetics and affinity

Functional Consequences Analysis:

• Deplete specific HSPGs using siRNA

• Overexpress CD82 in HSPG-deficient cells

• Analyze EGFR ubiquitylation and trafficking

These approaches help elucidate the critical role of CD82 in regulating communication between HSPGs and ligand-bound EGFR, affecting the activity of c-Cbl and subsequent receptor trafficking .

How can CD82 Antibody, HRP conjugated be utilized in cancer metastasis research?

CD82/KAI1 functions as a metastasis suppressor, making it an important target in cancer research. CD82 antibodies enable several experimental approaches:

Metastatic Potential Correlation Studies:

• Analyze CD82 expression across cancer cell lines with different metastatic potentials

• Correlate expression levels with invasive properties

• Create tissue microarrays from primary tumors and metastatic sites to examine CD82 expression patterns

Functional Mechanism Investigations:

• Examine CD82-dependent regulation of integrins (α4/ITA4, α6/ITGA6, β1/ITGB1)

• Analyze effect on cell-matrix adhesion

• Investigate interaction with urokinase-type plasminogen activator (PLAU) and its receptor (PLAUR)

Therapeutic Target Validation:

• Restore CD82 expression in metastatic cell lines

• Assess changes in invasive properties

• Evaluate effects on EGFR signaling pathways

• Examine impact on angiogenesis through CD82's binding and sequestration of VEGFA and PDGFB

Biomarker Development:

• Develop quantitative IHC protocols for CD82 detection in clinical samples

• Correlate CD82 expression with patient outcomes

• Evaluate CD82 as part of multi-marker prognostic panels

These approaches leverage CD82 antibodies to advance understanding of metastasis mechanisms and potentially identify new therapeutic strategies for metastatic disease.

How should researchers interpret varying molecular weight bands when detecting CD82 with specific antibodies?

CD82 protein often presents multiple bands on Western blots due to post-translational modifications, requiring careful interpretation:

Expected Band Patterns:

• Predicted molecular weight: 30-35 kDa (unmodified protein)

• Observed molecular weight: 40-60 kDa range (glycosylated forms)

• Multiple bands typically represent different glycosylation states

Band Interpretation Guidelines:

When evaluating Western blot results, investigators should note that specific cell lines show characteristic patterns. For example, U-87 MG and Jurkat cell lines display specific band patterns when probed with the EPR4112 monoclonal antibody .

Verification Approaches:

• Deglycosylation: Treat samples with PNGase F and compare band patterns

• Multiple antibodies: Use antibodies recognizing different epitopes

• CD82 knockdown: Compare with shRNA-depleted samples as demonstrated in 2.5.2A breast cancer cells

• Recombinant protein: Run alongside purified CD82 as size reference

What approaches provide robust quantification of CD82 expression for comparative studies?

Accurate quantification of CD82 expression requires rigorous methodological approaches:

Western Blot Quantification:

• Include dilution series of positive control (e.g., recombinant CD82) on each blot

• Use appropriate loading controls (β-actin, GAPDH, or total protein stains)

• Employ digital image acquisition with wide dynamic range

• Perform densitometry using software that accounts for background

• Generate standard curves to ensure measurements fall within linear range

• Normalize CD82 signal to loading control or total protein

• Perform at least three biological replicates for statistical validity

ELISA-Based Quantification:

• Develop sandwich ELISA using two non-competing CD82 antibodies

• Create standard curve using recombinant CD82 protein

• Validate assay linearity, sensitivity, and reproducibility

• Process samples in technical triplicates

• Include quality control samples across plates for inter-assay comparison

Flow Cytometry Quantification:

• Use calibrated beads with known antibody binding capacity

• Calculate molecules of equivalent soluble fluorochrome (MESF)

• Determine surface density of CD82 molecules per cell

• Compare expression across different cell populations

RT-qPCR for mRNA Quantification:

• Design specific primers spanning exon-exon junctions

• Validate primer efficiency using dilution series

• Use multiple reference genes for normalization

• Correlate mRNA with protein levels to identify post-transcriptional regulation

These approaches enable rigorous comparative studies of CD82 expression across different experimental conditions, cell types, or clinical samples.

How can researchers distinguish between CD82 and other tetraspanin family members in experimental systems?

Distinguishing CD82 from other tetraspanins requires careful methodological considerations:

Antibody Specificity Verification:

• Test antibodies against recombinant tetraspanin panel

• Use cell lines with defined tetraspanin expression profiles

• Employ knockout/knockdown models for validation

• Verify epitope specificity through peptide competition

Sequence Homology Analysis:
The tetraspanin family shares structural features but differs in key domains. CD82-specific antibodies like CSB-PA13269B0Rb target unique regions (amino acids 111-228 of human CD82) to minimize cross-reactivity.

Functional Discrimination Approaches:

• CD82-specific functions in EGFR regulation can distinguish it from other tetraspanins

• CD82 uniquely suppresses ubiquitylation of EGFR after HB-EGF or AR stimulation

• CD82's role in PKC-mediated phosphorylation of EGFR differs from other tetraspanins

Mass Spectrometry Verification:

• Immunoprecipitate with CD82 antibody

• Perform tryptic digestion

• Identify unique peptides by LC-MS/MS

• Compare against tetraspanin protein database

Co-localization Studies:

• Perform dual-color immunofluorescence with antibodies against different tetraspanins

• Analyze co-localization coefficients

• Use super-resolution microscopy to distinguish closely associated tetraspanins

These strategies enable confident discrimination between CD82 and other tetraspanin family members in experimental systems, ensuring specificity of findings in CD82-focused research.