ITGA8 Antibody, Biotin conjugated

What is ITGA8 and why is it a significant research target?

ITGA8 (Integrin subunit alpha 8) is a protein that functions as part of the integrin family of cell adhesion receptors. It plays crucial roles in cell-extracellular matrix interactions, brain development, and cellular adhesion processes. The human version has a canonical amino acid length of 1063 residues and a protein mass of approximately 117.5 kilodaltons . ITGA8 is particularly important in kidney research as it shows notable expression in renal tissues . The protein can be cleaved into two chains: Integrin alpha-8 heavy chain and Integrin alpha-8 light chain . Its involvement in signal transduction pathways makes it a valuable target for investigating developmental processes, tissue homeostasis, and various pathological conditions.

What are the structural characteristics of biotin-conjugated ITGA8 antibodies?

Biotin-conjugated ITGA8 antibodies consist of immunoglobulins (typically IgG isotype) that specifically recognize and bind to ITGA8 protein epitopes, with biotin molecules covalently attached to the antibody structure. These antibodies are generally supplied in liquid form, suspended in buffers containing preservatives such as 0.03% Proclin 300, and stabilizers like 50% glycerol in PBS at pH 7.4 . The biotin conjugation enables secondary detection using streptavidin-based systems while maintaining the antibody's specificity for ITGA8. For polyclonal versions, the antibodies are typically purified using Protein G chromatography with >95% purity . These structural characteristics ensure both specificity for the target and versatility in detection methods.

What applications are suitable for biotin-conjugated ITGA8 antibodies?

Biotin-conjugated ITGA8 antibodies are particularly useful for applications requiring signal amplification or multiple detection systems. The primary applications include:

• Enzyme-Linked Immunosorbent Assay (ELISA): Offering high sensitivity for detecting ITGA8 in solution or bound to plates .

• Immunocytochemistry (ICC): Detecting ITGA8 in cultured cells, as demonstrated with the detection of Integrin α8 in 4T1 mouse breast cancer cell lines using biotin-conjugated antibodies and NorthernLights™ 557-conjugated streptavidin .

• Immunohistochemistry (IHC): Localizing ITGA8 in tissue sections with enhanced sensitivity due to the biotin-streptavidin amplification system .

• Flow Cytometry: Measuring ITGA8 expression on cell surfaces with the advantage of signal amplification via streptavidin-fluorophore conjugates.

• Immunoprecipitation: Isolating ITGA8-containing complexes from cell or tissue lysates using streptavidin-based capture methods.

The versatility of these antibodies makes them valuable tools across multiple research platforms investigating ITGA8 biology.

How does epitope selection impact experimental outcomes when using biotin-conjugated ITGA8 antibodies?

Epitope selection is critical for experimental success with biotin-conjugated ITGA8 antibodies. Different antibodies target distinct regions of the ITGA8 protein, which can significantly influence experimental outcomes. For example, antibodies raised against the N-terminal region (such as amino acids 114-131 in human ITGA8) may detect the protein differently than those targeting other domains.

The significance of epitope selection is particularly evident when:

• Studying protein-protein interactions: Antibodies recognizing interaction domains may interfere with binding partners.

• Detecting cleaved forms: ITGA8 is cleaved into heavy and light chains ; antibodies specific to regions spanning the cleavage site may only detect uncleaved protein.

• Cross-species applications: Conserved epitopes increase the likelihood of cross-reactivity with homologous proteins in different species (human, mouse, rat) .

• Post-translational modifications: Epitopes containing phosphorylation, glycosylation, or other modification sites may yield variable results depending on the protein's modification state.

Researchers should carefully select antibodies targeting epitopes relevant to their specific experimental questions and validate them in their particular model system.

What are the critical parameters for validating specificity of biotin-conjugated ITGA8 antibodies in complex tissue samples?

Validating antibody specificity in complex tissues requires multiple complementary approaches:

• Positive and negative controls:

  • Known ITGA8-expressing tissues (e.g., kidney) should show positive staining

  • Tissues with minimal ITGA8 expression should show minimal signal

  • ITGA8 knockout tissues (if available) should show no specific signal

• Peptide competition assays:

  • Pre-incubation with the immunogen peptide should abolish specific staining

  • This is particularly relevant for antibodies raised against synthetic peptides corresponding to human ITGA8 residues

• Correlation with mRNA expression:

  • Antibody staining patterns should correlate with ITGA8 transcript levels as determined by in situ hybridization or RNA-seq data

• Multi-antibody validation:

  • Using multiple antibodies targeting different ITGA8 epitopes to confirm staining patterns

  • Discrepancies between antibodies may indicate non-specific binding or detection of specific isoforms

• Western blot correlation:

  • Confirming the antibody detects proteins of the expected molecular weight (~117.5 kDa for full-length ITGA8) in the same tissues showing immunostaining

• Signal-to-noise ratio assessment:

  • Evaluating background staining at multiple antibody concentrations to determine optimal working dilutions

  • Accounting for endogenous biotin in tissues that may cause background with streptavidin detection systems

Comprehensive validation using these approaches ensures reliable interpretation of ITGA8 expression patterns in complex biological samples.

How do heterodimeric interactions between ITGA8 and beta integrins affect antibody recognition and experimental design?

ITGA8 functions primarily as a heterodimer with beta-1 integrin (ITGB1) , which significantly impacts antibody recognition and necessitates careful experimental design:

• Conformational epitopes:

  • ITGA8's conformation changes when dimerized with ITGB1

  • Some antibodies may preferentially recognize free ITGA8 while others may specifically detect the ITGA8-ITGB1 heterodimer

  • Researchers should consider whether their antibody recognizes conformational epitopes dependent on the heterodimeric state

• Co-immunoprecipitation considerations:

  • When using biotin-conjugated ITGA8 antibodies for pull-down experiments, researchers must assess whether the antibody disrupts or preserves the ITGA8-ITGB1 interaction

  • Harsh lysis conditions may dissociate the heterodimer, potentially affecting antibody recognition

• Functional assays:

  • Antibodies that bind near the ITGA8-ITGB1 interface may have functional effects by disrupting dimerization

  • This property could be exploited for functional studies but may confound other experiments

• Detection system optimization:

  • The quaternary structure of the ITGA8-ITGB1 complex may sterically hinder streptavidin binding to biotin-conjugated antibodies

  • Longer linkers between the antibody and biotin may improve detection of ITGA8 in heterodimeric complexes

• Heterodimer-specific detection:

  • Some experimental designs may benefit from using antibodies that specifically recognize the ITGA8-ITGB1 interface

  • Commercial heterodimer-specific antibodies or recombinant proteins may serve as important controls

Understanding these interactions is crucial for accurate interpretation of results, particularly in experiments investigating integrin-mediated signaling or cell adhesion processes.

What are the optimal protocols for immunofluorescence staining using biotin-conjugated ITGA8 antibodies?

The following protocol has been optimized for immunofluorescence applications with biotin-conjugated ITGA8 antibodies:

Sample Preparation:

• Fix cells on coverslips using 4% paraformaldehyde (15 minutes at room temperature)

• Permeabilize with 0.1% Triton X-100 in PBS (10 minutes)

• Block with 5% normal serum from the same species as the secondary detection reagent in PBS (1 hour)

Staining Procedure:

• Incubate with biotin-conjugated anti-ITGA8 antibody (10 μg/mL) in blocking buffer for 3 hours at room temperature

• Wash 3× with PBS (5 minutes each)

• Incubate with fluorophore-conjugated streptavidin (e.g., NorthernLights™ 557-conjugated streptavidin) at manufacturer's recommended dilution (typically 1:200-1:500) for 1 hour

• Wash 3× with PBS (5 minutes each)

• Counterstain nuclei with DAPI (1 μg/mL) for 5 minutes

• Mount with anti-fade mounting medium

Critical Parameters:

• Antibody concentration may require optimization (typical range: 5-15 μg/mL)

• Include negative controls (omitting primary antibody) to assess background from streptavidin binding

• For tissues with high endogenous biotin (e.g., liver, kidney), consider using an avidin/biotin blocking kit prior to antibody incubation

• When examining membrane localization, omit permeabilization step to visualize only cell surface ITGA8

This protocol has successfully demonstrated specific staining of ITGA8 in cell lines, with signal localized to both cytoplasm and cell surface .

What are the recommended approaches for quantifying ITGA8 expression levels using biotin-conjugated antibodies in Western blotting?

Quantifying ITGA8 expression by Western blot using biotin-conjugated antibodies requires specialized protocols:

Sample Preparation:

• Lyse cells or tissues in RIPA buffer containing protease inhibitors

• Determine protein concentration using BCA or Bradford assay

• Prepare samples in non-reducing or reducing conditions depending on whether conformational epitopes need to be preserved

  • Note: ITGA8 migrates differently under reducing (117.5 kDa) vs. non-reducing conditions (140-150 kDa) due to its glycosylation pattern

Western Blot Protocol:

• Load 20-50 μg protein per lane on SDS-PAGE (7.5% gel recommended for high MW proteins)

• Transfer to PVDF membrane (overnight at 30V for better transfer of high MW proteins)

• Block with 5% BSA or milk in TBST (1 hour)

• Incubate with biotin-conjugated anti-ITGA8 antibody (1:500-1:1000 dilution) in blocking buffer (overnight at 4°C)

• Wash 3× with TBST (10 minutes each)

• Incubate with streptavidin-HRP (1:2000-1:5000) for 1 hour

• Wash 3× with TBST (10 minutes each)

• Develop using enhanced chemiluminescence (ECL) substrate

• Image using digital acquisition system

Quantification Guidelines:

• Include a standard curve using recombinant ITGA8 protein if absolute quantification is needed

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

• Normalize ITGA8 band intensity to loading control

• Analyze using ImageJ or similar software for densitometry

• Perform at least three biological replicates for statistical validity

Critical Considerations:

• Expected molecular weight should be 117.5 kDa for human ITGA8, but glycosylation may cause higher apparent molecular weight

• Validate antibody specificity using known positive controls (e.g., kidney tissue lysates)

• Test samples from multiple tissues or cell lines to confirm specificity, as demonstrated with HeLa, Raw264.7, and PC12 cell lysates

This approach provides reliable quantification of ITGA8 expression while leveraging the sensitivity of biotin-streptavidin detection systems.

What is the recommended experimental design for investigating ITGA8-mediated cell adhesion using biotin-conjugated antibodies?

A comprehensive experimental design for investigating ITGA8-mediated cell adhesion includes:

Cell Adhesion Assay:

• Substrate preparation:

  • Coat 96-well plates with ITGA8 ligands (e.g., nephronectin, fibronectin)

  • Include BSA-coated wells as negative controls

  • Block non-specific binding with 1% BSA (1 hour at 37°C)

• Cell preparation:

  • Harvest cells expressing ITGA8 (e.g., renal cells)

  • Label with fluorescent dye (e.g., Calcein-AM)

  • Pre-incubate subset of cells with biotin-conjugated anti-ITGA8 antibody (10-50 μg/mL) to block ITGA8-mediated adhesion

• Adhesion procedure:

  • Add cells to coated wells (30,000-50,000 cells/well)

  • Allow adhesion for 30-60 minutes at 37°C

  • Wash gently to remove non-adherent cells

  • Quantify adherent cells by fluorescence measurement

Functional Validation:

• Antibody blocking studies:

  • Compare adhesion of untreated cells vs. cells treated with biotin-conjugated anti-ITGA8 antibodies

  • Include isotype control antibodies to confirm specificity

  • Test concentration-dependent effects (dose-response curve)

• Signaling pathway analysis:

  • Examine phosphorylation of focal adhesion kinase (FAK) and other downstream molecules

  • Compare cells adhering to ITGA8 ligands vs. control substrates

  • Assess how biotin-conjugated anti-ITGA8 antibodies affect these signaling events

• Co-localization studies:

  • Perform immunofluorescence on adherent cells using:

    • Biotin-conjugated anti-ITGA8 antibody with streptavidin-fluorophore

    • Antibodies against focal adhesion components (vinculin, paxillin)

  • Analyze co-localization at adhesion sites

Data Analysis Framework:

This experimental design allows for comprehensive characterization of ITGA8's role in cell adhesion while leveraging the biotin-conjugated antibodies for both functional blocking and detection purposes.

How can researchers address non-specific binding issues when using biotin-conjugated ITGA8 antibodies in tissues with high endogenous biotin?

Non-specific binding due to endogenous biotin is a common challenge when using biotin-conjugated antibodies. Here's a systematic approach to address this issue:

Prevention Strategies:

• Endogenous biotin blocking:

  • Implement an avidin-biotin blocking step before antibody incubation

  • Incubate tissue sections with unconjugated avidin (15-30 minutes)

  • Follow with a biotin solution (15-30 minutes) to saturate avidin binding sites

  • Wash thoroughly before proceeding with antibody incubation

• Alternative detection systems:

  • Consider directly labeled primary antibodies instead of biotin-conjugated ones

  • Use zenon labeling technology to temporarily label the primary antibody

  • Employ non-biotin amplification systems (e.g., polymer-based detection)

• Sample-specific optimizations:

  • Reduce primary antibody concentration (starting with 1:1000 dilution)

  • Shorten incubation times for streptavidin reagents

  • Use streptavidin conjugates with minimal batch-to-batch variation

Diagnostic Approaches:

• Control experiments to identify the source of background:

  • Omit primary antibody but include streptavidin detection reagent

  • Include tissue sections known to be high or low in endogenous biotin

  • Test pre-treatment with enzymes that digest endogenous biotin-containing proteins

• Tissue-specific considerations:

  • Kidney tissues (where ITGA8 is highly expressed) often contain endogenous biotin

  • Adjust blocking protocols based on the specific tissue being examined

Validation Framework:

Implementing these strategies systematically will help researchers distinguish between specific ITGA8 staining and non-specific background, even in challenging tissues with high endogenous biotin.

What are the potential causes and solutions for signal variability when detecting ITGA8 using biotin-conjugated antibodies?

Signal variability is a common challenge when working with ITGA8 detection. Understanding the causes and implementing appropriate solutions ensures consistent and reliable results:

Biological Sources of Variability:

• ITGA8 expression heterogeneity:

  • ITGA8 expression varies significantly between tissues and cell types

  • Expression levels depend on developmental stage and physiological conditions

  • Solution: Include positive controls (kidney tissue) in each experiment to normalize between runs

• Post-translational modifications:

  • Glycosylation affects ITGA8 detection and may vary between samples

  • Phosphorylation status may impact epitope accessibility

  • Solution: Use antibodies targeting non-modified regions or consistent sample preparation methods

• Heterodimer formation:

  • ITGA8 forms heterodimers with ITGB1, affecting epitope accessibility

  • Solution: Standardize lysis conditions to maintain consistent heterodimer integrity

Technical Sources of Variability:

• Antibody-related factors:

  • Lot-to-lot variations in biotin conjugation efficiency

  • Freeze-thaw cycles affecting antibody activity

  • Solution: Purchase sufficient quantity of single lot; aliquot upon receipt to avoid freeze-thaw cycles

• Detection system variability:

  • Inconsistent streptavidin conjugate performance

  • Variable development times in colorimetric detection

  • Solution: Standardize reagent concentrations and incubation times

• Sample preparation inconsistencies:

  • Variable fixation affecting epitope preservation

  • Differences in antigen retrieval efficiency

  • Solution: Develop and strictly follow standardized protocols

Systematic Approach to Reducing Variability:

By systematically addressing these variables, researchers can achieve more consistent and reliable ITGA8 detection across experiments and between laboratories.

How should researchers interpret discrepancies between ITGA8 protein expression detected by biotin-conjugated antibodies versus mRNA expression data?

Discrepancies between protein and mRNA expression are common in biological systems and require careful interpretation, particularly for proteins like ITGA8:

Common Causes of Protein-mRNA Discrepancies:

• Post-transcriptional regulation:

  • miRNA-mediated suppression of ITGA8 translation

  • Variations in mRNA stability and translational efficiency

  • Solution: Examine miRNA profiles in tissues of interest to identify potential regulators

• Post-translational regulation:

  • Protein degradation rates affecting steady-state levels

  • Protein stability differences between tissues

  • Solution: Use proteasome inhibitors to assess degradation contribution

• Technical limitations:

  • Antibody epitope accessibility affected by protein conformation or interactions

  • Differences in detection sensitivity between protein and mRNA methods

  • Solution: Use multiple antibodies targeting different ITGA8 epitopes

• Biological compartmentalization:

  • Secretion or sequestration of ITGA8 in specific cellular compartments

  • Differential translation in subcellular locations

  • Solution: Combine immunostaining with subcellular fractionation

Analytical Framework for Resolving Discrepancies:

• Systematic comparison:

  • Quantify ITGA8 protein using both biotin-conjugated antibodies and alternative detection methods

  • Compare with mRNA quantification by RT-qPCR and RNA-seq

  • Calculate protein-mRNA correlation coefficients across samples

• Time-course studies:

  • Examine temporal relationships between mRNA and protein expression

  • Assess whether protein expression follows mRNA with a time delay

  • Determine protein half-life through pulse-chase experiments

• Tissue-specific considerations:

  • ITGA8 expression in kidney may have different protein-mRNA correlation than in other tissues

  • Solution: Analyze tissue-specific factors that might influence post-transcriptional regulation

Decision Tree for Interpreting Discrepancies:

By applying this systematic approach, researchers can determine whether discrepancies represent meaningful biological phenomena or technical limitations, leading to more accurate interpretations of ITGA8 expression patterns across experimental systems.

What are the optimized protocols for detecting ITGA8 in single-cell applications using biotin-conjugated antibodies?

Single-cell analysis of ITGA8 requires specialized approaches to maximize sensitivity while maintaining specificity:

Flow Cytometry Protocol:

• Cell preparation:

  • Harvest cells using enzyme-free dissociation buffer to preserve surface integrins

  • Filter through 40 μm strainer to obtain single-cell suspension

  • Resuspend in flow buffer (PBS with 2% FBS, 1 mM EDTA)

• Staining procedure:

  • Block Fc receptors with 10% normal serum (10 minutes)

  • Incubate with biotin-conjugated anti-ITGA8 antibody (5-10 μg/mL) for 30 minutes on ice

  • Wash twice with flow buffer

  • Incubate with fluorophore-conjugated streptavidin (1:200-1:500) for 30 minutes on ice

  • Wash twice with flow buffer

  • Add viability dye (e.g., 7-AAD) before analysis

• Controls and validation:

  • Include isotype control matched to anti-ITGA8 antibody

  • Use known ITGA8-positive and negative cell populations

  • Perform fluorescence-minus-one (FMO) controls

Single-Cell Imaging Cytometry:

• Sample preparation:

  • Prepare single-cell suspension as above

  • Cytospin onto slides or use imaging flow cytometry platform

• Staining approach:

  • Block with 10% normal serum (30 minutes)

  • Incubate with biotin-conjugated anti-ITGA8 (10 μg/mL) for 1-2 hours

  • Wash three times

  • Detect with fluorophore-conjugated streptavidin (1:200) for 30 minutes

  • Counterstain nuclei with DAPI

• Analysis considerations:

  • Quantify membrane vs. cytoplasmic ITGA8 localization

  • Correlate with cell morphology parameters

  • Measure co-expression with other markers

Single-Cell Sequencing Integration:

• CITE-seq adaptation:

  • Conjugate anti-ITGA8 antibodies to DNA barcodes instead of biotin

  • Follow standard CITE-seq protocols for simultaneous protein and RNA detection

  • Analyze correlation between ITGA8 protein and mRNA at single-cell level

• Data integration framework:

  • Correlate ITGA8 protein levels with transcriptional signatures

  • Identify cell subpopulations based on ITGA8 expression patterns

  • Map ITGA8-expressing cells in tissue context

This comprehensive approach enables researchers to study ITGA8 expression and localization with single-cell resolution, providing insights into cellular heterogeneity and function.

How can researchers leverage biotin-conjugated ITGA8 antibodies for proximity ligation assays to study protein-protein interactions?

Proximity Ligation Assay (PLA) offers a powerful approach for studying ITGA8 interactions with other proteins in situ. Here's how to optimize this technique using biotin-conjugated ITGA8 antibodies:

PLA Protocol Optimization:

• Primary antibody selection:

  • Biotin-conjugated anti-ITGA8 antibody (rabbit host)

  • Anti-interacting protein antibody (different host species, e.g., mouse)

  • Ensure antibodies recognize spatially distinct epitopes that don't interfere with the interaction

• Sample preparation:

  • Fix cells or tissues (4% paraformaldehyde, 10 minutes)

  • Permeabilize if needed (0.1% Triton X-100, 10 minutes)

  • Block with Duolink blocking solution (1 hour)

• PLA procedure:

  • Incubate with biotin-conjugated anti-ITGA8 (10 μg/mL) and anti-partner antibody overnight at 4°C

  • Wash 3× with Buffer A

  • Incubate with streptavidin-conjugated PLA probe and secondary antibody-conjugated PLA probe (1 hour at 37°C)

  • Proceed with ligation and amplification steps according to manufacturer's protocol

  • Counterstain and mount

Interaction Analysis Framework:

• Key ITGA8 interaction partners to investigate:

  • ITGB1 (forms functional heterodimer)

  • Extracellular matrix components (fibronectin, nephronectin)

  • Signaling molecules (FAK, ILK, Src)

• Controls and validation:

  • Negative control: Omit one primary antibody

  • Positive control: Known interacting proteins (e.g., ITGA8-ITGB1)

  • Competition control: Pre-incubate with immunizing peptide

• Quantification approach:

  • Count PLA puncta per cell

  • Measure distance of PLA signals from cell membrane

  • Correlate PLA signal intensity with functional outcomes

Advanced Applications:

• Multi-protein complex analysis:

  • Three-way PLA using biotin-conjugated anti-ITGA8 plus two other antibodies

  • Map spatial organization of ITGA8-containing adhesion complexes

• Conformational state detection:

  • Design PLA to detect specific ITGA8 conformational states

  • Study activation-dependent interactions

• Tissue-specific interaction mapping:

  • Apply PLA to tissue sections to map ITGA8 interactions in physiological context

  • Compare interaction patterns between normal and diseased tissues

This approach provides valuable insights into ITGA8's molecular interactions and signaling networks in their native cellular context.