PURA Antibody, Biotin conjugated

What is a PURA antibody with biotin conjugation?

A PURA antibody with biotin conjugation is an immunoglobulin that specifically recognizes and binds to the Purine-Rich Element Binding Protein A (PURA) and has been chemically modified through the attachment of biotin molecules. This conjugation enables detection through biotin-streptavidin interactions, which is widely utilized in research applications. For example, the PURA antibody targeting amino acids 131-230 (ABIN6982721) is a polyclonal antibody produced in rabbits that has been conjugated to biotin . The biotin tag serves as an affinity tag that can be detected by streptavidin, which has an extremely high binding affinity for biotin, allowing for signal amplification and improved detection sensitivity .

What are the primary applications for PURA biotin-conjugated antibodies?

PURA biotin-conjugated antibodies are versatile tools that can be employed in multiple research applications. According to the product specifications, these antibodies are suitable for ELISA, Immunohistochemistry on paraffin-embedded sections (IHC-P), and Immunohistochemistry on frozen sections (IHC-fro) . The biotin conjugation enhances detection sensitivity through signal amplification with streptavidin systems . This makes these antibodies particularly valuable for detecting low abundance PURA protein in complex biological samples. Additionally, biotin-conjugated antibodies can be used in proximity labeling experiments, where they help identify protein-protein interactions and protein localization within cellular compartments .

How does detection sensitivity differ between direct and indirect methods when using PURA biotin-conjugated antibodies?

Detection sensitivity varies significantly between direct and indirect methods when using PURA biotin-conjugated antibodies. In direct detection, the biotin-conjugated PURA antibody binds directly to the target antigen, followed by addition of a streptavidin-conjugated reporter molecule (fluorophore or enzyme). This approach offers a streamlined workflow but may provide limited signal amplification. In contrast, indirect detection employs a two-step process where an unconjugated primary PURA antibody binds to the target antigen, followed by detection with a biotin-conjugated secondary antibody . The indirect method can increase assay sensitivity through signal amplification without risking damage to the antigen recognition site on the primary antibody . Studies have shown that antibody-based enrichment of biotinylated peptides yields 30-fold more biotinylation sites compared to streptavidin-based biotinylated protein enrichment, demonstrating the superior sensitivity of antibody-based approaches in certain applications .

What are the optimal conditions for using PURA biotin-conjugated antibodies in immunohistochemistry?

For optimal immunohistochemistry results with PURA biotin-conjugated antibodies, several critical parameters must be considered:

• Tissue preparation: For paraffin-embedded sections (IHC-P), complete deparaffinization and proper antigen retrieval (typically heat-induced in citrate buffer pH 6.0 or EDTA buffer pH 9.0) are essential.

• Antibody concentration: Titration experiments should be performed to determine the optimal antibody concentration, typically starting with the manufacturer's recommended dilution (e.g., 1:100 to 1:500).

• Incubation conditions: Primary antibody incubation is typically performed overnight at 4°C or for 1-2 hours at room temperature in a humidified chamber.

• Detection system: A streptavidin-conjugated reporter system (e.g., streptavidin-HRP or streptavidin-fluorophore) is used following primary antibody incubation.

• Controls: Include positive and negative controls. For PURA antibodies, tissues known to express PURA protein should be used as positive controls, while omission of primary antibody serves as a negative control.

The PURA antibody (AA 131-230) with biotin conjugation has been validated for both paraffin-embedded and frozen section immunohistochemistry , making it versatile for different sample preparation methods.

How can PURA biotin-conjugated antibodies be effectively utilized in ELISA protocols?

Effective utilization of PURA biotin-conjugated antibodies in ELISA requires careful optimization of multiple parameters:

• Coating concentration: When using a capture antibody system, optimize the coating concentration of the capture antibody (typically 1-10 μg/mL).

• Sample preparation: Proper sample dilution in appropriate buffers (typically PBS with 0.05% Tween-20 and 1% BSA) to minimize background signal.

• Antibody dilution: Optimal dilution of the PURA biotin-conjugated antibody should be determined through titration experiments.

• Detection system: Utilize streptavidin conjugated to enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP) for signal generation . HRP acts on substrates like TMB for colorimetric detection, while AP acts on substrates like NPP .

• Signal development and measurement: Allow for appropriate substrate development time and measure signal using a spectrophotometer at the appropriate wavelength.

The advantage of using biotin-conjugated PURA antibodies in ELISA is the potential for signal amplification through the high-affinity biotin-streptavidin interaction, which can improve sensitivity for detecting low levels of PURA protein.

How should researchers validate the specificity of PURA biotin-conjugated antibodies?

Validation of PURA biotin-conjugated antibody specificity is crucial for reliable experimental results. A comprehensive validation approach should include:

• Positive and negative controls: Use tissues or cell lines known to express or lack PURA, respectively.

• Knockdown/knockout validation: Compare antibody signal in wild-type versus PURA knockdown or knockout samples to confirm specificity.

• Western blot analysis: Verify that the antibody detects a band of the expected molecular weight for PURA (approximately 35 kDa).

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide (in this case, a peptide corresponding to AA 131-230 of human PURA ) to demonstrate specific binding.

• Cross-reactivity assessment: Test the antibody against closely related proteins (e.g., PURB) to confirm target specificity.

• Multiple antibody comparison: Compare results with other PURA antibodies targeting different epitopes to ensure consistency.

It's worth noting that the PURA antibody (AA 131-230) is generated using a KLH conjugated synthetic peptide derived from human PURA , which should be considered when assessing potential cross-reactivity.

What controls should be included when using PURA biotin-conjugated antibodies in immunoassays?

When designing experiments with PURA biotin-conjugated antibodies, the following controls should be included:

• Primary antibody controls:

  • Positive control: Sample known to express PURA

  • Negative control: Sample known to lack PURA expression

  • Isotype control: Non-specific IgG (same isotype as the PURA antibody, which is IgG ) to assess non-specific binding

• Technical controls:

  • No primary antibody control: Omit primary antibody to assess background from secondary detection reagents

  • Biotin blocking control: Block endogenous biotin to prevent false-positive signals

  • Concentration gradient: Include a dilution series of the antibody to determine optimal concentration

• Specificity controls:

  • Peptide competition: Pre-incubate antibody with the immunizing peptide to block specific binding

  • PURA knockdown/knockout: Compare results in samples with reduced or eliminated PURA expression

• Signal development controls:

  • Substrate-only control: Assess background from detection reagents

  • Time course: Monitor signal development over time to determine optimal detection point

Including these controls helps distinguish specific signal from background and validates the reliability of experimental findings.

How does the choice of biotin conjugation affect PURA antibody performance in different applications?

The biotin conjugation method and degree of labeling significantly impact PURA antibody performance across different applications:

• Conjugation chemistry: Different biotin derivatives (NHS-biotin, maleimide-biotin, etc.) target different functional groups on antibodies (lysines vs. cysteines), affecting epitope accessibility. For PURA antibodies, the conjugation method should preserve the antigen-binding region integrity.

• Degree of labeling (DOL): Higher DOL increases detection sensitivity but may compromise antigen binding if biotin molecules are attached near the antigen-binding site. Optimal DOL typically ranges from 3-8 biotin molecules per antibody.

• Application-specific considerations:

  • For IHC: Moderate biotinylation is preferred to maintain tissue penetration while providing sufficient signal

  • For ELISA: Higher biotinylation can be beneficial for increased sensitivity

  • For Western blotting: Moderate biotinylation balances sensitivity and specificity

• Detection system compatibility: Different streptavidin conjugates (HRP, fluorophores, etc.) require different optimal biotin densities on the antibody.

The PURA antibody (AA 131-230) with biotin conjugation has been validated for multiple applications including ELISA and IHC , suggesting an appropriate biotin conjugation level for these diverse applications.

How can PURA biotin-conjugated antibodies be utilized in proximity labeling experiments?

PURA biotin-conjugated antibodies can be powerful tools in proximity labeling experiments to study protein-protein interactions and subcellular localization:

• BioID approach: PURA biotin-conjugated antibodies can be used to validate results from BioID experiments where a biotin ligase is fused to PURA to identify proximity partners.

• APEX proximity labeling: When combined with APEX peroxidase systems, biotinylated proteins can be enriched using anti-biotin antibodies for mass spectrometry analysis. This approach has shown a remarkable 30-fold increase in identification of biotinylation sites compared to streptavidin-based methods .

• Protein complex identification: Sequential immunoprecipitation using PURA antibodies followed by biotin-based purification can help identify stable protein complexes associated with PURA.

• ChIP-seq applications: Biotin-conjugated PURA antibodies can be used in chromatin immunoprecipitation followed by sequencing (ChIP-seq) to identify genomic binding sites of PURA, which is known to bind purine-rich elements.

• Super-resolution microscopy: The biotin-streptavidin system provides excellent signal amplification for detecting PURA localization with nanometer precision in super-resolution microscopy approaches.

Research has shown that anti-biotin antibody enrichment yields significantly more biotinylated peptides than traditional streptavidin-based enrichment methods, with one study identifying over 1,600 biotinylation sites compared to only 151 sites using streptavidin-based protein enrichment .

What are the advantages of using anti-biotin antibodies versus streptavidin for detection of biotin-conjugated PURA antibodies?

Anti-biotin antibodies offer several distinct advantages over streptavidin for detection of biotin-conjugated PURA antibodies:

Research has demonstrated that anti-biotin antibodies enable "unprecedented enrichment of biotinylated peptides from complex peptide mixtures" . This makes them particularly valuable for applications requiring site-specific identification of biotinylation, such as in proximity labeling experiments using APEX peroxidase followed by mass spectrometry .

How can mass spectrometry be integrated with PURA biotin-conjugated antibody techniques for protein interaction studies?

Integration of mass spectrometry with PURA biotin-conjugated antibody techniques enables comprehensive protein interaction studies:

• Sample preparation:

  • Cells expressing PURA are treated with proximity labeling enzymes (e.g., APEX2) and biotin-phenol

  • Alternatively, PURA biotin-conjugated antibodies can be used for immunoprecipitation

  • Proteins are digested into peptides using trypsin or other proteases

• Enrichment strategy:

  • Anti-biotin antibodies are used to enrich biotinylated peptides (50 μg antibody per 1 mg peptide input is optimal)

  • Enrichment is performed in buffer conditions of 50 mM MOPS pH 7.2, 10 mM sodium phosphate, and 50 mM NaCl with end-over-end rotation for 1 hour at 4°C

• Mass spectrometry analysis:

  • Enriched peptides are analyzed by LC-MS/MS

  • Data is processed to identify PURA-interacting proteins

  • Biotinylation sites provide direct evidence of proximity

• Data interpretation:

  • Proteins identified in multiple replicates are considered high-confidence interactors

  • Biotinylation sites can provide insights into protein topology and interaction interfaces

Studies have shown that anti-biotin antibody enrichment followed by mass spectrometry can yield over 1,600 biotinylation sites on hundreds of proteins, representing a >30-fold increase compared to streptavidin-based protein enrichment methods . This approach provides direct evidence of proximity labeling, potentially offering additional information on protein topologies and identifying proteins that might be missed using traditional streptavidin enrichment .

What are common sources of background when using PURA biotin-conjugated antibodies and how can they be mitigated?

Several factors can contribute to background when using PURA biotin-conjugated antibodies:

• Endogenous biotin interference:

  • Problem: Many tissues contain endogenous biotin that can bind to streptavidin detection reagents

  • Solution: Use commercial biotin blocking kits or pre-incubate samples with free streptavidin followed by free biotin

• Non-specific antibody binding:

  • Problem: The polyclonal nature of some PURA antibodies may lead to non-specific binding

  • Solution: Optimize blocking conditions (5% BSA or 5-10% normal serum from the same species as the secondary reagent); use more stringent washing buffers (increasing salt concentration or adding 0.1-0.3% Triton X-100)

• Cross-reactivity with related proteins:

  • Problem: PURA antibodies may cross-react with related proteins such as PURB

  • Solution: Validate antibody specificity using knockout/knockdown controls; consider using monoclonal alternatives for higher specificity

• Inappropriate detection system:

  • Problem: Excessive signal amplification leading to high background

  • Solution: Titrate detection reagents; consider using fluorescent rather than enzymatic detection for better signal-to-noise ratio

• Tissue autofluorescence or endogenous peroxidase activity:

  • Problem: Interference with detection systems

  • Solution: For IHC, quench endogenous peroxidase with H₂O₂ treatment; for fluorescence, use specialized quenching reagents or spectral unmixing

Careful optimization of antibody concentration is critical, as the polyclonal nature of the PURA antibody (AA 131-230) may contribute to background if used at excessive concentrations.

How can researchers optimize the signal-to-noise ratio when working with PURA biotin-conjugated antibodies?

Optimizing signal-to-noise ratio for PURA biotin-conjugated antibodies requires systematic optimization of multiple parameters:

• Antibody titration:

  • Perform serial dilutions of the PURA biotin-conjugated antibody to determine the optimal concentration that maximizes specific signal while minimizing background

  • For the PURA antibody (AA 131-230), start with the manufacturer's recommended dilution and test at least 3-4 dilutions above and below this concentration

• Blocking optimization:

  • Test different blocking agents (BSA, normal serum, commercial blockers) at varying concentrations

  • Add 0.1-0.3% Tween-20 to blocking buffers to reduce hydrophobic interactions

• Incubation conditions:

  • Compare room temperature versus 4°C incubation

  • Test different incubation times to optimize signal development

• Washing protocols:

  • Increase wash buffer volume and duration

  • Add detergents (0.05-0.1% Tween-20) to wash buffers

  • Consider using automated washers for consistent results

• Detection system selection:

  • For enzymatic detection, compare different substrates (TMB, DAB for HRP; BCIP/NBT for AP)

  • For fluorescent detection, select fluorophores with minimal spectral overlap with sample autofluorescence

• Signal amplification methods:

  • For low-abundance targets, employ tyramide signal amplification or other amplification techniques

  • When using enzyme conjugates, optimize substrate development time to achieve optimal signal before background development

Systematic optimization using a matrix approach, where multiple parameters are varied simultaneously, can efficiently identify optimal conditions for specific experimental systems.

What strategies can address cross-reactivity issues with PURA biotin-conjugated antibodies?

Cross-reactivity of PURA biotin-conjugated antibodies can complicate experimental interpretation. Several strategies can address this challenge:

• Epitope selection and antibody validation:

  • Choose antibodies targeting unique regions of PURA with minimal homology to related proteins

  • The PURA antibody (AA 131-230) targets a specific region ; compare its sequence homology with potential cross-reactive proteins

• Absorption controls:

  • Pre-absorb antibodies with recombinant proteins of potential cross-reactive targets

  • Perform peptide competition assays with both target and related protein peptides

• Genetic validation approaches:

  • Use PURA knockout/knockdown systems as definitive controls

  • Compare antibody reactivity in these systems to identify non-specific signals

• Orthogonal detection methods:

  • Validate findings using alternative PURA antibodies targeting different epitopes

  • Complement antibody-based detection with nucleic acid-based methods (e.g., RNA-seq)

• Species-specific considerations:

  • When working across species, align the target epitope sequence (AA 131-230) across species to predict potential cross-reactivity

  • The PURA antibody has confirmed reactivity with Mouse and Zebrafish and predicted reactivity with multiple other species

• Application-specific optimizations:

  • For immunohistochemistry: Use antigen retrieval optimization and more stringent washing

  • For Western blotting: Increase membrane blocking time and detergent concentration

  • For ELISA: Employ sandwich formats with two different PURA antibodies targeting distinct epitopes

By systematically addressing cross-reactivity through these approaches, researchers can increase confidence in the specificity of their PURA biotin-conjugated antibody results.