p37 Antibody, Biotin conjugated

What are the primary p37 proteins targeted by research antibodies?

Research antibodies target two main p37 proteins with distinct biological functions. The first is the p37 subunit of Replication Factor C (RFC), a critical component of DNA replication machinery. RFC is a heteropentameric protein consisting of p140, p40, p38, p37, and p36 subunits that participates in targeting DNA polymerase delta processivity factor (PCNA) onto primed DNA templates during DNA synthesis and repair . The second major target is the p37 protein of Mycoplasma hyorhinis, a cell surface-associated protein with a molecular mass of approximately 40 kDa that serves as a highly specific marker for M. hyorhinis infection . Understanding which p37 protein your antibody targets is essential for experimental design and interpretation of results.

What validation criteria should be applied before using biotin-conjugated p37 antibodies?

A comprehensive validation approach for biotin-conjugated p37 antibodies should include:

• Specificity testing: Comparative Western blot analysis using samples with varying p37 expression levels

• Sensitivity assessment: Titration experiments to determine optimal working concentrations

• Cross-reactivity evaluation: Testing against related proteins within the experimental system

• Application-specific validation: Function-specific testing for intended applications (e.g., immunoprecipitation, flow cytometry)

• Lot-to-lot consistency verification: Particularly important for long-term studies

For anti-Replication Factor C p37 antibodies, validation should include testing against both human and yeast (S. cerevisiae) samples to confirm the expected cross-reactivity . For anti-Mycoplasma p37 antibodies, validation should demonstrate specific binding to M. hyorhinis-infected cells but not to uninfected cells or cells infected with other Mycoplasma species .

What dilution ranges are optimal for biotin-conjugated p37 antibodies in different applications?

Optimal dilution ranges vary by application and specific antibody preparation. Based on available data for unconjugated p37 antibodies that can guide biotin-conjugated versions:

For biotin-conjugated versions, initial titration experiments starting at these ranges are recommended, with potential adjustment to 1.5-2× higher concentrations if signal strength is reduced compared to unconjugated antibodies .

How can biotin-conjugated p37 antibodies be effectively used in multi-color flow cytometry?

Multi-color flow cytometry with biotin-conjugated p37 antibodies requires strategic panel design to prevent fluorophore overlap and maximize signal separation:

• Use streptavidin conjugates with bright fluorophores (e.g., PE, APC) for detection of biotinylated p37 antibodies

• Implement a sequential staining protocol:

  • First apply all direct fluorophore-conjugated antibodies

  • Wash thoroughly to remove unbound antibodies

  • Apply the biotin-conjugated p37 antibody

  • Wash again thoroughly

  • Apply the streptavidin-fluorophore conjugate

• Include appropriate controls:

  • FMO (Fluorescence Minus One) control excluding the biotin-p37 antibody

  • Isotype-biotin control with matching streptavidin-fluorophore

  • Single-color compensation controls

This approach is particularly valuable for detecting p37 from M. hyorhinis in infection studies, where distinguishing infected from uninfected cells in heterogeneous populations is critical .

What are the critical buffer considerations when using biotin-conjugated p37 antibodies?

Buffer composition significantly impacts the performance of biotin-conjugated p37 antibodies. Consider these factors:

• Avoid biotin-containing buffers: Exclude biotin, BSA, or serum that may contain biotin from all buffers used with streptavidin detection systems

• Optimize blocking conditions: When detecting Replication Factor C p37, use 3-5% BSA in PBS for Western blotting applications

• pH considerations: Maintain pH 7.2-7.4 for optimal antibody-antigen binding

• Detergent selection: Use mild detergents (0.05% Tween-20 or 0.1% Triton X-100) to reduce background without affecting epitope recognition

• Salt concentration: 150mM NaCl typically provides optimal ionic strength, but may require adjustment for specific applications

For Mycoplasma p37 detection, researchers have successfully employed 5% BSA/DPBS buffer for blocking followed by antibody incubation in binding buffer at 200nM concentration .

How can biotin-conjugated p37 antibodies be employed in ChIP-seq experiments investigating replication factor dynamics?

ChIP-seq experiments using biotin-conjugated anti-RFC p37 antibodies offer several advantages for studying replication dynamics:

• Protocol optimization:

  • Crosslinking: 1% formaldehyde for 10 minutes at room temperature

  • Sonication: Adjust to generate 200-500bp DNA fragments

  • Immunoprecipitation: Use streptavidin magnetic beads for capture

  • Elution: Employ biotin competition or targeted protease digestion to release DNA-protein complexes

• Data analysis considerations:

  • Compare binding profiles with other replication factors (PCNA, RFC140)

  • Correlate with replication timing domains

  • Analyze enrichment at origins of replication versus elongating regions

• Validation approaches:

  • Perform parallel ChIP with antibodies against other RFC subunits

  • Conduct sequential ChIP (Re-ChIP) to identify co-occupancy with DNA polymerases

  • Compare results with nascent DNA synthesis mapping (e.g., EdU-seq)

This approach enables high-resolution mapping of RFC complex binding sites across the genome, providing insights into replication initiation and progression mechanisms .

What strategies exist for multiplexed detection of ubiquitinated proteins using biotin-conjugated p37 antibodies?

Advanced multiplexed detection of ubiquitinated proteins can be achieved through several sophisticated approaches:

• Multi-epitope targeting strategy:

  • Combine biotin-conjugated ubiquitin p37 antibody with antibodies recognizing different ubiquitin chain linkages (K48, K63, etc.)

  • Detect with spectrally distinct streptavidin conjugates and secondary antibodies

  • Analyze co-localization to determine ubiquitin chain topology on target proteins

• Proximity ligation assay (PLA) implementation:

  • Use biotin-conjugated ubiquitin p37 antibody with an antibody against a protein of interest

  • Apply streptavidin-oligonucleotide and secondary antibody-oligonucleotide conjugates

  • Perform rolling circle amplification and fluorescent probe hybridization

  • Quantify discrete fluorescent spots representing ubiquitinated target proteins

• Mass cytometry (CyTOF) approach:

  • Conjugate p37 antibody with biotin and detect with metal-tagged streptavidin

  • Combine with metal-tagged antibodies against potential ubiquitinated proteins

  • Analyze single-cell data to identify correlations and cell populations

These methodologies provide complementary information about ubiquitin-proteasome pathway regulation of target proteins like IκB, p53, cdc25A, and Bcl-2 in processes such as cell cycle progression, differentiation, and apoptosis .

How can biotin-conjugated p37 antibodies be utilized in high-resolution microscopy techniques?

Biotin-conjugated p37 antibodies offer significant advantages for super-resolution microscopy through molecular amplification and flexible labeling:

• STORM/PALM microscopy implementation:

  • Apply biotin-conjugated p37 antibody at 1:500-1:1000 dilution

  • Detect with streptavidin conjugated to photoswitchable fluorophores (e.g., Alexa Fluor 647)

  • Optimize imaging buffer with oxygen scavenger system and reducing agent

  • Achieve 10-20nm resolution of p37 protein localization

• Expansion microscopy protocol:

  • Apply biotin-conjugated p37 antibody before or after sample embedding in expandable polymer

  • Detect with streptavidin-fluorophore after expansion

  • Achieve effective resolution improvement proportional to expansion factor (typically 4-10×)

• Correlative light-electron microscopy (CLEM):

  • Apply biotin-conjugated p37 antibody

  • Detect with streptavidin-gold nanoparticles for EM visualization

  • Correlate with fluorescence microscopy data using fiducial markers

These approaches enable precise subcellular localization of p37 proteins, revealing their spatial relationship with other components of the DNA replication machinery or Mycoplasma infection processes .

How can non-specific binding be reduced when using biotin-conjugated p37 antibodies?

Non-specific binding is a common challenge with biotin-conjugated antibodies that can be addressed through systematic optimization:

• Endogenous biotin blocking:

  • Pre-block samples with unconjugated streptavidin (10-50 μg/ml)

  • Apply commercial biotin blocking kits before antibody incubation

  • Use avidin followed by biotin blocking for tissues with high endogenous biotin

• Secondary detection optimization:

  • Titrate streptavidin conjugate concentration to minimize background

  • Include 0.1-0.5% BSA in detection buffer to reduce non-specific interactions

  • Consider using directly labeled primary antibodies for particularly problematic samples

• Sample-specific considerations:

  • For Mycoplasma p37 detection, treatment with trypsin or proteinase K can be used as a negative control to confirm specific cell surface binding

  • For RFC p37 detection in nuclear extracts, include additional washes with higher salt concentration (300-500mM NaCl) to reduce non-specific nuclear protein interactions

A systematic approach comparing multiple blocking conditions is recommended for new experimental systems to identify optimal conditions.

How should researcher address epitope masking concerns when using biotin-conjugated p37 antibodies?

Epitope masking can significantly impact experimental results and requires careful consideration:

• Antigen retrieval optimization for fixed samples:

  • For formaldehyde-fixed samples: Test heat-induced epitope retrieval (citrate buffer pH 6.0 or Tris-EDTA pH 9.0)

  • For alcohol-fixed samples: Enzymatic retrieval with proteinase K may be more effective

  • Compare multiple retrieval methods with positive control samples

• Detection system selection:

  • For Mycoplasma p37 epitopes potentially masked by host cell interactions, consider membrane permeabilization optimization

  • For RFC p37 detection in chromatin contexts, test chromatin fragmentation methods (sonication vs. enzymatic digestion)

• Competitive binding analysis:

  • Evaluate whether unlabeled and biotin-conjugated antibodies compete for the same epitope

  • Test whether biotin conjugation alters epitope accessibility compared to unconjugated antibody

Interestingly, research shows that anti-p37 antibody and nucleic acid aptamers targeting Mycoplasma p37 can bind simultaneously to different sites on the protein, suggesting multiple accessible epitopes that may be differently affected by biotin conjugation .

What are the best practices for storage and handling of biotin-conjugated p37 antibodies to maintain optimal activity?

Biotin-conjugated antibodies require specific storage and handling practices to maintain functionality:

• Storage recommendations:

  • Temperature: Store at -20°C for long-term storage or 4°C for short-term (1-2 weeks)

  • Avoid freeze-thaw cycles (aliquot upon receipt)

  • Protect from light to prevent photodegradation of both fluorophores and biotin

  • Do not aliquot the antibody as indicated for some p37 antibody preparations

• Buffer considerations:

  • Maintain pH stability (pH 7.2-7.4)

  • Include carrier protein (0.1-1.0% BSA or gelatin) for dilute solutions

  • Consider adding sodium azide (0.02-0.05%) as preservative for non-live cell applications

  • Avoid prolonged exposure to solutions containing competing biotin molecules

• Quality control practices:

  • Implement routine functional testing schedule for stored antibodies

  • Monitor signal-to-noise ratio in standard samples over time

  • Document lot information and prepare for potential lot-to-lot variation

Following these practices helps ensure consistent experimental results and extends the useful life of valuable biotin-conjugated p37 antibodies.

How can biotin-conjugated p37 antibodies contribute to studying Mycoplasma-induced oncogenic transformation?

Biotin-conjugated anti-Mycoplasma p37 antibodies offer powerful tools for investigating the mechanisms of Mycoplasma-induced oncogenic transformation:

• Temporal-spatial analysis of infection:

  • Track p37 protein localization during different stages of cellular transformation

  • Correlate p37 binding patterns with activation of oncogenic signaling pathways

  • Implement time-lapse imaging with biotin-conjugated antibodies and compatible live-cell streptavidin detection systems

• Receptor interaction studies:

  • Investigate p37-TLR4 interactions through proximity-based detection methods

  • Map binding domains through epitope competition assays

  • Characterize downstream signaling cascade activation using phospho-specific antibodies

• Therapeutic intervention assessment:

  • Compare p37 binding inhibition efficiency between antibodies and aptamers like ZY3A

  • Evaluate combinatorial approaches targeting multiple Mycoplasma virulence factors

  • Monitor changes in p37 expression and localization during experimental treatments

These approaches build upon findings that p37 protein from M. hyorhinis interacts with host cell TLR4 receptors, and that blocking this interaction can inhibit Mycoplasma-mediated effects on host cells .

What are the considerations for using biotin-conjugated p37 antibodies in proteomics workflows?

Integrating biotin-conjugated p37 antibodies into proteomics workflows requires careful planning and optimization:

• Immunoprecipitation protocol design:

  • Use streptavidin-coated magnetic beads for capture

  • Implement stringent washing to reduce non-specific binding

  • Develop efficient elution strategies (biotin competition or on-bead digestion)

  • Consider crosslinking antibodies to beads to prevent antibody contamination in MS samples

• Sample preparation optimization:

  • Evaluate detergent compatibility with downstream MS applications

  • Test enzymatic digestion efficiency for different sample types

  • Consider filter-aided sample preparation (FASP) for complex samples

• Data analysis considerations:

  • Implement appropriate controls for background subtraction

  • Apply interaction scoring algorithms to distinguish specific from non-specific interactions

  • Validate novel interactions through orthogonal methods

This approach is particularly valuable for identifying protein complexes associated with RFC p37 during DNA replication and repair, potentially revealing novel components of the replication machinery or regulatory factors .

How can machine learning approaches enhance image analysis of biotin-conjugated p37 antibody staining patterns?

Advanced machine learning methods can significantly improve the analysis of complex staining patterns:

• Convolutional neural network applications:

  • Train models to distinguish between specific subcellular localization patterns

  • Implement automated quantification of colocalization with other factors

  • Develop pattern recognition for different stages of DNA replication or Mycoplasma infection

• Multi-parametric analysis integration:

  • Combine image features with other experimental parameters (e.g., cell cycle phase, expression levels)

  • Implement dimensionality reduction techniques to identify key pattern determinants

  • Develop predictive models for cellular responses based on p37 localization patterns

• Workflow implementation considerations:

  • Standardize image acquisition parameters for consistent model application

  • Develop validation protocols using known positive and negative controls

  • Create user-friendly interfaces for researchers without computational expertise

These approaches enable extraction of subtle information from imaging data that may not be apparent through visual inspection alone, potentially revealing novel aspects of p37 protein biology in both DNA replication and Mycoplasma infection contexts .