PH1536 Antibody

What is PH1536 Antibody and what is its target specificity?

PH1536 Antibody (CSB-PA026349ZA01FHX) is a rabbit polyclonal antibody raised against recombinant Pyrococcus horikoshii PH1536 protein. It specifically targets the PH1536 protein from Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3) . As a polyclonal antibody, it recognizes multiple epitopes on the target antigen, which can provide robust detection even if some epitopes are modified or masked in experimental conditions.

The antibody is produced through immunization of rabbits with the recombinant PH1536 protein and is subsequently purified using Antigen Affinity methods to ensure high specificity. This IgG isotype antibody is provided in liquid form and is primarily intended for research applications in archaeal protein studies.

What are the optimal storage conditions for PH1536 Antibody?

PH1536 Antibody should be stored at either -20°C or -80°C upon receipt to maintain optimal activity and stability . Importantly, repeated freeze-thaw cycles should be avoided as they can lead to antibody denaturation and loss of binding capacity.

For optimal storage practices:

• Aliquot the antibody into smaller volumes based on expected usage to minimize freeze-thaw cycles

• Store aliquots in polypropylene tubes rather than glass to prevent protein adsorption

• Ensure proper temperature monitoring of freezers to avoid temperature fluctuations

• When handling, keep the antibody on ice and return to storage promptly

• Record date of receipt, aliquoting, and first use for each vial

The storage buffer (0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4) has been formulated to enhance stability during storage, with glycerol acting as a cryoprotectant to minimize damage during freezing .

How should PH1536 Antibody be validated before experimental use?

Before incorporating PH1536 Antibody into critical experiments, thorough validation is essential to ensure reliable results. A comprehensive validation approach should include:

• Positive control testing: Use the provided recombinant immunogen protein (200μg) as a positive control to confirm antibody functionality

• Negative control testing: Utilize the provided pre-immune serum (1ml) as a negative control to establish background levels and confirm specificity

• Concentration titration: Perform a dilution series to determine optimal antibody concentration for each application

• Cross-reactivity assessment: Test against related proteins to confirm specificity for PH1536

• Reproducibility testing: Perform replicate experiments to ensure consistent results

Similar to the approach used in validating anti-HPA-15b antibodies in clinical settings, testing on multiple days with different antigen preparations can help establish reliability and consistency of results .

What are the validated applications for PH1536 Antibody?

PH1536 Antibody has been validated for the following applications:

This antibody has been specifically tested and validated for ELISA and Western Blot applications to ensure identification of the PH1536 antigen . Each application requires specific optimization for maximum sensitivity and specificity.

For novel applications not listed above, preliminary validation experiments are strongly recommended, following similar principles to those employed in validating other research antibodies, such as the approach used for anti-SARS-CoV-2 or anti-PhtD antibodies, which required extensive testing to confirm specificity and functionality .

What is the recommended protocol for using PH1536 Antibody in Western Blotting?

For optimal Western Blot results with PH1536 Antibody, follow this methodological approach:

• Sample preparation:

  • Extract proteins from Pyrococcus horikoshii or recombinant systems expressing PH1536

  • Denature samples in SDS-PAGE loading buffer (containing β-mercaptoethanol) at 95°C for 5 minutes

  • Load 20-50μg of total protein per lane

• Gel electrophoresis and transfer:

  • Separate proteins on 10-12% SDS-PAGE

  • Transfer to PVDF or nitrocellulose membrane (0.45μm pore size)

  • Confirm transfer efficiency with reversible staining (Ponceau S)

• Blocking and antibody incubation:

  • Block membrane with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • Incubate with PH1536 Antibody (1:1000 dilution) in blocking buffer overnight at 4°C

  • Wash 3× with TBST, 5 minutes each

  • Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000) for 1 hour at room temperature

  • Wash 3× with TBST, 5 minutes each

• Detection and analysis:

  • Apply ECL substrate and image using appropriate detection system

  • Include positive control (recombinant immunogen) and molecular weight markers

  • Expected molecular weight should be verified against database information for PH1536

• Controls and validation:

  • Run pre-immune serum at the same dilution on a duplicate blot as a negative control

  • Include lysate lacking PH1536 as a specificity control

This protocol draws on standard Western blotting principles, similar to those used for other research antibodies, while accounting for the specific properties of the PH1536 Antibody .

How can PH1536 Antibody be optimized for ELISA assays?

To optimize PH1536 Antibody for ELISA applications, consider the following methodological approach:

• Plate preparation:

  • Coat high-binding 96-well plates with purified recombinant PH1536 protein (2-5μg/ml) in carbonate buffer (pH 9.6) overnight at 4°C

  • Alternatively, for indirect ELISA, coat with samples containing the target protein

• Blocking and antibody addition:

  • Block with 1-3% BSA in PBS for 1-2 hours at room temperature

  • Create a dilution series of PH1536 Antibody (starting from 1:500 to 1:10,000) in blocking buffer

  • Add 100μl of each dilution to appropriate wells and incubate for 2 hours at room temperature

  • Wash 4× with PBST

• Detection:

  • Add HRP-conjugated anti-rabbit secondary antibody (1:5000) for 1 hour at room temperature

  • Wash 4× with PBST

  • Develop with TMB substrate and read absorbance at 450nm after stopping reaction with H₂SO₄

• Optimization parameters:

  • Test multiple antibody concentrations to determine optimal signal-to-noise ratio

  • Vary blocking agents (BSA, casein, non-fat milk) to minimize background

  • Test different incubation times and temperatures

  • Optimize washing steps to reduce background while maintaining signal

• Quality control:

  • Always include a standard curve using recombinant PH1536 protein

  • Include wells with pre-immune serum as negative controls

  • Calculate coefficient of variation between replicate wells (target CV < 10%)

This approach incorporates best practices from antibody-based assay development, similar to those used in developing sensitive detection methods for other research antibodies .

What are common causes of false positives/negatives when using PH1536 Antibody?

When working with PH1536 Antibody, several factors can contribute to false results:

Causes of false positives:

• Cross-reactivity: As a polyclonal antibody, PH1536 Antibody may recognize epitopes on proteins structurally similar to PH1536. Always include appropriate negative controls and pre-immune serum comparisons .

• Non-specific binding: Insufficient blocking or inadequate washing can lead to high background. Optimize blocking conditions and consider adding 0.05-0.1% Tween-20 to wash buffers.

• Denatured protein interactions: The antibody may recognize normally inaccessible epitopes exposed in denatured proteins. Verify results using multiple detection methods.

• Secondary antibody issues: HRP-conjugated secondary antibodies can bind non-specifically to certain samples. Include secondary-only controls in each experiment.

Causes of false negatives:

• Epitope masking: Post-translational modifications or protein-protein interactions may block antibody access to epitopes. Consider different extraction or denaturation methods.

• Insufficient antigen: Low expression levels of PH1536 may result in signals below detection threshold. Increase sample concentration or use more sensitive detection methods.

• Antibody degradation: Improper storage or repeated freeze-thaw cycles can reduce antibody activity. Store according to recommendations and use fresh aliquots .

• Incompatible buffers: Certain buffer components may interfere with antibody-antigen binding. Test alternative buffer systems if negative results persist.

How can cross-reactivity issues with PH1536 Antibody be addressed?

Cross-reactivity is a common challenge with polyclonal antibodies like PH1536 Antibody. Here are methodological approaches to address this issue:

• Pre-absorption techniques:

  • Incubate the antibody with lysates lacking PH1536 (from related species) to absorb cross-reactive antibodies

  • Remove bound antibodies by centrifugation or affinity methods

  • Test the pre-absorbed antibody against your target sample

• Epitope competition assays:

  • Pre-incubate the antibody with excess recombinant PH1536 protein

  • Apply this mixture to your assay; specific signals should be significantly reduced

  • Non-specific signals that persist indicate cross-reactivity

• Validation across multiple assays:

  • Confirm results using orthogonal methods (e.g., if positive in Western blot, verify with ELISA)

  • Cross-reactivity patterns often differ between native and denatured detection methods

• Comparative analysis:

  • Test samples from PH1536-knockout or depleted systems as negative controls

  • Use recombinant PH1536 protein as positive control

  • Compare signal patterns and intensities between controls and test samples

• Increased stringency protocols:

  • Use higher dilutions of primary antibody (1:2000-1:5000)

  • Increase salt concentration in wash buffers (up to 500mM NaCl)

  • Add non-ionic detergents (0.1-0.3% Triton X-100) to reduce non-specific interactions

This approach draws on techniques similar to those used to validate the specificity of other research antibodies, like those developed for pneumococcal protein detection or platelet antigens .

What controls are essential when using PH1536 Antibody in experimental protocols?

Robust experimental design with PH1536 Antibody requires comprehensive controls:

Essential positive controls:

• Recombinant PH1536 protein: The supplied recombinant immunogen (200μg) serves as the ideal positive control, confirming antibody functionality

• Pyrococcus horikoshii lysate: Wild-type organism expressing native PH1536 provides validation in the natural context

• Concentration gradient: Serial dilutions of positive control samples establish the detection range and sensitivity

Essential negative controls:

• Pre-immune serum: The supplied pre-immune serum (1ml) establishes background and non-specific binding levels

• Secondary antibody only: Reveals background from secondary antibody binding

• Lysates from organisms lacking PH1536: Demonstrates specificity across species barriers

Procedural controls:

• Loading/extraction controls: Housekeeping proteins verify equal loading and extraction efficiency

• Blocking peptide competition: Antibody pre-incubated with excess target peptide should show reduced specific binding

• Technical replicates: Minimum of three replicates to establish reproducibility and calculate statistical significance

Application-specific controls:

This control strategy incorporates approaches similar to those used in validating other research antibodies, such as the anti-HPA-15b antibodies evaluated in international collaborative studies .

How can PH1536 Antibody be applied in archaeal protein research?

PH1536 Antibody offers valuable capabilities for investigating archaeal proteins, particularly those from hyperthermophilic organisms like Pyrococcus horikoshii. Methodological approaches include:

• Comparative proteomics:

  • Use PH1536 Antibody to track protein expression across growth conditions

  • Compare protein levels between wild-type and mutant strains

  • Investigate protein expression changes in response to environmental stressors

  • Correlate PH1536 levels with biochemical or phenotypic data

• Protein localization studies:

  • Employ immunofluorescence microscopy with PH1536 Antibody to determine subcellular localization

  • Combine with organelle markers to confirm localization patterns

  • Compare localization under different growth or stress conditions

  • Use cell fractionation followed by Western blotting to biochemically validate microscopy findings

• Protein-protein interaction analysis:

  • Perform co-immunoprecipitation with PH1536 Antibody to identify interaction partners

  • Use cross-linking approaches to capture transient interactions

  • Validate interactions through reciprocal pull-downs and mass spectrometry

  • Map interaction domains through truncated protein constructs

• Evolutionary biology investigations:

  • Examine PH1536 conservation across archaeal species using cross-reactivity testing

  • Correlate structural conservation with functional conservation

  • Investigate adaptive changes in protein structure across extremophiles

These approaches build upon strategies used for other research antibodies in microbial research, such as those developed for specific bacterial antigens , while accounting for the unique properties of archaeal systems.

What considerations are important when designing co-immunoprecipitation experiments with PH1536 Antibody?

Co-immunoprecipitation (Co-IP) with PH1536 Antibody requires careful methodological planning:

• Lysate preparation optimization:

  • Use gentle lysis buffers (e.g., 50mM Tris-HCl pH 7.5, 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate) to preserve protein-protein interactions

  • Include protease inhibitors (PMSF, leupeptin, aprotinin) to prevent degradation

  • Consider detergent selection carefully: NP-40 or Triton X-100 (0.5-1%) preserves most interactions, while harsher detergents may disrupt them

  • For thermophilic archaeal proteins, optimize lysis temperature to maintain native conformations

• Antibody coupling strategies:

  • Direct approach: Couple PH1536 Antibody to protein A/G beads or magnetic beads

  • Indirect approach: Add antibody to lysate, then capture with protein A/G beads

  • Pre-clear lysates with beads alone to reduce non-specific binding

  • Cross-link antibody to beads (using BS3 or DMP) to prevent antibody co-elution

• Control experiments:

  • Input control: Save a fraction of pre-IP lysate

  • Negative control: Pre-immune serum or irrelevant IgG

  • Reciprocal IP: If possible, IP with antibodies against suspected interaction partners

  • Validation: Confirm identified interactions using alternative methods (e.g., proximity ligation assay)

• Washing and elution optimization:

  • Optimize wash stringency to remove non-specific binders while retaining specific interactions

  • Test gradient of salt concentrations (150-500mM NaCl)

  • Consider competing peptide elution for increased specificity

  • For sensitive interactions, include stabilizing agents (e.g., 5-10% glycerol)

• Detection methods:

  • Western blot: Probe for specific suspected interaction partners

  • Mass spectrometry: For unbiased identification of all potential interactors

  • Functional assays: Test activity of co-immunoprecipitated complexes

This protocol incorporates best practices from co-IP methodologies used with other research antibodies, such as those applied in malaria research for protein complex identification .

How can epitope mapping be performed with PH1536 Antibody?

Epitope mapping with PH1536 polyclonal antibody requires specialized approaches to identify the specific binding regions:

• Peptide array analysis:

  • Generate overlapping peptides (15-20 amino acids) spanning the entire PH1536 protein sequence

  • Synthesize peptides on membranes or glass slides

  • Probe with PH1536 Antibody at optimized dilution (1:1000-1:3000)

  • Detect binding using HRP-conjugated secondary antibody and chemiluminescence

  • Positive signals identify epitope-containing regions

• Recombinant fragment analysis:

  • Create a series of truncated PH1536 protein constructs

  • Express and purify fragments using bacterial or cell-free systems

  • Perform Western blot analysis with PH1536 Antibody

  • Compare binding patterns to identify epitope-containing regions

  • Further refine by testing smaller sub-fragments

• Hydrogen/deuterium exchange mass spectrometry (HDX-MS):

  • Expose PH1536 protein to deuterated buffer with and without antibody binding

  • Monitor changes in deuteration patterns by mass spectrometry

  • Regions protected from exchange represent potential epitopes

  • Validate findings using site-directed mutagenesis

• Computational epitope prediction and validation:

  • Use algorithms to predict surface-exposed, hydrophilic regions of PH1536

  • Generate synthetic peptides based on predictions

  • Test antibody binding to these peptides via ELISA

  • Confirm findings through competitive binding assays

• Cross-competition assays:

  • For polyclonal antibodies like PH1536 Antibody, epitope clustering can be determined

  • Pre-incubate protein with one set of antibodies, then probe with labeled PH1536 Antibody

  • Reduced binding indicates overlapping epitopes

This approach draws on methodologies similar to those used for epitope mapping of other functional antibodies, such as those targeting malaria invasion proteins or pneumococcal antigens .

How does PH1536 Antibody compare to other methods for studying archaeal proteins?

When considering research approaches for archaeal proteins like PH1536, multiple methodologies can be evaluated:

This comparative analysis helps researchers select complementary methods for comprehensive study of archaeal proteins, similar to the multi-method approaches used in other microbial research fields .

What alternative approaches can complement PH1536 Antibody-based detection?

To build a comprehensive understanding of PH1536 protein, researchers should consider these complementary approaches:

• Transcriptomic analysis:

  • RT-qPCR to quantify PH1536 mRNA levels

  • RNA-Seq to examine expression in different conditions

  • Compare protein levels (via PH1536 Antibody) with mRNA levels to identify post-transcriptional regulation

  • Methodology: Extract total RNA using TRIzol or equivalent methods optimized for archaeal samples, perform reverse transcription with random hexamers, and quantify using gene-specific primers

• Structural biology techniques:

  • X-ray crystallography or cryo-EM for high-resolution structure

  • Circular dichroism spectroscopy for secondary structure analysis

  • Hydrogen-deuterium exchange mass spectrometry for dynamics

  • These approaches provide context for epitope locations detected by PH1536 Antibody

• Activity assays:

  • Develop biochemical assays based on predicted PH1536 function

  • Use PH1536 Antibody to immunodeplete protein and assess functional impact

  • Compare wild-type activity with recombinant protein activity

• Protein-protein interaction screens:

  • Yeast two-hybrid or bacterial two-hybrid systems

  • Proximity labeling approaches (BioID, APEX)

  • Validate interactions identified through these methods using co-IP with PH1536 Antibody

• In silico analysis:

  • Homology modeling based on related proteins

  • Molecular dynamics simulations to predict functional domains

  • Evolutionary analysis to identify conserved regions

  • Guide experimental design by predicting antibody-accessible regions

This multi-method approach incorporates strategies similar to those used in comprehensive studies of other microbial proteins, such as those in pneumococcal and malaria research .

What are the limitations of PH1536 Antibody and how can they be addressed?

Like all research tools, PH1536 Antibody has inherent limitations that researchers should recognize and address:

• Batch-to-batch variability:

  • Limitation: As a polyclonal antibody, PH1536 Antibody may show batch-to-batch differences in epitope recognition and affinity

  • Solution: Validate each new lot against previous lots using standardized samples and protocols

  • Methodology: Perform side-by-side Western blots or ELISAs with previous and new lots, calculating relative sensitivities and specificities

• Limited species cross-reactivity:

  • Limitation: PH1536 Antibody is raised against Pyrococcus horikoshii protein and may not recognize orthologs from distantly related species

  • Solution: Test cross-reactivity systematically against proteins from related archaeal species

  • Methodology: Create a panel of lysates from diverse archaeal species and test recognition patterns via Western blot

• Potential non-specific binding:

  • Limitation: Polyclonal antibodies may recognize epitopes on unrelated proteins

  • Solution: Implement rigorous controls and pre-absorption protocols

  • Methodology: Pre-incubate antibody with lysates from organisms lacking PH1536 to absorb cross-reactive antibodies

• Limited application validation:

  • Limitation: PH1536 Antibody is primarily validated for ELISA and Western Blot

  • Solution: Perform step-wise validation for additional applications

  • Methodology: Start with well-established conditions from validated applications, then systematically optimize for new applications

• Epitope masking in native conditions:

  • Limitation: Some epitopes may be inaccessible in certain experimental conditions

  • Solution: Use multiple detection methods that access different epitopes

  • Methodology: Compare results from native (immunoprecipitation) and denaturing (Western blot) conditions to identify context-dependent recognition

These approaches to addressing limitations draw on strategies similar to those employed in other antibody validation studies, such as the international collaborative studies for antibody standardization .

What future research directions might benefit from PH1536 Antibody?

PH1536 Antibody represents a valuable tool for archaeal protein research with several promising future applications:

• Extremophile adaptation studies: PH1536 Antibody can help track protein expression and modifications in Pyrococcus horikoshii under extreme conditions, providing insights into archaeal adaptation mechanisms at the molecular level.

• Evolutionary proteomics: By examining potential cross-reactivity with homologous proteins across archaeal species, researchers can map evolutionary conservation and divergence patterns of this protein family.

• Structural biology integration: Combining antibody epitope mapping with structural studies could reveal functionally significant domains within PH1536, particularly in relation to thermostability mechanisms.

• Archaeal systems biology: PH1536 Antibody could serve as one component in larger-scale studies mapping protein interaction networks in archaeal systems, similar to approaches used in bacterial and eukaryotic systems.

• Biotechnology applications: Understanding PH1536's properties through antibody-based studies may reveal biotechnologically valuable features, potentially applicable to enzyme engineering for industrial processes requiring thermostable proteins.

These future directions build upon established approaches in antibody-based research while addressing the unique challenges and opportunities presented by archaeal systems research .