SPAC688.16 Antibody
Description
Production and Purification
The production process of SPAC688.16 Antibody involves several sophisticated steps to ensure its quality and specificity. The immunogen used is a recombinant protein corresponding to SPAC688.16 from Schizosaccharomyces pombe (strain 972 / ATCC 24843). This protein serves as the antigen for immunizing rabbits, which then produce polyclonal antibodies against various epitopes of the target protein .
Following immunization and serum collection, the antibody undergoes antigen affinity purification. This critical purification step ensures that only antibodies specifically binding to the target protein are isolated, significantly reducing background signals in experimental applications. The purification process yields an IgG isotype antibody with high specificity for the SPAC688.16 protein . This rigorous production methodology contributes to the antibody's reliability in research applications.
Target Protein Analysis
Understanding the target of SPAC688.16 Antibody requires examining the characteristics of the SPAC688.16 protein itself. This uncharacterized membrane protein in S. pombe represents an intriguing research subject with potential implications for understanding membrane protein function in eukaryotic systems.
SPAC688.16 Protein Structure
SPAC688.16 is classified as an uncharacterized membrane protein in Schizosaccharomyces pombe, with a UniProt identification number of C6Y4A4. The full-length protein consists of 109 amino acids and has a predicted membrane localization, suggesting its involvement in cellular processes that occur at or within membranes . The amino acid sequence of the full-length protein is:
MSCLNLHVPKNPVGKYIPLVVLLQMYIIYVEPYYGLHYFESVRQFLGPKILYGTVYFLVICHSIESAIAFLLCLKKGLPFCSSMKWIVSTFIFGGPTLAMLNKQKKHIA
Analysis of this sequence indicates the presence of hydrophobic regions consistent with transmembrane domains, supporting its classification as a membrane protein. The protein's relatively small size (109 amino acids) suggests it may function as part of a larger protein complex or have a specialized role within the membrane environment .
Expression and Localization
The SPAC688.16 protein is naturally expressed in Schizosaccharomyces pombe (fission yeast), a model organism widely used in molecular and cellular biology research. For research purposes, recombinant versions of the protein can be produced in expression systems such as E. coli, which facilitates both antibody production and protein characterization studies .
Applications and Experimental Usage
SPAC688.16 Antibody serves as a versatile research tool applicable across multiple experimental techniques. Understanding its validated applications and experimental conditions is essential for researchers planning to incorporate this antibody into their studies of S. pombe membrane proteins.
Validated Applications
The SPAC688.16 Antibody has been validated for several critical research applications, particularly in protein detection and characterization studies. The primary validated applications include:
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Western Blotting (WB): The antibody effectively detects the target protein in denatured samples separated by gel electrophoresis, allowing researchers to identify the protein of interest and estimate its molecular weight .
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Enzyme-Linked Immunosorbent Assay (ELISA): The antibody is suitable for detecting and potentially quantifying the target protein in ELISA-based assays, providing a sensitive method for protein detection .
These validated applications make the antibody valuable for studies involving protein expression analysis, protein purification verification, and comparative studies of SPAC688.16 expression under different experimental conditions. The antibody's specificity ensures reliable identification of the target protein in complex biological samples .
Experimental Considerations
When utilizing SPAC688.16 Antibody in experimental settings, several technical considerations should be addressed to optimize results. The antibody specifically reacts with Schizosaccharomyces pombe (strain 972 / ATCC 24843), and cross-reactivity with proteins from other species has not been extensively characterized .
For Western blotting applications, standard protocols for polyclonal antibodies can be followed, typically using dilutions determined by pilot experiments. The antibody's ability to recognize the denatured form of the protein makes it suitable for SDS-PAGE-based Western blotting .
It's important to note that this antibody is exclusively intended for research applications and should not be used in diagnostic or therapeutic procedures . This restriction highlights the antibody's specialized nature and its primary utility in basic science and exploratory research rather than clinical applications.
Production and Availability
The production process and commercial availability of SPAC688.16 Antibody influence its accessibility to researchers investigating S. pombe membrane proteins. This section examines the manufacturing timeline, quality control processes, and ordering considerations.
Manufacturing Timeline
SPAC688.16 Antibody is produced on a made-to-order basis with a lead time of approximately 14-16 weeks . This extended production timeline reflects the meticulous process required to generate high-quality polyclonal antibodies, including animal immunization, serum collection, and antigen affinity purification. The made-to-order nature of this product allows for customization options but requires advance planning by researchers .
The production process involves using a recombinant version of the SPAC688.16 protein as an immunogen in rabbits, followed by careful purification to isolate antibodies specifically targeting the protein of interest. This methodical approach ensures the resulting antibody preparation meets quality standards for research applications .
Quality Control Measures
Quality control testing for SPAC688.16 Antibody includes verification of specificity and functionality. The antibody undergoes testing in Western blotting and ELISA applications to confirm its ability to specifically recognize the target protein . These quality control measures ensure that researchers receive a product capable of reliably detecting SPAC688.16 protein in appropriate experimental contexts.
The manufacturer ensures purity greater than 90% as determined by SDS-PAGE analysis, confirming that the antibody preparation is largely free from contaminating proteins that could potentially interfere with experimental results . This high purity standard contributes to the antibody's reliability in sensitive research applications.
Related Research and Context
While specific research utilizing SPAC688.16 Antibody is not extensively documented in the provided search results, understanding the broader context of antibody research and potential applications provides valuable perspective for researchers considering this reagent.
Antibody Databases and Research Resources
The development and characterization of antibodies like SPAC688.16 Antibody contribute to the growing field of antibody research resources. The Patent and Literature Antibody Database (PLAbDab) represents one such resource, containing over 150,000 paired antibody sequences and 3D structural models from various research studies . While SPAC688.16 Antibody is not specifically mentioned in the PLAbDab context, such databases illustrate the importance of comprehensive antibody information for research purposes.
PLAbDab and similar resources allow researchers to search for antibodies based on sequence identity, structural similarity, or keywords related to the source material. This capability facilitates the identification of potentially relevant antibodies for specific research questions . The availability of such databases highlights the value of well-characterized antibodies in advancing scientific research across multiple disciplines.
Membrane Protein Research
As SPAC688.16 is classified as a membrane protein, SPAC688.16 Antibody holds particular relevance for membrane protein research. The study of membrane proteins presents unique challenges due to their hydrophobic nature and often complex structural arrangements within lipid bilayers. Specific antibodies provide valuable tools for investigating membrane protein expression, localization, and potential functions .
The full-length recombinant SPAC688.16 protein has been expressed in E. coli with an N-terminal His tag, enabling purification and characterization . This recombinant protein may serve as both an immunogen for antibody production and a positive control in experiments utilizing SPAC688.16 Antibody. Such resources facilitate comprehensive studies of this uncharacterized membrane protein.
Comparison with Other Research Antibodies
To provide context for evaluating SPAC688.16 Antibody, it is helpful to compare its properties with other research antibodies, particularly those targeting proteins in yeast or other model organisms. This comparison illuminates the antibody's distinctive characteristics and potential advantages for specific research applications.
Antibody Types and Production Methods
SPAC688.16 Antibody is a polyclonal antibody raised in rabbits using a recombinant protein immunogen . Polyclonal antibodies offer certain advantages for research applications, including recognition of multiple epitopes on the target protein, which can increase detection sensitivity. This characteristic potentially makes SPAC688.16 Antibody more robust for detecting its target protein under various experimental conditions compared to monoclonal alternatives .
The antigen affinity purification method used for SPAC688.16 Antibody represents a standard approach for improving antibody specificity. This purification step selectively isolates antibodies that bind to the target protein, reducing potential cross-reactivity with unrelated proteins . While monoclonal antibodies offer potentially higher specificity due to their single-epitope recognition, well-purified polyclonal antibodies like SPAC688.16 Antibody provide an effective balance of specificity and sensitivity.
Applications in Model Organism Research
Antibodies targeting proteins in model organisms like Schizosaccharomyces pombe play crucial roles in fundamental research. S. pombe has served as an important model organism for studying basic cellular processes such as cell division, DNA replication, and membrane dynamics. SPAC688.16 Antibody contributes to this research landscape by enabling studies of an uncharacterized membrane protein that may have undiscovered functions in cellular processes .
The specificity of SPAC688.16 Antibody for the target protein in S. pombe (strain 972 / ATCC 24843) makes it particularly valuable for research using this specific yeast strain . This strain specificity ensures reliable protein detection in the appropriate experimental context, although it may limit the antibody's utility in studies using significantly different S. pombe strains or other organisms.
Technical Data Table
The following comprehensive technical data table summarizes the key specifications and properties of SPAC688.16 Antibody to provide a quick reference for researchers considering its use:
Practical Guidelines for Use
To maximize the utility of SPAC688.16 Antibody in research settings, practical considerations regarding handling, experimental optimization, and troubleshooting are essential. This section provides guidance for researchers planning to incorporate this antibody into their experimental protocols.
Reconstitution and Dilution
If SPAC688.16 Antibody is supplied in lyophilized form, proper reconstitution is critical for maintaining its activity. The manufacturer recommends briefly centrifuging the vial before opening to ensure the content is at the bottom. The antibody should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL .
For long-term storage after reconstitution, adding glycerol to a final concentration of 5-50% is recommended, with 50% being the default concentration suggested by manufacturers. After reconstitution, the solution should be aliquoted to avoid repeated freeze-thaw cycles and stored at -20°C or -80°C . These handling practices help preserve the antibody's activity and specificity for extended periods.
Optimizing Experimental Conditions
When using SPAC688.16 Antibody in Western blotting or ELISA applications, optimizing experimental conditions is essential for reliable results. For Western blotting, standard protocols for polyclonal antibodies can be followed, typically beginning with a moderate dilution (e.g., 1:1000) and adjusting based on signal intensity in preliminary experiments .
For ELISA applications, similar optimization may be necessary to determine the appropriate antibody concentration that provides specific signal while minimizing background. The antibody's non-conjugated format may require a secondary antibody for detection, typically an anti-rabbit IgG conjugated to an appropriate detection system such as horseradish peroxidase (HRP) for chemiluminescent detection .
Product Specs
Target Background
KEGG: spo:SPAC688.16
Q&A
How should researchers validate the specificity of SPAC688.16 antibodies?
Antibody specificity validation is critical given recent findings that many antibodies lack true conformation specificity. Based on rigorous studies of α-synuclein antibodies, which found most allegedly conformation-specific antibodies reacted with multiple forms of the protein , researchers should implement multiple validation methods for SPAC688.16 antibodies:
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Western blot analysis: Test against wild-type S. pombe extracts, SPAC688.16 deletion mutants, and recombinant protein preparations.
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Immunoprecipitation followed by mass spectrometry: Verify that pulled-down proteins match the expected target.
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Cross-reactivity testing: Examine binding to related proteins within the S. pombe proteome.
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Epitope mapping: Determine the specific binding regions to understand potential cross-reactivity.
What controls are essential when using SPAC688.16 antibodies in immunofluorescence experiments?
When performing immunofluorescence with SPAC688.16 antibodies in S. pombe, the following controls are essential:
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Negative control: Include SPAC688.16 deletion strains to confirm signal specificity.
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Blocking peptide control: Pre-incubate antibody with excess purified antigen to confirm signal suppression.
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Secondary antibody-only control: Verify absence of signal without primary antibody.
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Methanol fixation comparison: When investigating membrane proteins, compare methanol fixation results with other fixation methods as demonstrated in S. pombe studies .
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Wild-type vs. tagged protein comparison: If using antibodies against tagged versions, compare localization patterns between wild-type and tagged strains.
For membrane or cell wall proteins, methanol fixation followed by proper immunofluorescence labeling provides reliable results in S. pombe, as demonstrated in studies of transmembrane proteins .
What are the optimal methods for using SPAC688.16 antibodies in proteomic studies of S. pombe?
Based on methodologies established for membrane protein analysis in S. pombe:
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Membrane preparation: Use spheroblasting of S. pombe cells followed by differential centrifugation as described in standardized protocols . This method preserves membrane protein architecture while removing cell wall components that may interfere with antibody access.
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Protein extraction optimization:
| Buffer Component | Recommended Concentration | Purpose |
|---|---|---|
| Tris-HCl pH 7.5 | 50 mM | pH stabilization |
| NaCl | 150 mM | Ionic strength |
| Glycerol | 10% | Protein stabilization |
| Triton X-100 | 1% | Membrane solubilization |
| Protease inhibitors | 1X complete cocktail | Prevent degradation |
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Antibody purification: For polyclonal antibodies, perform affinity purification against the target epitope, as demonstrated for yeast membrane proteins . This increases specificity and reduces background.
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Topological analysis: Employ proteinase K protection assays when studying transmembrane proteins to determine membrane topology and accessibility of epitopes .
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Mass spectrometry validation: Use LC-MS/MS to confirm antibody-precipitated proteins, applying analysis tools like Mascot for accurate protein identification .
How can researchers optimize SPAC688.16 antibody-based immunoprecipitation from S. pombe lysates?
Based on successful immunoprecipitation strategies for yeast proteins:
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Cell lysis optimization: For S. pombe, use glass bead lysis in buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and protease inhibitors.
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Pre-clearing: Remove non-specific binding proteins by pre-incubating lysates with protein A/G beads before adding the antibody.
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Antibody coupling: Consider covalently coupling the antibody to beads using dimethyl pimelimidate to prevent antibody leaching during elution.
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Antigen elution strategies: Use a comparison of different elution methods for optimal recovery:
| Elution Method | Advantages | Limitations | Recommended For |
|---|---|---|---|
| Low pH (glycine pH 2.5) | Efficient elution | May denature some proteins | Stable proteins |
| Competitive elution with peptide | Maintains native structure | Lower yield, requires peptide | Conformation-sensitive applications |
| SDS elution | Highest recovery | Denatures proteins | Mass spectrometry |
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Validation by mass spectrometry: Confirm the identity of immunoprecipitated proteins using techniques similar to those applied in SpA5 antibody validation studies .
How should researchers address inconsistent results when antibodies recognize multiple forms of SPAC688.16 protein?
Recent studies of α-synuclein antibodies revealed that antibodies claimed to be specific for particular protein conformations often recognize multiple forms with differing affinities . To address similar issues with SPAC688.16 antibodies:
Studies have shown that antibodies presumed to distinguish between protein conformations often bind multiple forms equally well, necessitating caution when interpreting results solely based on antibody reactivity .
What approaches can resolve discrepancies between antibody-based detection and genetic expression data for SPAC688.16?
When faced with conflicting results between antibody detection and genetic expression:
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Quantitative analysis calibration: Establish standard curves using recombinant protein to accurately quantify protein levels detected by antibodies.
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Post-translational modification assessment: Investigate whether modifications affect antibody recognition using techniques such as:
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Transcript vs. protein half-life analysis: Measure mRNA and protein stability using actinomycin D and cycloheximide chase experiments to identify discrepancies in turnover rates.
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Alternative splicing investigation: Use RT-PCR and qPCR to detect splice variants that might lack the antibody epitope .
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Technical validation:
| Validation Method | Purpose | Expected Outcome |
|---|---|---|
| Multiple antibodies to different epitopes | Confirm presence/absence of protein | Consistent detection pattern |
| RNA-seq validation | Confirm transcript presence | Correlation with protein levels |
| Tagged protein expression | Independent detection method | Agreement with antibody detection |
| Alternative detection methods (e.g., mass spectrometry) | Antibody-independent validation | Confirmation of protein presence |
How can high-throughput single-cell analyses be combined with SPAC688.16 antibodies for studying protein heterogeneity in S. pombe populations?
Drawing from advanced antibody-based techniques used in recent clinical research :
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Single-cell immunostaining protocol:
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Fix cells with 4% paraformaldehyde
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Permeabilize with 0.1% Triton X-100
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Block with 3% BSA
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Incubate with fluorophore-conjugated SPAC688.16 antibodies
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Analyze using flow cytometry or high-content imaging
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Correlation with gene expression:
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Multiplexed detection:
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Use spectral unmixing for simultaneous detection of multiple proteins
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Implement sequential staining protocols for proteins requiring incompatible fixation methods
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Correlate SPAC688.16 localization with other cellular markers
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Recent studies using high-throughput single-cell RNA and VDJ sequencing have successfully identified antigen-binding profiles in complex cell populations, providing a methodological framework for similar analyses in yeast cells .
What strategies enable structural epitope mapping of SPAC688.16 antibodies for improving specificity and affinity?
Advanced epitope mapping strategies can be implemented based on recent antibody characterization techniques:
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Computational prediction and validation:
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Hydrogen-deuterium exchange mass spectrometry (HDX-MS):
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Compare deuterium uptake patterns of free protein versus antibody-bound protein
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Identify regions with reduced exchange rates as potential binding sites
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Create detailed maps of conformational epitopes
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X-ray crystallography or Cryo-EM:
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Attempt co-crystallization of antibody Fab fragments with SPAC688.16
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Use structural data to guide affinity maturation
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Phage display epitope mapping:
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Express protein fragments on phage surface
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Screen for antibody binding
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Narrow down the minimal epitope sequence
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Recent studies have successfully employed structure prediction and molecular docking to identify antigenic epitopes that bind to antibodies with nanomolar affinity , providing a template for similar analyses of SPAC688.16 antibodies.
How can researchers ensure reproducibility when using SPAC688.16 antibodies across different experimental platforms?
Based on challenges identified in antibody research :
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Standardized reporting:
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Document complete antibody validation data
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Report exact experimental conditions including buffer compositions, incubation times, and temperatures
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Specify lot numbers and sources of antibodies used
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Cross-platform validation matrix:
| Platform | Validation Method | Expected Outcome | Common Pitfalls |
|---|---|---|---|
| Western blot | Test multiple protein amounts | Linear signal response | Saturated signal masking differences |
| Immunofluorescence | Compare fixation methods | Consistent localization pattern | Fixation artifacts affecting epitope |
| ELISA | Standard curve with recombinant protein | Linear detection range | Matrix effects from complex samples |
| Flow cytometry | Titration of antibody concentrations | Optimal signal-to-noise ratio | Autofluorescence interference |
| IP-MS | Comparison to input sample | Enrichment of target protein | Non-specific binding |
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Interlaboratory validation:
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Establish reference standards for antibody performance
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Implement round-robin testing between collaborating laboratories
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Document batch-to-batch variation in antibody performance
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Studies of α-synuclein antibodies demonstrated that antibodies previously reported as specific often showed unexpected cross-reactivity under systematic testing conditions, highlighting the importance of rigorous validation across multiple platforms .
What methodological advances could improve SPAC688.16 antibody development for detecting post-translational modifications?
Drawing from recent advances in antibody technology:
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Targeted immunization strategies:
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Generate antibodies against synthetic peptides containing specific modifications
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Use modified recombinant protein fragments as immunogens
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Implement negative selection against unmodified epitopes
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Single B-cell sequencing approach:
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Validation requirements for modification-specific antibodies:
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Test against unmodified protein to confirm specificity
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Validate with enzymatically modified versus unmodified substrates
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Use mass spectrometry to confirm modification state of recognized proteins
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Application of phage display technology:
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Screen synthetic antibody libraries against modified epitopes
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Select high-affinity binders through multiple rounds of panning
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Engineer selected antibodies for improved specificity
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Recent studies have successfully used high-throughput sequencing to identify antibodies with nanomolar affinity , providing a methodological framework for developing highly specific antibodies against modified forms of SPAC688.16.
