SPBC365.11 Antibody

What is SPBC365.11 and what are the best methods for validating an antibody against this fission yeast protein?

SPBC365.11 is a gene in the fission yeast Schizosaccharomyces pombe located on chromosome 2. To properly validate an antibody against this protein, multiple approaches should be employed:

• Western blot validation: Use wild-type and SPBC365.11 deletion mutant (SPBC365.11Δ) strains to confirm specificity. The absence of signal in the deletion strain provides strong evidence of specificity .

• Multiple detection methods: Employ at least two independent methods such as western blotting and immunofluorescence to confirm protein localization .

• Genetic tagging: Create a strain expressing tagged SPBC365.11 (e.g., with GFP, myc, or HA tags) as a positive control. The antibody signal should correspond to the molecular weight of the tagged protein .

• Knockdown verification: If gene deletion is lethal, use regulatable promoters like nmt1 to control expression levels and verify corresponding changes in antibody signal intensity .

• Recombinant protein: Test antibody against purified recombinant SPBC365.11 protein to verify recognition of the target .

What controls are essential when using antibodies against S. pombe proteins in experimental procedures?

When working with antibodies against fission yeast proteins like SPBC365.11, the following controls are essential:

Positive controls:

• Wild-type S. pombe strain expressing the protein of interest

• Strains with tagged versions of the protein (e.g., SPBC365.11-GFP, SPBC365.11-myc)

• Purified recombinant protein as a standard

Negative controls:

• Gene deletion strain (SPBC365.11Δ)

• Isotype control antibodies for immunoprecipitation experiments

• Secondary antibody-only controls to assess background

• Pre-immune serum (for polyclonal antibodies)

For western blot analysis, always include:

• Full blot images (not cropped) showing molecular weight markers

• Loading controls such as anti-α-tubulin (as seen in studies using antibody B-5-1-2)

• Control for non-specific binding by performing the experiment in deletion strains

How should I optimize sample preparation for detecting SPBC365.11 protein in different experimental contexts?

Optimizing sample preparation is crucial for successful detection of fission yeast proteins:

For Western blotting:

• Use a lysis buffer containing 20 mM HEPES (pH 7.9), 100 mM NaCl, 1 mM EDTA, 10% glycerol, protease inhibitors, and 1% Triton X-100

• Include phosphatase inhibitors (e.g., 0.1 mM Na₃VO₄) if studying phosphorylation states

• Denature samples in loading buffer containing β-mercaptoethanol, SDS, urea, and thiourea

• Resolve proteins on 6-12% SDS-polyacrylamide gels depending on the molecular weight of SPBC365.11

For immunoprecipitation:

• Use EBC buffer (140 mM NaCl, 0.5% NP-40, 100 mM NaF, 200 mM sodium orthovanadate, 50 mM Tris-HCl, pH 8.0)

• Pre-clear lysates to reduce non-specific binding

• For protein complexes, consider gentler lysis conditions and crosslinking approaches

For immunofluorescence:

• Fix cells with either 3.7% formaldehyde or cold methanol depending on epitope sensitivity

• For cell wall proteins, consider enzymatic digestion with zymolyase to improve antibody accessibility

• Use appropriate permeabilization methods (e.g., 1% Triton X-100) for nuclear proteins

What strategies can be employed to resolve contradictory results when using antibodies against SPBC365.11 across different S. pombe strains or experimental conditions?

When faced with contradictory results using antibodies against fission yeast proteins like SPBC365.11, consider these advanced troubleshooting strategies:

Potential sources of variation:

• Strain-specific genetic differences affecting protein expression or modification

• Growth media effects (YES vs. EMM2) can significantly alter gene expression patterns

• Cell cycle phase differences (synchronize cells using cdc25-22 or cdc10-129 temperature-sensitive mutants)

• Stress conditions (heat shock, nitrogen starvation, oxidative stress) dramatically alter expression profiles

Resolution approaches:

• Genetic verification: Sequence the SPBC365.11 locus in your strains to identify potential mutations or polymorphisms

• Expression profiling: Use RNA-seq or CAGE data to determine if SPBC365.11 is differentially expressed under your experimental conditions

• Epitope accessibility assessment: Test multiple antibodies targeting different regions of SPBC365.11 to identify if protein conformation or modifications affect epitope recognition

• Multiple detection methods: Employ orthogonal techniques (ChIP-seq, mass spectrometry) to validate protein presence, localization, or interactions

• Strain reconstruction: Generate new deletion or tagged strains using standardized protocols to eliminate strain background effects

• Comprehensive controls: Include parallel tests of antibody performance in wild-type, deletion, and tagged strains under identical conditions

A study found that nearly 50% of commercially available antibodies failed validation, with failure rates among vendors ranging from 0-100% . This highlights the importance of rigorous validation specific to your experimental system.

How can I determine if post-translational modifications of SPBC365.11 affect antibody recognition, and what methods should I use to characterize these modifications?

Post-translational modifications (PTMs) can significantly affect antibody recognition of proteins like SPBC365.11:

Detection strategies:

• Phosphorylation: Use phospho-specific antibodies if available, or employ general phospho-protein detection methods:

  • Lambda phosphatase treatment to remove phosphorylation

  • Mobility shift assays in western blots (phosphorylated proteins often migrate slower)

  • Phos-tag™ acrylamide gels to separate phosphorylated from non-phosphorylated species

• Ubiquitination/SUMOylation:

  • Immunoprecipitate SPBC365.11 and probe with anti-ubiquitin or anti-SUMO antibodies

  • Use tagged versions of ubiquitin/SUMO in your strains for detection

  • Treat samples with deubiquitinating enzymes to confirm modification

• Glycosylation:

  • Treat samples with glycosidases (PNGase F, Endo H) to remove glycosylation and observe mobility shifts

  • Use lectin-based affinity methods to enrich for glycosylated forms

Advanced characterization approaches:

• Mass spectrometry: The gold standard for PTM identification and mapping. Immunoprecipitate SPBC365.11 and analyze by LC-MS/MS

• Site-directed mutagenesis: Mutate potential modification sites and observe effects on antibody recognition

• Protein domain-specific antibodies: Use multiple antibodies targeting different regions to identify which domains are affected by modifications

When presenting such data, include comprehensive controls showing specificity of PTM detection and the effects of enzymatic removal of modifications on antibody recognition .

What are the most effective strategies for optimizing immunoprecipitation of SPBC365.11 to identify novel protein interaction partners?

Optimizing immunoprecipitation (IP) of SPBC365.11 for interaction studies requires careful consideration of multiple parameters:

Advanced IP optimization strategies:

• Crosslinking approaches:

  • Formaldehyde crosslinking (1%) for detecting transient interactions

  • DSP (dithiobis[succinimidyl propionate]) for reversible crosslinking

  • Compare results with and without crosslinking to distinguish direct vs. indirect interactions

• Buffer optimization:

  • Test multiple salt concentrations (100-500 mM NaCl) to balance specificity vs. sensitivity

  • Evaluate different detergents (NP-40, Triton X-100, digitonin) for membrane-associated complexes

  • Consider phosphatase inhibitors (Na₃VO₄, NaF) and protease inhibitor cocktails

• Cell synchronization:

  • Use temperature-sensitive mutants (cdc25-22) to synchronize cells at specific cell cycle phases

  • Monitor interactions across the cell cycle to identify temporal regulation

• Purification tags:

  • Compare performance of different tags (TAP, FLAG, HA, myc) for SPBC365.11 purification

  • Consider dual-tagging approaches for tandem purification to increase specificity

Identification and validation approaches:

• Mass spectrometry: Use quantitative approaches like SILAC or TMT labeling to distinguish specific from non-specific interactors

• Reciprocal IP: Confirm interactions by performing IP with antibodies against identified partners

• Co-localization: Validate interactions through microscopy using differentially tagged proteins

• Functional studies: Test if deletion/mutation of interacting partners affects SPBC365.11 function

A practical example from the search results shows how GST pull-down assays were used to study protein interactions in fission yeast: "For the binding assays, ∼500 ng of the GST fusion protein loaded on beads was incubated with 2–4 μl of in vitro translated proteins in a final volume of 200 μl in EBC buffer at 4°C on a rotating platform" .

How can I determine if SPBC365.11 expression and localization changes during the cell cycle or in response to environmental stresses in S. pombe?

Analyzing dynamic changes in SPBC365.11 expression and localization requires integration of multiple techniques:

Cell cycle regulation analysis:

• Synchronization methods:

  • Temperature-sensitive cdc mutants (cdc25-22 or cdc10-129)

  • Nitrogen starvation and release

  • Size selection by centrifugal elutriation

• Expression analysis across cell cycle:

  • Western blot time-course (take samples every 20 minutes after synchronous release)

  • qRT-PCR to measure mRNA levels at different cell cycle phases

  • Promoter activity using reporter constructs (e.g., under nmt1 promoter)

• Localization studies:

  • Live-cell imaging with SPBC365.11-GFP tagged strains

  • Fixed-cell immunofluorescence at different cell cycle stages

  • Co-localization with known cell cycle-regulated markers

Stress response analysis:

• Stress conditions to test:

  • Heat shock (temperature shift from 25°C to 37°C)

  • Oxidative stress (H₂O₂ treatment)

  • Nitrogen starvation

  • DNA damage (MMS, UV, or CPT treatment)

• Expression analysis under stress:

  • Compare SPBC365.11 levels before and after stress application

  • Monitor time-course of expression changes following stress induction

  • Compare with known stress-responsive genes as positive controls

Data analysis and presentation:

• Quantify western blot or immunofluorescence signals across multiple experiments

• Present normalized data as fold-change relative to t=0 or unstressed controls

• Include appropriate statistical analysis (typically 3+ independent experiments)

A study in fission yeast demonstrated how to analyze protein expression changes: "The cultures were then either left untreated (−) or treated with Dip (250 μM) or FeCl₃ (Fe, 100 μM) for 3 h. Whole cell extracts were analyzed by immunoblot assays with anti-GFP and anti-α-tubulin antibodies" .

What techniques can be employed to study the role of SPBC365.11 in specific cellular pathways using antibody-based approaches beyond standard western blotting and immunofluorescence?

Advanced antibody-based techniques can provide deeper insights into SPBC365.11 function:

Chromatin and DNA interaction analysis:

• Chromatin Immunoprecipitation (ChIP):

  • Use to determine if SPBC365.11 binds to specific genomic regions

  • Protocol example: "ChIP with an antibody against nuclear pore complex proteins (Mab414) and quantified using real-time PCR"

  • Can be coupled with sequencing (ChIP-seq) for genome-wide binding profile

• DNA-protein interaction studies:

  • Electrophoretic Mobility Shift Assay (EMSA) if SPBC365.11 binds DNA

  • DNA pull-down assays using biotinylated DNA sequences

Protein complex and pathway analysis:

• Proximity-dependent labeling:

  • BioID or TurboID fusion to SPBC365.11 to identify proximal proteins

  • Can reveal transient or weak interactions missed by standard IP

• Co-immunoprecipitation coupled with specific pathway inhibitors:

  • Treat cells with inhibitors of kinases, phosphatases, or other regulatory enzymes before IP

  • Reveals regulation of interaction networks

• Subcellular fractionation with immunoblotting:

  • Separate nuclear, cytoplasmic, membrane, and organelle fractions

  • Example: "Vacuoles were purified from cultures, and sample aliquots were examined by fluorescence microscopy"

Functional assays with antibody perturbation:

• Antibody microinjection:

  • Inject anti-SPBC365.11 antibodies into live cells to acutely block function

  • Monitor phenotypic consequences (cell division, morphology)

• Degron-mediated protein depletion combined with antibody detection:

  • Create auxin-inducible or temperature-sensitive degron tagged SPBC365.11

  • Use antibodies to monitor depletion kinetics and phenotypic consequences

Multi-dimensional analysis:

• Multiplexed immunofluorescence:

  • Use different fluorophore-conjugated antibodies to simultaneously detect multiple proteins

  • Analyze co-localization and potential functional relationships

• Mass cytometry (CyTOF):

  • Use metal-conjugated antibodies for high-dimensional protein analysis

  • Requires adaptation of protocols for yeast cells

When implementing these advanced techniques, "Optimal dilutions should be determined by each laboratory for each application" , and appropriate controls must be included to validate specificity in each experimental context .

What are the most common causes of non-specific binding when using antibodies against S. pombe proteins, and how can these issues be mitigated?

Non-specific binding is a common challenge when working with antibodies against fission yeast proteins:

Common causes and solutions:

• Cross-reactivity with related proteins:

  • Caused by: Conserved domains across protein families

  • Solution: Use peptide-derived antibodies targeting unique regions of SPBC365.11

  • Validation: Test specificity against recombinant proteins of related family members

• Cell wall interference:

  • Caused by: Glucans and other cell wall components binding antibodies

  • Solution: Include 1-3% BSA or 5% non-fat dry milk in blocking and antibody dilution buffers

  • Additional approach: Pre-clear antibodies with cell wall preparations from SPBC365.11Δ strains

• Protein A/G in S. pombe:

  • Caused by: Native proteins that bind immunoglobulins

  • Solution: Pre-incubate lysates with unconjugated agarose beads before immunoprecipitation

  • Alternative: Use only Fab fragments for detection

• Post-translational modifications masking epitopes:

  • Caused by: Phosphorylation, glycosylation, or other modifications

  • Solution: Test antibody recognition under different cell conditions or after treatment with modification-removing enzymes

• Denaturation-dependent epitope recognition:

  • Caused by: Antibodies recognizing linear vs. conformational epitopes

  • Solution: Compare native vs. denaturing conditions for IP and western blotting

  • Example: "For immunoblotting, proteins were analyzed by immunoblot assays with anti-GFP and anti-α-tubulin antibodies"

Experimental approaches to reduce non-specific binding:

• Buffer optimization:

  • Increase salt concentration (150-500 mM NaCl) to reduce non-specific ionic interactions

  • Add mild detergents (0.1% Tween-20, 0.1% Triton X-100) to reduce hydrophobic interactions

• Blocking optimization:

  • Test different blocking agents (BSA, milk, fish gelatin, commercial blockers)

  • Extend blocking time (overnight at 4°C vs. 1 hour at room temperature)

• Antibody concentration titration:

  • Perform dilution series to identify optimal concentration with maximal signal-to-noise ratio

  • "Optimal dilutions should be determined by each laboratory for each application"

• Pre-adsorption of antibodies:

  • Incubate primary antibody with lysate from SPBC365.11Δ strain to remove cross-reactive antibodies

Research has shown that "nearly 50% of commercially available antibodies failed validation" , highlighting the importance of thorough validation and optimization.

How can I adapt immunoprecipitation protocols to study interactions of SPBC365.11 with specific cellular components like chromatin, membranes, or the cytoskeleton?

Adapting immunoprecipitation (IP) protocols for different cellular contexts requires specific modifications:

For chromatin interactions:

• Chromatin Immunoprecipitation (ChIP) protocol:

  • Crosslink cells with 1% formaldehyde for 10-15 minutes

  • Lyse cells and sonicate chromatin to 200-500 bp fragments

  • IP with anti-SPBC365.11 antibody using specific ChIP buffers

  • Example from research: "ChIP with an antibody against nuclear pore complex proteins (Mab414) and quantified using real-time PCR"

• Chromatin fractionation with IP:

  • Separate soluble, chromatin-bound, and nuclear matrix fractions

  • Perform IP from each fraction to determine SPBC365.11 distribution

  • Use DNase treatment to distinguish DNA-dependent from protein-protein interactions

For membrane associations:

• Membrane fractionation protocol:

  • Lyse cells using gentle methods (e.g., freeze-thaw, spheroplasting)

  • Separate membrane fractions by ultracentrifugation on sucrose gradients

  • Use digitonin (0.2-1%) or CHAPS (0.5-1%) instead of stronger detergents

  • Example: "Vacuoles were purified from cultures and analyzed for their iron content by a BPS-based spectrophotometric method"

• In situ proximity labeling:

  • Create SPBC365.11 fusion with BioID or TurboID

  • Identify proximal membrane proteins through streptavidin pull-down and mass spectrometry

For cytoskeletal interactions:

• Cytoskeleton preservation protocol:

  • Include cytoskeleton stabilizing agents (taxol for microtubules, phalloidin for actin)

  • Use specialized buffers that maintain cytoskeletal integrity

  • Example: "The γ-tubulin complex, via its ability to organize microtubules, is critical for accurate chromosome segregation and cytokinesis in S. pombe"

• Differential extraction approach:

  • Extract cells sequentially with increasing detergent concentrations

  • Fractionate into soluble, cytoskeletal, and insoluble components

  • Perform IP from each fraction to determine SPBC365.11 association

General adaptation strategies:

• Crosslinking optimization:

  • Test different crosslinkers (formaldehyde, DSP, BS3) and concentrations

  • Optimize crosslinking time (5-30 minutes) and quenching conditions

• Buffer modifications for specific compartments:

  • Nuclear extraction: High salt (>300 mM NaCl) and DNase treatment

  • Membrane proteins: Mild detergents and reducing agents

  • Cytoskeletal proteins: Specialized extraction buffers with stabilizing agents

• Antibody immobilization approaches:

  • Direct conjugation to beads for cleaner IP and less antibody contamination

  • Orientation-specific coupling to maintain antibody activity

A study of γ-tubulin complex components demonstrates how specialized IP can reveal protein functions: "Mass spectrometric analyses of the purified complex revealed known components and identified two novel proteins with homology to γ-tubulin–associated proteins from other organisms" .

What quantitative approaches can be used to determine the expression levels of SPBC365.11 under different conditions, and how should antibody-based quantification be validated?

Accurate quantification of protein expression requires rigorous methodology:

Quantitative western blotting approaches:

• Linear dynamic range determination:

  • Create standard curves using purified recombinant SPBC365.11 or dilution series of positive control samples

  • Determine upper and lower detection limits for your antibody

  • Example protocol: "Equal concentrations of each sample were resuspended in loading buffer and proteins were resolved by electrophoresis on 6% SDS-polyacrylamide gels"

• Loading control selection and validation:

  • Traditional controls: α-tubulin, actin, GAPDH

  • Verify that control protein levels remain constant under your experimental conditions

  • Consider total protein staining methods (Ponceau S, SYPRO Ruby) as alternatives

• Image acquisition optimization:

  • Use CCD camera-based systems rather than film for wider linear range

  • Avoid saturated pixels that prevent accurate quantification

  • Capture multiple exposure times to ensure linearity

Validation of quantitative measurements:

• Orthogonal method comparison:

  • Correlate western blot results with mRNA levels (qRT-PCR, RNA-seq)

  • Compare with fluorescence intensity from SPBC365.11-GFP fusion proteins

  • Validate with mass spectrometry-based quantification when possible

• Statistical analysis requirements:

  • Perform at least three independent biological replicates

  • Include appropriate statistical tests (t-test, ANOVA)

  • Report variability measures (standard deviation, standard error)

  • Example from literature: "Data from three separate experiments are shown"

• Standards for reporting quantitative antibody data:

  • Provide full antibody information: "name, vendor, lot number, antibody concentration and dilution, and incubation time"

  • Include positive and negative controls in all published data

  • Show representative images of full blots alongside quantification

Advanced quantitative approaches:

• Multiplexed western blotting:

  • Use differently labeled secondary antibodies to detect multiple proteins in one sample

  • Fluorescent western blotting for more accurate quantification

• Quantitative immunofluorescence:

  • Include calibration standards in microscopy experiments

  • Control for photobleaching and background autofluorescence

  • Use automated image analysis software for unbiased quantification

• Flow cytometry for single-cell quantification:

  • Particularly useful for heterogeneous populations or cell cycle studies

  • Requires cell wall digestion and membrane permeabilization optimization

"Validation needs to focus on the precise application and tissue/cell type for which the antibody will be used, and all verification data must be reported openly" . This principle is especially important for quantitative applications.

What are the considerations for epitope selection when generating new antibodies against SPBC365.11, and how does this affect antibody performance in different applications?

Epitope selection is critical for developing effective antibodies against S. pombe proteins:

Strategic epitope selection considerations:

• Sequence analysis for optimal epitope regions:

  • Identify unique sequences with low homology to other S. pombe proteins

  • Select regions with high predicted antigenicity and surface exposure

  • Avoid transmembrane domains, signal peptides, and highly conserved functional motifs

  • Use structural information if available to identify accessible regions

• Application-specific epitope considerations:

  • Western blotting: Linear epitopes from internal regions often work well

  • Immunoprecipitation: Surface-exposed epitopes in the native protein

  • Immunofluorescence: Accessible epitopes in fixed cells

  • Functional blocking: Target catalytic or interaction domains

• Post-translational modification awareness:

  • Avoid regions with known or predicted PTM sites unless specifically targeting modified forms

  • Consider generating modification-specific antibodies for phosphorylation sites

  • Example: "Immunoprecipitate SPBC365.11 and probe with anti-ubiquitin or anti-SUMO antibodies"

Antibody format selection:

• Polyclonal vs. monoclonal considerations:

  • Polyclonal: Better for detection, recognizes multiple epitopes

  • Monoclonal: More specific, consistent lot-to-lot, better for quantification

  • Example from literature: "The mouse anti-influenza haemagglutinin (HA) monoclonal HA-11 was used at 1 µg/ml"

• Full-length vs. domain-specific antibodies:

  • Full-length: Good for multiple applications but higher risk of cross-reactivity

  • Domain-specific: More selective but may have limited application range

  • Peptide-derived: Highest specificity but may have lower affinity

Validation strategies for new antibodies:

• Comprehensive specificity testing:

  • Test against recombinant SPBC365.11 protein

  • Verify absence of signal in SPBC365.11Δ strains

  • Test cross-reactivity against related proteins

• Application-specific validation:

  • For each intended application, validate separately with proper controls

  • "Each antibody must be verified based on the content of the product sheet, and subsequently through experimentation to confirm integrity, specificity and selectivity"

• Documentation requirements:

  • Record complete information about immunogen, production method, and purification

  • Document all validation experiments with appropriate controls

  • Specify optimal conditions for each successful application

The importance of proper validation is highlighted by research showing that "about 50% of commercially available antibodies failed validation, with failure rates among vendors ranging from 0-100%" , emphasizing the need for rigorous testing of new antibodies.

What information should be included in publications when reporting results obtained using antibodies against S. pombe proteins like SPBC365.11?

Comprehensive reporting is essential for reproducibility in antibody-based research:

Essential antibody information:

• Complete antibody identification:

  • Full antibody name and clone number (for monoclonals)

  • Vendor name and catalog number

  • Lot number (critical as different lots may vary in performance)

  • For custom antibodies: immunogen sequence, host species, production method

• Experimental conditions details:

  • Antibody concentration and dilution used for each application

  • Incubation time and temperature

  • Buffer composition and blocking reagents

  • Example from literature: "Incubated with appropriate horseradish peroxidase-conjugated secondary antibodies, developed with enhanced chemiluminescence (ECL) reagents"

Validation data to include:

• Application-specific controls:

  • Positive control: Wild-type strain or tagged SPBC365.11 expression

  • Negative control: SPBC365.11Δ strain or siRNA knockdown

  • Loading or staining controls: α-tubulin, total protein

• Supporting evidence for specificity:

  • Full, uncropped blot images showing molecular weight markers

  • Side-by-side comparison with genetic knockouts when available

  • Additional validation approaches used (peptide blocking, multiple antibodies)

• Quantification methodology:

  • Image acquisition parameters (exposure time, gain settings)

  • Software used for quantification

  • Statistical analysis methods and number of independent replicates

Additional best practices:

• Method transparency:

  • Detailed protocols or references to previously published methods

  • Any modifications to standard protocols should be clearly described

  • Troubleshooting information that may help others reproduce results

• Data availability:

  • Raw data deposition in public repositories when appropriate

  • Sharing of custom antibodies or providing detailed production information

  • Example: "This work was supported by the Netherlands Organization for Scientific Research"

The Antibody Society recommends: "Each antibody must be verified based on the content of the product sheet, and subsequently through experimentation to confirm integrity, specificity and selectivity. Verification needs to focus on the precise application and tissue/cell type for which the antibody will be used, and all verification data must be reported openly" .

How should discrepancies in results obtained with different antibodies against the same protein be addressed and reported in scientific publications?

Addressing antibody discrepancies requires systematic investigation and transparent reporting:

Investigation of discrepancies:

• Systematic comparison approach:

  • Test multiple antibodies side-by-side under identical conditions

  • Compare antibodies targeting different epitopes of SPBC365.11

  • Include all relevant controls for each antibody

• Technical variables to consider:

  • Epitope accessibility differences in various applications

  • Sensitivity differences between antibodies

  • Lot-to-lot variability within the same antibody

  • Buffer and protocol compatibility issues

• Biological variables that explain discrepancies:

  • Post-translational modifications affecting epitope recognition

  • Protein isoforms or cleavage products

  • Complex formation masking epitopes

  • Cell-cycle or stress-dependent changes in protein localization

Reporting guidelines for discrepancies:

• Transparent documentation:

  • Report all antibodies tested, not just those that "worked"

  • Describe observed discrepancies in detail with supporting data

  • Provide possible explanations based on epitope location or technical factors

• Resolution approaches to include:

  • Additional validation using genetic tools (knockout, tagged lines)

  • Orthogonal techniques that confirm results (mass spectrometry, RNA expression)

  • Independent confirmation by multiple methods

• Limitations acknowledgment:

  • Clearly state the conditions under which each antibody performs reliably

  • Acknowledge unresolved discrepancies

  • Discuss implications for interpretation of results

Case examples from literature:

Studies have shown that "previously published data as the primary metric for assessing antibody quality" can be problematic . In one example from the search results: "The EpoR and CUZD1 examples not only demonstrate the devastating effect poorly performing antibodies can have on a research program, they also emphasize... the lack of enforced standards for antibody validation" .

When presenting western blot results, "Proteins were then transferred to nitrocellulose membranes, and the following antibodies were used for immunodetection: monoclonal anti-GFP antibody B-2 and monoclonal anti-α-tubulin antibody B-5-1-2" , providing clear identification of all antibodies used.