At5g52610 Antibody

What is the At5g52610 gene and why are antibodies developed against its protein product?

At5g52610 is a gene located on chromosome 5 of Arabidopsis thaliana that encodes a protein of significant research interest. Antibodies against this protein are developed to study its expression patterns, localization, and functional interactions within plant cellular networks . Researchers typically develop these antibodies by first analyzing the protein sequence for immunogenic regions that are unique and accessible. The antibody production process involves either polyclonal approaches using synthetic peptides or monoclonal approaches if more specific epitope targeting is required . When selecting target epitopes, researchers must consider protein folding, post-translational modifications, and sequence conservation to ensure the antibody will recognize the native protein in experimental conditions.

What validation methods are essential before using At5g52610 antibodies in experimental workflows?

Validating At5g52610 antibodies requires a multi-step approach to ensure specificity and reproducibility. Researchers should first perform Western blot analysis using positive controls (tissues known to express the target) and negative controls (knockout mutants or tissues where expression is absent) . Immunoprecipitation followed by mass spectrometry can confirm antibody-target interactions, which is especially important for plant proteins where cross-reactivity can be problematic. Additionally, comparative analysis with alternative antibodies targeting different epitopes of the same protein can provide convergent validation. Signal specificity can be further verified through peptide competition assays, where pre-incubation with the immunizing peptide should abolish the antibody signal . These validation steps must be performed in the specific experimental context (fixation methods, buffer systems) that will be used in the planned research.

How should researchers optimize protein extraction protocols for At5g52610 detection?

Optimization of protein extraction for At5g52610 detection requires careful consideration of plant tissue characteristics. Begin with a buffer system containing appropriate detergents (typically 0.1-1% Triton X-100 or NP-40) for membrane disruption, protease inhibitors to prevent degradation, and reducing agents to maintain protein integrity . The extraction protocol should consider the subcellular localization of the target protein—membrane-associated proteins may require different detergent concentrations compared to soluble proteins. For Arabidopsis tissues, grinding in liquid nitrogen followed by buffer extraction typically yields good results. Researchers should test multiple extraction conditions (varying pH, salt concentration, and detergent types) to determine optimal recovery of the target protein while minimizing interfering compounds from plant materials such as phenolics and polysaccharides . Following extraction, centrifugation parameters should be optimized to remove cellular debris while retaining the protein fraction containing At5g52610.

What control samples are necessary when performing immunohistochemistry with At5g52610 antibodies?

Immunohistochemistry with At5g52610 antibodies requires rigorous controls to ensure valid interpretation. Essential controls include:

• Primary antibody omission: Tissues processed identically but without primary antibody application to identify non-specific binding of secondary antibodies

• Isotype controls: Using matched concentration of irrelevant antibodies of the same isotype to detect non-specific binding

• Absorption controls: Pre-incubating the antibody with excess target peptide to confirm signal specificity

• Biological controls: Including tissues from knockout mutants lacking At5g52610 expression

• Positive reference tissues: Including samples known to express the target at varying levels

Additionally, researchers should include internal controls within the same tissue section, using structures or cell types with known expression patterns to confirm staining protocols are functioning properly . When detecting subtle expression differences, standardizing fixation times, antibody concentrations, and development periods becomes critical for generating reproducible and comparable results.

How can researchers troubleshoot weak or inconsistent At5g52610 antibody signals in Western blot analyses?

Troubleshooting weak or inconsistent At5g52610 antibody signals requires systematic evaluation of multiple parameters. Begin by confirming protein transfer efficiency using reversible total protein stains like Ponceau S. If transfer is adequate, optimize antibody concentration through titration experiments, typically testing ranges from 1:500 to 1:5000 for primary antibodies . For plant proteins like At5g52610, signal enhancement can be achieved by employing extended blocking periods (2-3 hours) with plant-optimized blocking reagents containing non-fat milk or BSA supplemented with 0.05-0.1% plant-derived proteins to reduce background. The membrane washing protocol may require optimization, with PBST (0.1% Tween-20) typically providing good results, though some plant proteins benefit from TBS-based systems when phospho-specific detection is needed.

For particularly challenging targets, signal amplification systems such as biotin-streptavidin can be employed, or enhanced chemiluminescence (ECL) reagents with longer activation periods may improve detection. If protein abundance is the limiting factor, immunoprecipitation before Western blotting can concentrate the target protein . Additionally, researchers should evaluate sample preparation methods, as some plant proteins require specialized extraction buffers containing chaotropic agents to maintain solubility and epitope accessibility.

What approaches can resolve epitope masking issues when detecting At5g52610 in fixed tissues?

Epitope masking is a common challenge when detecting plant proteins in fixed tissues. To resolve this issue with At5g52610 antibodies, researchers should implement antigen retrieval methods optimized for plant samples. Heat-induced epitope retrieval using citrate buffer (pH 6.0) at 95°C for 10-20 minutes often recovers masked epitopes in formalin-fixed plant tissues . For particularly recalcitrant samples, enzymatic retrieval using proteinase K (1-10 μg/mL for 5-15 minutes) may expose additional binding sites.

The fixation protocol itself can be modified to preserve epitope accessibility—replacing traditional aldehyde fixatives with alcohol-based ones or reducing fixation time can maintain epitope integrity. For At5g52610 detection, a fixation screening experiment comparing 4% paraformaldehyde, Carnoy's fluid, and 70% ethanol can identify the optimal preservation method that balances morphological preservation with epitope accessibility. Additionally, the use of shorter peptide linkers in primary antibody design can improve penetration and binding in dense plant tissues. Post-fixation treatment with sodium borohydride (0.1% for 10 minutes) can reduce autofluorescence while also breaking some protein crosslinks that mask epitopes .

What methodological strategies can distinguish between specific isoforms or post-translationally modified versions of the At5g52610 protein?

Distinguishing between specific isoforms or post-translationally modified versions of the At5g52610 protein requires sophisticated immunological approaches. Researchers should first conduct bioinformatic analysis to identify unique peptide sequences in each isoform, followed by generation of isoform-specific antibodies targeting these regions . When post-translational modifications (PTMs) are the focus, modification-specific antibodies should be developed against the modified peptide sequence.

For validation, two-dimensional gel electrophoresis followed by Western blotting can separate protein species based on both molecular weight and isoelectric point, allowing visualization of distinct isoforms and modified variants. Complementary approaches include:

• Immunoprecipitation followed by mass spectrometry to identify specific PTMs

• Sequential probing with pan-specific and modification-specific antibodies

• Treatment of samples with enzymes that remove specific modifications (e.g., phosphatases, deglycosylases)

• Comparative analysis of wild-type and mutant plant lines with altered PTM machinery

When interpreting results, researchers should consider that PTMs may affect antibody binding affinity, potentially leading to quantitative differences that don't reflect actual protein abundance variations . Cross-validation with non-antibody methods like targeted mass spectrometry can provide orthogonal confirmation of isoform or modification status.

How should researchers approach quantitative analysis when using At5g52610 antibodies for immunofluorescence microscopy?

Quantitative analysis of At5g52610 immunofluorescence requires rigorous standardization and appropriate controls. Begin by establishing consistent image acquisition parameters—fixed exposure times, detector gains, and pinhole settings for confocal microscopy—based on samples with the highest signal to prevent saturation . For accurate quantification, researchers should:

• Include calibration standards in each imaging session

• Apply flat-field correction to account for illumination heterogeneity

• Correct for background using multiple background ROIs

• Normalize signal to constitutive markers or total protein content

• Analyze multiple biological and technical replicates

Statistical approaches should account for the typically non-normal distribution of fluorescence intensity data. Methods like integrated density measurements (area × mean intensity) often provide more reliable quantification than simple intensity measurements. When comparing experimental groups, multiple samples should be processed simultaneously with identical protocols to minimize batch effects. For subcellular localization studies, colocalization analysis with organelle markers requires calculation of Pearson's or Mander's coefficients rather than visual assessment alone . Finally, automated image analysis algorithms can reduce investigator bias but require careful validation against manual measurements before implementation.

What are the optimal conditions for using At5g52610 antibodies in chromatin immunoprecipitation (ChIP) experiments?

Chromatin immunoprecipitation with At5g52610 antibodies requires careful optimization tailored to plant chromatin characteristics. The crosslinking protocol is critical—while 1% formaldehyde for 10 minutes is standard, plant tissues often benefit from vacuum infiltration to ensure fixative penetration through cell walls . Chromatin fragmentation should be optimized by testing sonication cycles incrementally (typically 10-30 seconds pulses with 30-second cooling periods) to achieve fragments averaging 200-500 bp, as verified by agarose gel electrophoresis.

For the immunoprecipitation step, researchers should determine the minimal antibody amount needed through titration experiments, typically testing 1-10 μg per reaction. Pre-clearing with protein A/G beads (1-2 hours at 4°C) significantly reduces background in plant samples. The washing stringency requires careful balance—too stringent and specific interactions may be lost, too gentle and background increases. A step gradient of salt concentrations (150 mM to 500 mM NaCl) can identify optimal conditions.

Controls must include:

• Input chromatin (pre-immunoprecipitation sample)

• IgG control (matched concentration of non-specific IgG)

• Positive control regions (known binding sites)

• Negative control regions (genomic regions not expected to interact)

For plant ChIP experiments, including an additional control using tissues where the target is not expressed can help distinguish true binding events from technical artifacts that can occur in complex plant chromatin .

How can researchers develop a quantitative immunoassay for measuring At5g52610 protein levels across different plant tissues?

Developing a quantitative immunoassay for At5g52610 requires methodical optimization of multiple parameters. For an ELISA-based approach, researchers should begin by determining the optimal antibody pair through cross-reactivity testing—typically one antibody for capture and another recognizing a different epitope for detection . For sandwich ELISA development:

• Screen multiple antibody concentrations in a checkerboard titration (typically 1-10 μg/mL for capture antibody)

• Optimize blocking conditions using plant-specific blocking buffers containing 3-5% non-fat milk or BSA supplemented with 0.1% plant protein extract

• Develop a suitable standard curve using recombinant At5g52610 protein (covering 0.1-100 ng/mL)

• Determine sample dilution ranges that position readings in the linear portion of the standard curve

For plant samples specifically, extraction buffers should include compounds to neutralize interfering substances—PVPP (polyvinylpolypyrrolidone) at 1-2% effectively removes phenolic compounds that can interfere with antibody binding. Signal detection systems should be selected based on sensitivity requirements, with chemiluminescence typically providing 10-100 fold greater sensitivity than colorimetric methods.

Validation should include spike-recovery experiments (adding known amounts of recombinant protein to samples) and dilution linearity tests across multiple tissue types . For absolute quantification, researchers should calibrate the assay against a mass spectrometry-based absolute quantification method.

What strategies can overcome matrix effects when detecting At5g52610 in complex plant extracts?

Matrix effects present significant challenges when detecting At5g52610 in complex plant extracts due to the presence of interfering compounds. Effective strategies include:

• Sequential extraction methods that separate classes of interfering compounds—initial extraction with a non-ionic detergent buffer followed by a more stringent buffer can improve target protein recovery while reducing interference

• Implementing two-phase extraction systems such as Tris-buffered phenol/SDS extraction followed by protein precipitation with ammonium acetate/methanol

• Addition of specific compounds to neutralize common interferents:

  • 2% PVPP to remove phenolic compounds

  • 5-10 mM DTT to prevent oxidation of proteins

  • 1-5% PEG to reduce interference from polysaccharides

  • Protease inhibitor cocktails optimized for plant tissues

Sample clean-up through size exclusion chromatography, immunoaffinity purification, or protein precipitation can further reduce matrix effects prior to immunodetection . When developing wash protocols, researchers should test multiple detergent types and concentrations, as certain plant compounds require specific detergents for effective removal.

For particularly complex tissues, background subtraction using pre-immune serum controls becomes essential, and signal amplification methods such as tyramide signal amplification can improve detection of low-abundance targets without amplifying background . Finally, researchers should validate results using orthogonal detection methods such as mass spectrometry to confirm findings obtained with antibody-based approaches.

How do environmental growth conditions affect At5g52610 protein expression and antibody detection efficiency?

Environmental growth conditions significantly impact At5g52610 protein expression and can affect antibody detection efficiency. Researchers must consider these variables when designing experiments and interpreting results. Light conditions (intensity, photoperiod, spectral quality) can alter expression levels of many plant proteins, necessitating standardized growth conditions when comparing different treatment groups . Temperature fluctuations may affect protein folding and epitope accessibility—plants grown at different temperatures may require adjusted extraction protocols to maintain protein solubility and preserve epitope integrity.

Nutrient availability also influences protein expression profiles, particularly for proteins involved in stress responses or nutrient utilization pathways. For reproducible antibody detection, researchers should standardize:

• Growth media composition (macro and micronutrients)

• Watering regimen and humidity levels

• Plant age and developmental stage at sampling

• Time of day for tissue collection (accounting for circadian effects)

When comparing plants grown under different environmental conditions, extraction buffers may require condition-specific optimization. For instance, plants grown under high light or drought stress often accumulate higher levels of interfering compounds requiring more stringent extraction protocols . Additionally, certain environmental stressors can induce post-translational modifications that alter epitope accessibility or antibody binding affinity. In such cases, researchers should perform epitope mapping under different growth conditions to ensure consistent antibody performance across experimental conditions.

How should researchers interpret contradictory results when using different antibodies targeting the same At5g52610 protein?

Contradictory results from different antibodies targeting At5g52610 require systematic investigation rather than immediate dismissal of either finding. First, researchers should characterize the specific epitopes recognized by each antibody through epitope mapping or manufacturer documentation review . Different results may reflect epitope-specific biological phenomena rather than technical artifacts—antibodies targeting different domains may reveal protein isoforms, post-translational modifications, or conformation-specific states.

To resolve contradictions, researchers should implement a multi-step approach:

• Cross-validate with orthogonal non-antibody methods (mass spectrometry, RNA expression)

• Perform antibody validation using knockout/knockdown models

• Test antibody performance under varied sample preparation conditions

• Examine epitope conservation across closely related proteins to assess potential cross-reactivity

• Investigate whether differences appear in specific tissues/conditions, suggesting context-dependent protein modifications

When reporting contradictory findings, researchers should clearly document all experimental conditions, antibody sources, epitope information, and validation methods . This transparency allows the scientific community to properly interpret seemingly conflicting results, which often provide valuable insights into protein biology once the underlying causes are understood.

What statistical approaches are recommended for analyzing semi-quantitative immunoblot data for At5g52610?

Semi-quantitative immunoblot analysis of At5g52610 requires appropriate statistical approaches that account for the technical limitations of the method. Researchers should begin with proper experimental design—including biological replicates (minimum n=3) and technical replicates to assess variation . Normalization is critical and should utilize loading controls appropriate for the experimental context (housekeeping proteins for stable conditions, total protein stains like Ponceau S for variable conditions).

For statistical analysis:

• Use densitometry software that allows background subtraction and defines clear band boundaries

• Test data for normality using Shapiro-Wilk or similar tests before selecting parametric or non-parametric approaches

• Apply appropriate transformations (log or square root) if data distribution is skewed

• Implement ANOVA with post-hoc tests for multiple group comparisons or t-tests for two-group comparisons

• Consider repeated measures approaches when analyzing time-course experiments

When reporting results, researchers should present both normalized band intensities and representative blot images, including molecular weight markers and loading controls . For accurate interpretation, the linear dynamic range of detection should be determined through standard curve analysis, and all quantified samples should fall within this range. Signal saturation can be assessed by testing multiple exposure times or dilution series of representative samples. Finally, effect size calculations should accompany p-values to communicate biological significance beyond statistical significance.

How can researchers distinguish between specific antibody binding and autofluorescence in plant tissues during immunofluorescence microscopy?

Distinguishing specific antibody binding from autofluorescence in plant tissues represents a significant challenge in immunofluorescence microscopy. Plant tissues naturally contain autofluorescent compounds such as chlorophyll, phenolics, and cell wall components that can confound interpretation . To address this challenge, researchers should:

• Acquire spectral profiles of unstained tissues across multiple excitation/emission wavelengths to characterize natural autofluorescence

• Select fluorophores with excitation/emission spectra that minimize overlap with plant autofluorescence (far-red fluorophores often provide better signal-to-noise ratios)

• Implement spectral unmixing algorithms that can separate overlapping fluorescence signals based on their spectral signatures

• Use chemical treatments to reduce autofluorescence:

  • Sodium borohydride (0.1% for 10-15 minutes) to reduce aldehyde-induced fluorescence

  • Sudan Black B (0.1-0.3% in 70% ethanol) to quench lipofuscin autofluorescence

  • 0.1% Toluidine Blue in aqueous solution to reduce cell wall autofluorescence

Control samples are essential and should include:

• Primary antibody omission controls

• Absorption controls using excess antigen

• Tissues known to lack the target protein

• Comparative analysis with different fixation methods that preserve antigenicity while reducing autofluorescence

Advanced microscopy techniques such as fluorescence lifetime imaging (FLIM) can further distinguish between specific antibody binding and autofluorescence based on fluorescence decay characteristics . When analyzing results, researchers should document autofluorescence reduction methods and control images alongside experimental samples to demonstrate specificity of observed signals.

What bioinformatic tools can predict potential cross-reactivity of At5g52610 antibodies with other plant proteins?

Predicting potential cross-reactivity of At5g52610 antibodies requires comprehensive bioinformatic analysis using multiple complementary approaches. Researchers should begin with sequence-based tools to identify proteins sharing epitope similarity:

• BLAST and FASTA searches against plant proteome databases using the immunizing peptide sequence (typical threshold: >70% identity over the epitope region)

• Multiple sequence alignment tools (MUSCLE, Clustal Omega) to visualize conservation across protein families

• Epitope prediction algorithms (BepiPred, DiscoTope) to identify potential linear and conformational epitopes

Structure-based approaches provide additional insights:

• Protein structure prediction tools (AlphaFold, SWISS-MODEL) to model the 3D conformation of potential cross-reactive proteins

• Molecular docking simulations to predict antibody-antigen interactions

• Solvent accessibility analysis to determine if homologous sequences are exposed in native proteins

For experimental validation of predictions, researchers should perform:

• Peptide arrays containing predicted cross-reactive epitopes

• Western blot analysis against recombinant proteins with predicted similarity

• Immunoprecipitation followed by mass spectrometry to identify all proteins captured by the antibody

When evaluating cross-reactivity predictions, researchers should consider not just sequence similarity but also tissue-specific expression patterns and subcellular localization of potential cross-reactive proteins. This integrated approach allows researchers to anticipate potential specificity issues and design appropriate controls for experimental validation.

What protocol modifications are needed to use At5g52610 antibodies for co-immunoprecipitation studies?

Co-immunoprecipitation (co-IP) with At5g52610 antibodies requires specific protocol modifications to preserve protein-protein interactions while achieving efficient target capture. The lysis buffer composition is critical—researchers should use a mild non-ionic detergent (0.1-0.5% NP-40 or 0.1-1% Digitonin) to solubilize membranes while maintaining protein complexes . Buffer ionic strength should be moderate (typically 100-150 mM NaCl) with neutral pH (7.2-7.5) to preserve most interactions.

For plant tissues specifically:

• Incorporate 5-10% glycerol to stabilize protein complexes during extraction

• Add 1 mM EDTA to inhibit metalloproteases common in plant tissues

• Include plant-specific protease inhibitor cocktails supplemented with PMSF (1 mM)

• Consider crosslinking with formaldehyde (0.1-0.3% for 5-10 minutes) for capturing transient interactions

• Perform extraction at 4°C throughout the procedure

The antibody binding step requires optimization—direct antibody conjugation to beads often provides cleaner results than protein A/G-based capture. For plant co-IP, pre-clearing samples with beads alone for 1-2 hours significantly reduces non-specific binding . Washing conditions must balance removal of contaminants against preservation of interactions—a stepped washing approach using decreasing detergent concentrations (0.1% to 0.01%) often yields optimal results.

For elution, native conditions using excess competing peptide allow recovery of intact complexes for further functional studies, while harsher elution with SDS is preferred for subsequent mass spectrometry analysis. Validation should include reciprocal co-IP experiments and controls using tissues where the target protein is absent or depleted.

How can At5g52610 antibodies be adapted for use in plant tissue clearing and 3D imaging methods?

Adapting At5g52610 antibodies for tissue clearing and 3D imaging requires modifications to standard immunohistochemistry protocols to accommodate thick plant tissue sections. Researchers should begin by selecting appropriate clearing methods compatible with immunolabeling—PEA-CLARITY and ClearSee systems have been successfully adapted for plant tissues . The workflow should follow this sequence:

• Fixation: 4% paraformaldehyde with vacuum infiltration to ensure penetration through plant cell walls

• Pre-clearing: Gradually replace water with clearing solution over 3-5 days (using 25%, 50%, 75%, 100% steps)

• Permeabilization: Extend treatment with cell wall digesting enzymes (1% cellulase, 0.2% macerozyme) for 1-3 hours to improve antibody penetration

• Blocking: Extended blocking (overnight at 4°C) with higher concentrations of blocking agents (5-10% BSA, 1-2% normal serum)

• Primary antibody incubation: Extend to 3-7 days at 4°C with gentle agitation

• Washing: Multiple extended washes (4-6 hours each)

• Secondary antibody: Fluorophores with far-red emission spectra reduce interference from plant autofluorescence

For samples thicker than 100 μm, antibody fragments (Fab or single-domain) often provide better penetration than full IgG molecules. Alternatively, electrophoretic immunolabeling can improve antibody penetration by applying a mild electric field to drive antibodies into the tissue . For 3D reconstruction, confocal z-stacks should be acquired with step sizes not exceeding half the axial resolution, and deconvolution algorithms should be applied to improve image quality. Finally, reference anatomical markers should be included to provide spatial context for the At5g52610 localization pattern within the tissue architecture.

What considerations are important when using At5g52610 antibodies for super-resolution microscopy techniques?

Super-resolution microscopy with At5g52610 antibodies requires specific considerations to achieve optimal resolution while maintaining signal specificity. For sample preparation, standard immunofluorescence protocols require modification:

• Fixation should preserve ultrastructure while maintaining epitope accessibility—2% paraformaldehyde with 0.05% glutaraldehyde provides a good balance for plant tissues

• Sections should be thinner than for conventional microscopy (5-10 μm optimal) to reduce out-of-focus fluorescence

• Background reduction becomes critical—additional blocking with Image-iT FX (for lipofuscin autofluorescence) and 1% acetylated BSA can improve signal-to-noise ratio

For specific super-resolution techniques, optimizations include:

STED Microscopy:

• Select fluorophores with appropriate photostability (ATTO 647N, Abberior STAR RED)

• Mount samples in specialized media with matched refractive index (ProLong Glass, TDE mounting media)

• Use two-color STED to correlate At5g52610 with known subcellular markers at nanoscale resolution

STORM/PALM:

• Use photoconvertible fluorophores (Alexa Fluor 647, mEos) conjugated to secondary antibodies

• Prepare oxygen-scavenging imaging buffer (glucose oxidase/catalase system) with optimal thiol concentration

• Acquire 10,000-30,000 frames for reconstruction with fiducial markers for drift correction

For all super-resolution approaches, antibody concentration should be carefully titrated—excessive labeling density can compromise resolution due to overlapping point spread functions . Direct labeling of primary antibodies rather than using secondary antibodies can improve localization precision. Additionally, researchers should validate super-resolution findings with complementary approaches such as electron microscopy or proximity ligation assays to confirm nanoscale protein distributions observed with optical super-resolution techniques.

How can researchers develop multiplexed immunoassays to study At5g52610 alongside other proteins of interest?

Developing multiplexed immunoassays for simultaneous detection of At5g52610 and other proteins requires careful planning to ensure antibody compatibility and signal discrimination. Researchers should begin with antibody selection, choosing antibodies raised in different host species to enable species-specific secondary antibody detection . For fluorescence-based multiplexing:

• Select fluorophores with minimal spectral overlap (typically 50+ nm separation between emission peaks)

• Include single-color controls to establish spectral profiles for compensation

• Implement linear unmixing algorithms to separate overlapping signals

• Consider sequential detection using antibody stripping or quenching between rounds

For multiplex Western blotting, researchers can employ:

• Size-based multiplexing for proteins of different molecular weights

• Fluorescent detection systems with spectrally distinct fluorophores

• Sequential reprobing with antibody stripping between detections (verify complete stripping with secondary-only controls)

When developing bead-based multiplexed assays (e.g., Luminex platform), each antibody pair should be validated individually before combination, with careful attention to cross-reactivity between capture and detection antibodies . For all multiplexed approaches, researchers should perform extensive validation including:

• Comparison of single-plex vs. multiplex results to identify interference

• Spike-recovery experiments across a concentration gradient to detect matrix effects

• Cross-reactivity testing using recombinant proteins

• Specificity verification using genetic knockouts or CRISPR-edited lines

Data analysis for multiplexed assays requires normalization strategies that account for differences in antibody affinity and detection sensitivity. Finally, researchers should acknowledge the technical limitations of multiplexed assays when interpreting complex protein interaction networks or expression patterns.