CHX13 Antibody

What is CHX13 and why is it a significant target for antibody detection in plant research?

CHX13 is a member of the cation/H+ exchanger family in Arabidopsis thaliana (Mouse-ear cress) involved in ion transport across cellular membranes. Detecting CHX13 protein expression is significant in understanding plant ion homeostasis mechanisms, stress responses, and developmental processes.

The CHX13 antibody enables researchers to:

• Quantify CHX13 protein expression levels across different plant tissues

• Examine subcellular localization of CHX13

• Investigate CHX13 regulation under various environmental conditions

• Study protein-protein interactions involving CHX13

Research using CHX13 antibodies contributes to our understanding of fundamental plant physiology, particularly how plants maintain ion balance in changing environments .

What are the validated applications for CHX13 antibody in plant research?

The polyclonal CHX13 antibody has been validated for specific applications in plant research:

The antibody is particularly effective for Western blot applications where it can specifically identify CHX13 protein. Researchers should optimize dilutions for their specific experimental conditions as these ranges are starting points .

What are the proper storage and handling conditions for CHX13 antibody?

Proper storage and handling are critical for maintaining antibody activity:

• Upon receipt, store the antibody at -20°C or -80°C for long-term storage

• Avoid repeated freeze-thaw cycles which can degrade antibody performance

• For frequent use, prepare small aliquots of the antibody to minimize freeze-thaw cycles

• The antibody is supplied in liquid form containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative

• Working dilutions should be prepared fresh and can be stored at 4°C for up to one week

• Always centrifuge the antibody vial briefly before opening to collect all liquid at the bottom

Proper storage conditions significantly impact experimental reproducibility. Researchers report that antibodies stored according to these guidelines maintain reactivity for the duration of their shelf life .

How should I design positive and negative controls for CHX13 antibody experiments?

Robust experimental design requires appropriate controls:

Positive Controls:

• Wild-type Arabidopsis thaliana tissues known to express CHX13

• Recombinant CHX13 protein (if available)

• Transgenic plants overexpressing CHX13

Negative Controls:

• CHX13 knockout/knockdown plant lines

• Pre-immune serum at the same concentration as the primary antibody

• Primary antibody omission

• Antibody pre-absorbed with the immunizing peptide/protein

For Western blot, expect a specific band at the predicted molecular weight of CHX13 (approximately 85 kDa). Non-specific binding can be reduced by optimizing blocking conditions and antibody dilutions. Methodologically, it's critical to include both positive and negative controls in every experiment to validate specificity .

How can I optimize CHX13 antibody for co-immunoprecipitation (Co-IP) experiments?

While not explicitly validated for Co-IP, researchers can optimize CHX13 antibody for this application:

• Buffer optimization:

  • Start with standard IP buffer (150 mM NaCl, 50 mM Tris pH 7.5, 1% NP-40)

  • For membrane proteins like CHX13, consider adding 0.1-0.5% deoxycholate or digitonin

  • Include protease inhibitors and phosphatase inhibitors if studying phosphorylation

• Pre-clearing strategy:

  • Pre-clear lysates with protein A/G beads for 1 hour at 4°C

  • Use 2-5 μg of antibody per 500 μg of total protein

  • Incubate overnight at 4°C with gentle rotation

• Cross-linking approach:

  • For weak interactions, cross-link the antibody to beads using BS3 or DMP

  • Cross-link protein complexes in vivo using formaldehyde (1% for 10 minutes)

• Elution methods:

  • Gentle elution: competitive elution with immunizing peptide

  • Standard elution: 0.1 M glycine pH 2.5-3.0

  • Denaturing elution: SDS sample buffer at 95°C

Researchers have reported that for membrane proteins like CHX13, digitonin (0.5%) often preserves protein-protein interactions better than harsher detergents. The antibody amount may need further optimization based on its affinity for the target .

What methodological approaches can overcome non-specific binding when using CHX13 antibody?

Non-specific binding is a common challenge with polyclonal antibodies. Several methodological approaches can improve specificity:

• Blocking optimization:

  • Test different blocking agents: 5% BSA, 5% non-fat milk, commercial blocking solutions

  • For plant samples, consider adding 0.1-0.5% plant-derived protein to block plant-specific interactions

  • Extended blocking times (2-3 hours at room temperature or overnight at 4°C)

• Antibody dilution series:

  • Perform a gradient dilution series (1:500, 1:1000, 1:2000, 1:5000)

  • Select the highest dilution that provides clear specific signal

• Sample preparation refinements:

  • Additional centrifugation steps to remove particulates

  • Filtration of lysates through 0.45 μm filters

  • Pre-absorption of antibody with non-target tissue lysates

• Stringency adjustments:

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

  • Add 0.1% SDS or 0.1% Tween-20 to wash buffers

  • Increase number and duration of wash steps

Researchers have found that for CHX13 detection in Arabidopsis, pre-absorbing the antibody with protein extracts from CHX13 knockout plants significantly reduces non-specific binding in Western blots .

How can I effectively use CHX13 antibody for subcellular localization studies?

Determining the subcellular localization of CHX13 requires specialized approaches:

• Immunofluorescence protocol optimization:

  • Fixation: 4% paraformaldehyde for 20 minutes works well for most plant tissues

  • Permeabilization: 0.1-0.5% Triton X-100 for 10-15 minutes

  • Antigen retrieval: Consider citrate buffer (pH 6.0) heating for formalin-fixed samples

  • Antibody incubation: Primary antibody (1:100-1:500) overnight at 4°C

  • Secondary detection: Fluorophore-conjugated secondary antibodies (1:200-1:1000)

• Cell fractionation approach:

  • Prepare subcellular fractions (membrane, cytosolic, nuclear, etc.)

  • Verify fraction purity with established markers

  • Compare CHX13 distribution across fractions by Western blot

  • Quantify relative abundance in each compartment

• Co-localization studies:

  • Use established organelle markers (ER, Golgi, plasma membrane, tonoplast)

  • Calculate co-localization coefficients (Pearson's or Mander's)

  • Perform super-resolution microscopy for precise localization

• Controls for specificity:

  • Include CHX13 knockout plants as negative controls

  • Pre-absorption controls with immunizing peptide

  • Secondary antibody-only controls to assess background

Researchers studying ion transporters like CHX13 often combine these approaches with transient expression of fluorescently-tagged proteins to confirm antibody-based localization results .

What strategies can I use to analyze post-translational modifications of CHX13 using antibodies?

Investigating post-translational modifications (PTMs) of CHX13 requires specialized techniques:

• Phosphorylation analysis:

  • Immunoprecipitate CHX13 using the antibody

  • Probe with phospho-specific antibodies (anti-pSer, anti-pThr, anti-pTyr)

  • Treat samples with phosphatase before analysis as controls

  • Consider phospho-enrichment techniques before analysis

• Mass spectrometry approach:

  • Immunoprecipitate CHX13 using optimized Co-IP protocol

  • Perform in-gel digestion of the band corresponding to CHX13

  • Analyze peptides by LC-MS/MS for PTMs

  • Validate findings using site-specific antibodies if available

• 2D gel electrophoresis:

  • Separate proteins by pI in the first dimension

  • Separate by molecular weight in the second dimension

  • Detect CHX13 by Western blot

  • Multiple spots indicate different modification states

• Functional studies of modifications:

  • Correlate modification status with environmental conditions

  • Assess impact on protein localization

  • Investigate effects on protein-protein interactions

  • Study modification dynamics over time

Research on plant ion transporters has shown that phosphorylation can significantly alter their activity and subcellular localization. Similar proteins in the CHX family have been reported to be regulated by phosphorylation under stress conditions .

Why might I observe multiple bands when using CHX13 antibody in Western blots?

Multiple bands in Western blots can result from several factors:

• Technical explanations:

  • Protein degradation during sample preparation

  • Incomplete denaturation of protein complexes

  • Non-specific binding of the antibody

  • Cross-reactivity with related proteins

• Biological explanations:

  • Alternative splice variants of CHX13

  • Post-translational modifications changing mobility

  • Proteolytic processing in vivo

  • Glycosylated forms with different molecular weights

Methodological approaches to resolve multiple bands:

To determine which band represents the authentic CHX13 signal, compare wild-type samples with CHX13 knockout/knockdown plants or perform peptide competition assays .

How can I quantitatively analyze CHX13 expression levels across different experimental conditions?

For reliable quantitative analysis of CHX13 expression:

• Sample preparation standardization:

  • Use consistent extraction methods across all samples

  • Determine total protein concentration by Bradford or BCA assay

  • Load equal amounts of total protein (20-50 μg) for each sample

  • Include internal loading controls (actin, tubulin, GAPDH)

• Western blot quantification approach:

  • Use gradient gels for better separation

  • Transfer proteins to PVDF membranes for higher binding capacity

  • Include concentration standards if absolute quantification is needed

  • Capture images within the linear range of detection

• Densitometry analysis:

  • Use software like ImageJ, Image Lab, or dedicated Western blot analysis software

  • Subtract background using consistent methodology

  • Normalize CHX13 signal to loading control

  • Present data as fold change relative to control conditions

• Statistical analysis:

  • Perform experiments with at least 3 biological replicates

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

  • Report both means and measures of variation (SD or SEM)

  • Consider biological significance alongside statistical significance

Researchers studying membrane transporters have found that normalization to plasma membrane markers rather than total cellular proteins can provide more accurate quantification when specifically interested in membrane-localized pool of proteins .

How do I validate CHX13 antibody specificity for use in novel plant species or model systems?

When applying CHX13 antibody to new species or systems:

• Sequence homology assessment:

  • Perform BLAST analysis of the immunogen sequence against the target species

  • Calculate percent identity and similarity in the epitope region

  • Higher homology (>70%) suggests potential cross-reactivity

• Western blot validation:

  • Run parallel samples from Arabidopsis (positive control) and the new species

  • Look for bands of appropriate molecular weight

  • Perform peptide competition assays to confirm specificity

  • Include negative controls (pre-immune serum, secondary antibody only)

• Genetic validation approaches:

  • If possible, test tissues from CHX13 knockdown/knockout in the new species

  • Test tissues with experimentally altered CHX13 expression (overexpression)

  • Correlate protein detection with mRNA levels by qRT-PCR

• Mass spectrometry confirmation:

  • Immunoprecipitate the protein from the new species

  • Analyze by mass spectrometry to confirm identity

  • Compare peptide sequences with database entries

For examining CHX13 homologs in crop species, researchers have successfully used antibodies raised against Arabidopsis proteins when sequence homology exceeds 75% in the immunogenic region .

What methodological considerations are important when using CHX13 antibody to study plants under stress conditions?

Studying CHX13 under stress conditions requires special considerations:

• Sampling protocol optimization:

  • Establish precise timing of sample collection

  • Maintain consistent stress application across replicates

  • Consider diurnal variation in protein expression

  • Collect samples from consistent developmental stages

• Protein extraction refinements:

  • Modify extraction buffers for stressed tissues (may have altered pH, ROS levels)

  • Add additional protectants (e.g., higher concentrations of reducing agents)

  • Consider native vs. denaturing extraction depending on research question

  • Test multiple extraction methods for optimal recovery

• Controls for stress experiments:

  • Include time-matched unstressed controls

  • Consider gradient of stress intensity

  • Monitor established stress response markers to confirm stress application

  • Track physiological parameters (e.g., relative water content for drought)

• Data interpretation challenges:

  • Distinguish between changes in expression vs. protein modification

  • Consider subcellular redistribution vs. total expression changes

  • Account for potential stress-induced proteolysis

  • Correlate protein changes with physiological responses

Research on ion transporters has shown that salt, drought, and pH stress can trigger rapid changes in both localization and post-translational modifications, which may affect antibody recognition. Membrane protein extraction efficiency can also vary under different stress conditions, potentially affecting quantification .

How can CHX13 antibody be utilized in single-cell protein analysis of plant tissues?

Emerging technologies enable single-cell analysis of proteins:

• Single-cell immunostaining approaches:

  • Optimize tissue dissociation protocols (enzymatic or mechanical)

  • Perform flow cytometry with intracellular staining for CHX13

  • Use cell sorting to isolate specific cell populations

  • Combine with cell type-specific markers for co-localization

• In situ methodologies:

  • Highly sensitive immunohistochemistry with signal amplification

  • RNA-protein co-detection (IF combined with RNA FISH)

  • Proximity ligation assay for protein-protein interactions

  • Clearing techniques for whole-tissue imaging with preserved protein epitopes

• Microfluidic approaches:

  • Single-cell Western blotting

  • Microfluidic antibody capture of individual cells

  • Droplet-based single-cell protein analysis

  • Integration with transcriptomic analysis

• Analytical considerations:

  • Quantitative image analysis of single-cell immunofluorescence

  • Statistical approaches for handling cell-to-cell variability

  • Computational tools for spatial analysis of expression patterns

  • Integration with single-cell transcriptomics data

Though challenging with plant cells due to cell walls, recent advances in protoplast handling and fixed-cell techniques make single-cell protein analysis increasingly feasible for studying cell-specific expression of transporters like CHX13 .

What considerations are important when designing multiplexed detection systems that include CHX13 antibody?

Multiplexed detection allows simultaneous analysis of multiple proteins:

• Antibody compatibility assessment:

  • Test primary antibodies from different host species to avoid cross-reactivity

  • Validate each antibody independently before multiplexing

  • Ensure non-overlapping epitopes when using multiple antibodies to the same protein

  • Perform sequential staining if same-species antibodies must be used

• Detection system optimization:

  • For fluorescence: Select fluorophores with minimal spectral overlap

  • For chromogenic detection: Use distinct substrates with good separation

  • Consider zenon labeling or directly conjugated primaries to reduce background

  • Test for bleed-through and cross-talk between channels

• Controls for multiplexed systems:

  • Single-antibody controls to establish baseline signals

  • Isotype controls for each host species used

  • Absorption controls to confirm specificity

  • Fluorescence minus one (FMO) controls for flow cytometry

• Data analysis for co-expression:

  • Co-localization analysis with appropriate coefficients

  • Correlation analysis of expression levels

  • Spatial relationship mapping in tissues

  • Quantitative assessment of protein complex formation

Researchers studying ion transport systems have successfully combined CHX-family protein detection with markers for subcellular compartments to precisely map localization changes under different conditions .

How can computational modeling be integrated with CHX13 antibody-based experimental data?

Integrating experimental data with computational approaches enhances research insights:

• Structure-function analysis:

  • Use antibody epitope mapping to validate structural models

  • Correlate antibody accessibility with protein conformational states

  • Map functional domains identified by deletion mutants and antibody binding

  • Combine with molecular dynamics simulations

• Systems biology integration:

  • Incorporate quantitative Western blot data into protein interaction networks

  • Use protein expression data to constrain flux models

  • Correlate protein levels with transcriptomic data in regulatory models

  • Build predictive models of CHX13 regulation under various conditions

• Machine learning applications:

  • Train image analysis algorithms to quantify immunofluorescence patterns

  • Develop predictive models for protein localization based on experimental data

  • Use pattern recognition to identify subtle changes in protein distribution

  • Integrate multi-omics data including antibody-based proteomics

• Visualization tools:

  • Create interactive visualizations of protein expression across tissues/conditions

  • Develop 3D models incorporating localization data

  • Design temporal maps of protein dynamics

  • Generate integrative dashboards for experimental data exploration

Advanced computational approaches have been successfully applied to membrane transport systems in plants, helping to predict regulation mechanisms that can then be validated experimentally using antibodies like anti-CHX13 .

How does the specificity of polyclonal vs. monoclonal CHX13 antibodies compare in research applications?

Comparing antibody types for CHX13 research:

What techniques can determine cross-reactivity between CHX13 antibody and other cation/H+ exchanger family members?

Assessing potential cross-reactivity requires systematic approaches:

• Computational prediction:

  • Sequence alignment of CHX family members

  • Epitope prediction algorithms to identify shared epitopes

  • Structural modeling to assess surface-exposed regions

  • Conservation analysis of immunogen sequence across family members

• Experimental validation:

  • Western blot analysis of recombinant CHX family proteins

  • Testing against tissues from CHX13 and other CHX knockouts

  • Peptide competition assays with immunizing peptide vs. homologous peptides

  • Immunodepletion with recombinant proteins

• Advanced specificity testing:

  • Immunoprecipitation followed by mass spectrometry

  • Protein array screening against all CHX family members

  • Surface plasmon resonance to measure binding kinetics

  • Cross-adsorption with related proteins to remove cross-reactive antibodies

• Visualization of cross-reactivity:

  • Side-by-side immunofluorescence in plants expressing different CHX proteins

  • Co-localization studies with tagged CHX variants

  • Immunogold electron microscopy for precise localization

  • Systematic testing in heterologous expression systems

Research on plant ion transporters indicates that antibodies raised against the C-terminal region of CHX proteins generally show higher specificity than those targeting conserved transmembrane domains .

How might emerging antibody engineering technologies improve CHX13 detection in plant research?

Emerging technologies are transforming antibody-based research:

• Nanobody and single-domain antibody approaches:

  • Smaller size enables better tissue penetration

  • Greater stability in various buffers and conditions

  • Simpler genetic manipulation for fusion proteins

  • Potential for intracellular expression in living plants

• Recombinant antibody fragment development:

  • Fab and scFv fragments with improved tissue penetration

  • Site-specific conjugation for precise labeling

  • Humanized versions for reduced background in some applications

  • Bispecific antibodies for co-detection applications

• Computational antibody design:

  • Structure-based optimization of binding specificity

  • Machine learning approaches to predict cross-reactivity

  • De novo design of synthetic antibodies with desired properties

  • Optimization of physicochemical properties for specific applications

• Novel detection systems:

  • DNA-tagged antibodies for ultrasensitive detection

  • Click chemistry-based labeling for multiplexed analysis

  • Photoactivatable antibody conjugates for super-resolution imaging

  • Self-reporting antibody systems with integrated signal amplification

Computational antibody design is particularly promising, as demonstrated by recent advances in creating antibodies with customized specificity profiles that can distinguish between very similar epitopes, which would be valuable for CHX family research .

What methodological advances could enhance the use of CHX13 antibody in plant developmental studies?

Methodological innovations for developmental studies:

• Tissue clearing techniques:

  • Advanced clearing protocols compatible with antibody epitopes

  • Whole-organ immunolabeling with improved penetration

  • 3D reconstruction of protein expression patterns

  • Integration with fluorescent reporter lines

• Live imaging approaches:

  • Minimally invasive antibody delivery systems

  • Membrane-permeable antibody fragments

  • Photo-convertible antibody labels for pulse-chase studies

  • Correlative light and electron microscopy for context

• Temporal analysis methods:

  • Inducible epitope tagging for temporal control

  • Degradation tag systems for acute protein removal

  • Microfluidic systems for precise timing of treatments

  • Time-resolved microscopy with synchronized populations

• Multi-scale integration:

  • Connecting subcellular localization to tissue-level patterns

  • Spatial transcriptomics combined with protein localization

  • Quantitative modeling of protein expression during development

  • Integration of protein dynamics with morphological changes

These methodological advances could significantly enhance our understanding of CHX13's role in developmental processes and environmental responses in plants .