CHX21 Antibody

What is CHX21 and why is it important in plant research?

CHX21 is a putative sodium transporter protein found in Arabidopsis thaliana, encoded by the AtCHX21 (At2g31910) gene. This protein belongs to the cation/H+ exchanger (CHX) family and plays a significant role in ion homeostasis within plant cells. Understanding CHX21 function is critical for elucidating plant responses to salt stress and ion transport mechanisms across cellular membranes. Research has demonstrated that CHX21 is part of a largely unstudied gene family with potential functional diversification among its members, making it an important target for researchers investigating plant adaptation to environmental stresses . The protein has been studied through knockout experiments and immunolocalization to determine its role in sodium transport within plant tissues, particularly in root cells which are critical for nutrient uptake and stress response .

What applications are recommended for CHX21 antibody use?

CHX21 antibody has been validated for use in enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) applications . These techniques are essential for detecting and quantifying CHX21 protein in plant tissue samples. For Western blot applications, the polyclonal antibody can detect CHX21 in membrane protein extracts from Arabidopsis tissues. The antibody has been successfully used in immunolocalization studies to determine the spatial distribution of CHX21 within plant tissues, particularly in root sections . This application is valuable for understanding the subcellular localization and tissue-specific expression patterns of the protein, providing insights into its functional role.

How is CHX21 antibody specificity validated?

Validation of CHX21 antibody specificity involves multiple approaches to ensure reliable experimental results. First, immunogen design is critical – the antibody described in the literature was raised against a unique peptide sequence (SKSGVRNEMLPYSL) from CHX21 to minimize cross-reactivity . Dot blot assays are typically performed to optimize antibody concentration using alkaline phosphatase-conjugated secondary antibodies. Specificity can be further confirmed through comparative analysis between wild-type and CHX21 knockout mutant plants, where positive signals should be present in wild-type samples but absent in knockout mutants . Additionally, pre-immune serum is used as a negative control to establish baseline signal levels and identify any non-specific binding. Western blot analysis with appropriate positive and negative controls (such as the recombinant immunogen protein provided as a positive control with commercial antibodies) offers definitive confirmation of antibody specificity .

What are the recommended storage conditions for CHX21 antibody?

For optimal stability and performance, CHX21 antibody should be stored at -20°C or -80°C . The antibody is typically shipped on blue ice to maintain its integrity during transport. When working with the antibody, it's recommended to aliquot the stock solution into smaller volumes upon receipt to minimize freeze-thaw cycles, which can degrade antibody performance. For short-term usage (within a week), antibody aliquots can be stored at 4°C. The antibody solution should not be subjected to repeated freeze-thaw cycles, as this can lead to protein denaturation and reduced binding efficiency. Commercial CHX21 antibody typically includes the recombinant immunogen protein as a positive control, which should be stored according to the same conditions to maintain its integrity for validation experiments .

How can CHX21 antibody be used to study CHX21 localization in different plant tissues?

Immunolocalization using CHX21 antibody provides valuable insights into the tissue-specific distribution and subcellular localization of the protein. The following methodology has been successfully employed in research: Plant roots are sectioned and fixed in 2% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M sodium cacodylate buffer overnight . After fixation, samples undergo dehydration through an alcohol series to 90% ethanol, followed by resin embedding using LR White hard grade resin. Root bundles are transferred to gelatin capsules and polymerized under UV light for approximately 48 hours . Ultrathin sections (2 μm) are cut using an ultramicrotome for immunostaining.

For fluorescence immunolocalization, sections are incubated with CHX21 primary antibody at a 1:1000 dilution in blocking buffer (1% BSA, 0.1% Triton X-100 in PBS), followed by a fluorescein isothiocyanate (FITC)-conjugated secondary antibody at 1:30 dilution . Visualization is performed using fluorescence microscopy, which allows for the precise determination of CHX21 distribution within different cell types and cellular compartments. This approach has revealed specific localization patterns in Arabidopsis root tissues, providing functional insights into the role of CHX21 in ion transport processes across different cellular membranes and tissues.

What controls should be included when using CHX21 antibody in experimental designs?

A robust experimental design for CHX21 antibody applications requires several carefully selected controls. For genotype controls, compare wild-type plants with CHX21 knockout mutants (chx21) to verify antibody specificity – knockout plants should show absence or significant reduction of signal . Commercial CHX21 antibody kits often include pre-immune serum that should be used as a negative control at the same concentration as the primary antibody to establish baseline non-specific binding levels .

For protein loading controls in Western blots, use antibodies against constitutively expressed membrane proteins (e.g., plasma membrane H+-ATPase for membrane fractions) to normalize CHX21 signal intensity. When performing immunolocalization, include blocking peptide controls by pre-incubating the antibody with excess immunogenic peptide (SKSGVRNEMLPYSL) before applying to tissues – this should abolish specific binding .

Additionally, positive control materials such as the recombinant immunogen protein/peptide provided with commercial antibodies can verify detection sensitivity . For cross-reactivity assessment, test the antibody against related CHX family proteins if available, particularly those with high sequence homology, to confirm the specificity for CHX21 rather than related proteins within the cation/H+ exchanger family.

How can CHX21 antibody be used to study protein-protein interactions in the CCR5 signalosome?

While CHX21 is a plant protein, the methodology used for studying protein-protein interactions can be informed by approaches used for other membrane proteins such as CCR5. In CCR5 signalosome studies, co-immunoprecipitation (co-IP) assays have been successfully employed to identify interacting partners . Similarly, for CHX21 research, co-IP can be performed by incubating plant membrane protein extracts with CHX21 antibody pre-adsorbed to protein A/G beads, followed by Western blot analysis to detect potential interacting proteins.

Biolayer interferometry (BLI) has been used to confirm antibody class assignments for other membrane proteins and could be adapted for CHX21 interaction studies . This approach allows real-time monitoring of protein binding kinetics without labeling requirements. Additionally, methods such as proximity ligation assay (PLA) can provide in situ visualization of protein-protein interactions with high specificity and sensitivity. This technique uses antibodies against two potentially interacting proteins along with oligonucleotide-conjugated secondary antibodies that generate fluorescent signals when in close proximity.

For temporal analysis of protein interactions, researchers can apply the model demonstrated with CCR5, where cells are collected at different time points after stimulation (e.g., t1 at 48h and t2 at 48h plus 24h additional incubation) to monitor dynamic changes in protein complex formation . These approaches can reveal how CHX21 may function within larger protein complexes involved in ion transport and stress response signaling networks.

How does the CHX21 knockout affect downstream signaling pathways?

Analysis of CHX21 knockout effects on signaling pathways requires a multi-faceted approach. RT-PCR analysis can confirm the absence of CHX21 transcript in knockout mutants, as demonstrated in previous research where cDNA of the predicted size was evident in wild-type plants but undetectable in chx21 mutants . The knockout verification can be further confirmed through Southern blot analysis using gene-specific probes, which has shown single bands in mutant DNA samples, confirming single insertion events .

For downstream signaling analysis, researchers should consider:

• Transcriptome profiling: RNA sequencing comparing wild-type and chx21 knockout plants under various stress conditions (particularly salt stress) to identify differentially expressed genes

• Ion flux measurements: Using ion-selective microelectrodes to measure Na+/H+ exchange activity in different tissues

• Phosphoproteomic analysis: To identify changes in protein phosphorylation patterns that may indicate altered signaling cascades

• Measurement of second messengers: Calcium imaging or quantification of other signaling molecules that may be affected by altered ion homeostasis

The chx21 knockout may display altered expression patterns of genes involved in salt stress response pathways or other ion transport mechanisms. These changes can be quantified using real-time PCR for selected genes or global transcriptome analysis. Phenotypic analysis under different stress conditions provides functional insights into the role of CHX21 in stress adaptation mechanisms .

What protein extraction protocols are optimal for detecting CHX21 in plant tissues?

Efficient extraction of membrane-bound proteins like CHX21 requires specialized protocols to preserve protein integrity while maximizing yield. A recommended extraction protocol begins with harvesting fresh plant tissue (preferably roots, where CHX21 is predominantly expressed) and grinding in liquid nitrogen to a fine powder. The ground tissue is then homogenized in extraction buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton X-100, and protease inhibitor cocktail .

After centrifugation at 1,000 × g for 10 minutes to remove cell debris, the supernatant undergoes ultracentrifugation at 100,000 × g for 1 hour to isolate the membrane fraction. The resulting pellet, containing membrane proteins including CHX21, is resuspended in solubilization buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% dodecyl maltoside or other appropriate detergent, and protease inhibitors) .

Protein concentration should be quantified using the Bradford Bio-Rad assay prior to Western blot analysis . For plants grown under different experimental conditions, it's crucial to standardize the extraction protocol and loading amounts to ensure comparable results. When extracting proteins from different plant tissues or developmental stages, optimization of detergent type and concentration may be necessary to account for variations in membrane composition.

What are the recommended parameters for Western blot detection of CHX21?

For optimal Western blot detection of CHX21 protein, the following protocol parameters are recommended based on research applications:

For CHX21 detection, it's important to note that membrane proteins can form aggregates when boiled, so sample heating at 37°C is preferred. Signal specificity can be verified using wild-type versus chx21 knockout samples as controls . For challenging samples, signal enhancement systems or more sensitive detection methods like fluorescent secondary antibodies may improve results. The expected molecular weight should be compared with the theoretical molecular weight calculated from the amino acid sequence, accounting for potential post-translational modifications or protein processing.

How can CHX21 antibody be optimized for immunofluorescence applications?

Optimizing CHX21 antibody for immunofluorescence applications requires careful attention to fixation, permeabilization, and antibody incubation conditions. Based on successful protocols, a combination of 2% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M sodium cacodylate provides effective fixation while preserving antigen accessibility . For plant tissues, embedding in LR White resin and polymerization under UV light produces sections suitable for immunostaining.

Antigen retrieval methods may improve signal detection, particularly with aldehyde-fixed samples. Citrate buffer (10 mM sodium citrate, pH 6.0) heating at 95°C for 10-20 minutes or enzymatic treatment with proteases may unmask epitopes. Optimization of permeabilization is critical – 0.1% Triton X-100 in the blocking buffer provides a starting point, but concentration may need adjustment based on tissue type .

For antibody incubation, a 1:1000 dilution of primary antibody in blocking buffer (1% BSA, 0.1% Triton X-100 in PBS) has been successful, with overnight incubation at 4°C providing optimal binding . FITC-conjugated secondary antibodies at 1:30 dilution provide sufficient signal, but fluorophore selection should consider tissue autofluorescence characteristics. Signal-to-noise ratio can be improved through extended washing steps (3× 15 minutes) and inclusion of 0.05% Tween-20 in wash buffers. Counterstaining with DAPI allows nuclear visualization for subcellular localization context.

What approaches can be used to study CHX21 protein turnover and degradation pathways?

Investigating CHX21 protein turnover and degradation pathways requires techniques that can track protein synthesis, stability, and degradation over time. Cycloheximide (CHX) chase assays have been successfully employed to study protein synthesis inhibition and subsequent degradation of CHX21 . In this approach, plant tissues or cell cultures are treated with cycloheximide to block new protein synthesis, followed by sample collection at various time points (e.g., 48h, 48h+24h, 48h+72h) to monitor protein degradation rates .

Pulse-chase experiments using metabolic labeling can provide quantitative data on CHX21 turnover rates. Cells are briefly exposed to media containing radioactively labeled amino acids (pulse), followed by incubation in normal media (chase). At various time points, CHX21 is immunoprecipitated and the radioactive signal is measured to determine degradation kinetics.

To identify specific degradation pathways, selective inhibitors can be employed:

• MG132 for proteasome-mediated degradation

• Concanamycin A for vacuolar/lysosomal degradation

• 3-methyladenine for autophagy inhibition

Changes in CHX21 levels after treatment with these inhibitors can reveal the primary degradation pathways. For in vivo studies, transgenic plants expressing CHX21 fused to fluorescent timer proteins that change color with age can provide spatial and temporal information about protein turnover within living tissues. Real-time RT-PCR analysis comparing mRNA and protein levels can distinguish between transcriptional and post-transcriptional regulation mechanisms .

How can researchers address non-specific binding when using CHX21 antibody?

Non-specific binding is a common challenge when working with polyclonal antibodies like those against CHX21. Several strategies can effectively minimize this issue:

First, optimize blocking conditions by testing different blocking agents (BSA, non-fat milk, normal serum, commercial blocking buffers) at various concentrations (3-5%). Extending the blocking time to 2 hours at room temperature or overnight at 4°C can significantly reduce background signal. When performing Western blots, adding 0.1-0.3% Tween-20 to wash buffers and increasing both the number (3-5) and duration (10-15 minutes) of washes helps eliminate non-specific binding .

Pre-adsorption of the antibody with non-target tissues (e.g., chx21 knockout plant extract) can remove antibodies that bind to non-target epitopes. For immunohistochemistry applications, include 0.1-0.3% Triton X-100 in antibody dilution buffers to reduce hydrophobic interactions while ensuring adequate permeabilization .

Titrating primary antibody concentration is essential – begin with the manufacturer's recommended dilution (typically 1:1000) and test a range of concentrations to determine optimal signal-to-noise ratio . Using a more dilute secondary antibody (1:5000-1:10000) can also reduce background. For particularly challenging samples, consider using more specific detection systems such as tyramide signal amplification, which provides signal enhancement while maintaining specificity.

How should researchers interpret contradictory results between transcript and protein levels of CHX21?

Discrepancies between CHX21 transcript and protein levels are not uncommon and may reveal important biological regulatory mechanisms. When interpreting such contradictions, researchers should consider several possible explanations:

Post-transcriptional regulation may significantly impact CHX21 protein levels. MicroRNAs or RNA-binding proteins could affect mRNA stability or translation efficiency, leading to reduced protein despite abundant transcript. Post-translational modifications, such as phosphorylation or ubiquitination, can affect protein stability and turnover rates, potentially causing rapid degradation of the protein despite high transcript levels .

Protein localization effects should be considered – CHX21 might be sequestered in specific cellular compartments that are poorly represented in whole-cell extracts, leading to apparently low protein levels despite high transcript abundance. Technical considerations include extraction efficiency (membrane proteins like CHX21 may require specialized extraction protocols) and antibody sensitivity (the detection limit of the antibody may not be sufficient for low-abundance proteins) .

To resolve these discrepancies, researchers should employ complementary approaches:

• Use multiple antibodies targeting different epitopes of CHX21

• Perform subcellular fractionation to enrich for membrane proteins

• Employ pulse-chase experiments to assess protein synthesis and degradation rates

• Utilize proteasome or lysosome inhibitors to test if protein degradation explains low protein levels

• Analyze polysome-associated mRNA to determine translation efficiency

These discrepancies often reveal important regulatory mechanisms and should be viewed as opportunities for deeper investigation rather than experimental failures.

What are the key considerations when comparing CHX21 expression across different experimental conditions?

When comparing CHX21 expression across different experimental conditions, several key factors must be carefully controlled to ensure reliable and interpretable results:

Standardization of growth conditions is paramount – plants should be grown under identical conditions (light intensity, photoperiod, temperature, humidity, nutrient composition) up until the point where experimental treatments are applied. Age-matched plants at the same developmental stage should be used, as CHX21 expression may vary throughout development .

For transcript analysis, consistent RNA extraction methods, DNase treatment protocols, and reverse transcription conditions are essential. Multiple reference genes (at least 3) should be validated for stability across all experimental conditions for accurate normalization of RT-PCR data. For protein analysis, standardized protein extraction protocols specifically optimized for membrane proteins should be employed consistently .

Biological variability should be addressed through sufficient biological replicates (minimum n=3, ideally n≥5) and appropriate statistical analysis. Technical replicates (minimum n=2-3) help identify measurement variation. Time-course experiments rather than single time-point analyses provide more comprehensive understanding of expression dynamics, particularly for stress responses where timing of induction/repression is critical .

When interpreting results, consider that CHX21 may respond differently across tissues – tissue-specific analysis may reveal localized responses not apparent in whole-plant studies. Additionally, cross-talk between signaling pathways means that CHX21 responses to one stress may be influenced by other environmental factors, necessitating carefully controlled single-variable experiments or factorial designs that account for interactions.

How can bispecific antibody approaches be adapted to study CHX21 interaction with other membrane proteins?

Bispecific antibody approaches, while more commonly used in immunotherapy research, offer innovative possibilities for studying CHX21 interactions with other membrane proteins. Drawing from principles used in PD-1/CTLA-4 bispecific antibody development , researchers can develop custom bispecific antibodies that simultaneously bind CHX21 and potential interacting partners.

To implement this approach, researchers would first identify candidate interaction partners through preliminary co-immunoprecipitation or proximity labeling experiments. Next, they would design a bispecific antibody construct with one arm targeting CHX21 and another targeting the candidate interacting protein. Surface plasmon resonance (SPR) validation, as used for therapeutic bispecific antibodies, would confirm binding to both target proteins .

The bispecific construct could be used for:

• Pull-down assays to capture intact protein complexes while maintaining native membrane architecture

• Flow cytometry analysis of protoplasts or membrane vesicles to quantify co-localization frequency

• Super-resolution microscopy to visualize protein interactions in situ with minimal disruption

• Förster resonance energy transfer (FRET) applications when conjugated with appropriate fluorophores