At1g14686 Antibody

What is the At1g14686 antibody and what protein does it target?

The At1g14686 antibody is a polyclonal antibody specifically raised against a peptide sequence (amino acids 223–235) of the Arabidopsis thaliana Phosphoribulokinase (PRK) protein, which is encoded by the gene At1g14686. Phosphoribulokinase functions as a chloroplast-localized enzyme that plays an essential role in the Calvin-Benson cycle, where it catalyzes the critical phosphorylation of ribulose-5-phosphate to ribulose-1,5-bisphosphate. The antibody demonstrates high specificity by targeting a unique epitope within the PRK protein's conserved region, which significantly minimizes cross-reactivity with other plant proteins. This specificity makes it particularly valuable for researchers investigating photosynthetic efficiency and carbon fixation mechanisms in Arabidopsis and related model plant systems. The antibody has been validated predominantly with Arabidopsis thaliana samples, and researchers should note that reactivity has primarily been confirmed only in this species.

What are the primary experimental applications of the At1g14686 antibody?

The At1g14686 antibody serves multiple experimental purposes in plant biology research focused on photosynthesis and carbon metabolism. Primarily, researchers utilize this antibody to detect Phosphoribulokinase expression in chloroplasts through Western blotting and ELISA techniques, enabling quantitative assessment of protein levels across different experimental conditions. The antibody is particularly valuable for investigating photosynthetic efficiency and carbon fixation mechanisms in Arabidopsis mutants, allowing researchers to correlate phenotypic changes with alterations in PRK expression or localization. Additionally, the At1g14686 antibody serves as an effective validation tool for gene-editing outcomes, such as verifying CRISPR/Cas9 modifications targeting the At1g14686 locus in transgenic plant lines. The technical flexibility of this antibody makes it compatible with multiple experimental workflows, including various types of plant tissue lysate analysis methods, from crude extracts to purified chloroplast fractions. Research applications extend to stress response studies examining PRK activity under drought or high-light conditions, metabolic engineering approaches aimed at enhancing crop yield through modulation of Calvin cycle enzymes, and protein localization studies investigating subcellular distribution in chloroplast stroma or thylakoid membranes.

How does the At1g14686 antibody compare to other plant protein-targeting antibodies?

The At1g14686 antibody possesses distinct characteristics that differentiate it from other plant protein-targeting antibodies, particularly when compared to antibodies targeting transcription factors or transport proteins. As detailed in comparative studies, the At1g14686 antibody (catalog No. CSB-PA862848) specifically targets the PRK enzyme involved in carbon fixation, while other antibodies like BRN2 (CSB-PA920279) target transcription factors, and BOR1 (CSB-PA837778) target proteins involved in boron transport. The technical applications also differ, with the At1g14686 antibody optimized for ELISA and Western blotting applications, whereas the BRN2 antibody is suitable for Western blotting and immunohistochemistry, and the BOR1 antibody is designed for Western blotting and immunofluorescence. The following table provides a comparative analysis of these antibodies:

This comparison illustrates how the choice of antibody depends significantly on the specific research question and experimental approach.

What is the recommended protocol for Western blotting using the At1g14686 antibody?

The optimal Western blotting protocol for the At1g14686 antibody begins with proper sample preparation from plant tissues, typically involving grinding leaf tissue in liquid nitrogen followed by extraction in a buffer containing phosphatase and protease inhibitors to preserve protein integrity. Following standard SDS-PAGE separation (typically on 10-12% polyacrylamide gels), proteins should be transferred to PVDF or nitrocellulose membranes using either wet or semi-dry transfer systems. Blocking should be performed with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature, after which the membrane should be incubated with the At1g14686 antibody at a recommended dilution of 1:1000 to 1:2000 in blocking buffer overnight at 4°C. After washing with TBST (3-5 times for 5 minutes each), the membrane should be incubated with HRP-conjugated anti-rabbit secondary antibody at a 1:5000 dilution for 1 hour at room temperature. The Phosphoribulokinase protein (approximately 45 kDa) can be visualized using ECL detection reagents following manufacturer's protocols, with expected band intensity correlating with expression levels. For quantitative analysis, researchers should include appropriate loading controls such as anti-actin or anti-tubulin antibodies, and consider using fluorescent secondary antibodies for more precise quantification if available.

How should researchers optimize ELISA procedures when using the At1g14686 antibody?

For ELISA optimization with the At1g14686 antibody, researchers should begin by coating high-binding microplates with purified protein extracts or crude chloroplast fractions (typically diluted in carbonate-bicarbonate coating buffer pH 9.6) overnight at 4°C. After washing with PBS-T (PBS with 0.05% Tween-20), plates should be blocked with 2-5% BSA or non-fat dry milk for 1-2 hours at room temperature. The At1g14686 primary antibody should be applied at various dilutions (starting with 1:500 to 1:2000) in blocking buffer to determine optimal concentration, and incubated for 2 hours at room temperature or overnight at 4°C. Following thorough washing (4-6 times with PBS-T), an HRP-conjugated anti-rabbit secondary antibody should be added at a 1:5000 to 1:10000 dilution and incubated for 1 hour at room temperature. After final washing steps, substrate solution (typically TMB for HRP detection) should be added and allowed to develop for the appropriate time (5-30 minutes) before stopping the reaction with sulfuric acid or other suitable stopping solution. Absorbance values should be measured at the appropriate wavelength (typically 450 nm for TMB) using a microplate reader, with signal intensity correlating with Phosphoribulokinase concentration in the samples. To ensure reliability, researchers should always include positive controls (purified PRK protein if available), negative controls (samples from PRK-knockout plants), and standard curves using recombinant protein when performing quantitative analysis.

What sample preparation techniques maximize antibody performance for plant tissue analysis?

Optimal sample preparation for plant tissue analysis using the At1g14686 antibody requires several critical considerations to preserve protein integrity and maximize detection sensitivity. For most applications, fresh plant tissue should be flash-frozen in liquid nitrogen immediately after collection and ground to a fine powder using a pre-chilled mortar and pestle or mechanical homogenizer to prevent protein degradation. Extraction buffers should contain 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, supplemented with protease inhibitor cocktail and phosphatase inhibitors (1 mM sodium orthovanadate, 10 mM sodium fluoride) to prevent proteolysis and preserve post-translational modifications. When specifically targeting chloroplast proteins like Phosphoribulokinase, researchers may benefit from chloroplast isolation protocols using Percoll gradient centrifugation prior to protein extraction, which can significantly enhance signal-to-noise ratios by enriching for the target compartment. Following extraction, samples should be centrifuged at 12,000-15,000 × g for 15 minutes at 4°C to remove cell debris, and protein concentration should be determined using Bradford or BCA assays prior to experimental use. For applications requiring higher purity, researchers may implement additional purification steps such as ammonium sulfate precipitation or ion exchange chromatography, which can further improve specific detection of the PRK protein in complex samples.

How can the At1g14686 antibody be utilized in studies of plant stress responses?

The At1g14686 antibody offers valuable insights into plant stress response mechanisms by enabling researchers to monitor Phosphoribulokinase expression and activity changes under various environmental stressors. In drought stress studies, researchers can use the antibody to quantify PRK protein levels via Western blotting at different time points following water withdrawal, correlating changes with physiological measurements such as relative water content, stomatal conductance, and photosynthetic efficiency measured by chlorophyll fluorescence. For high-light stress investigations, the antibody allows researchers to monitor PRK expression changes during photoacclimation processes, providing insights into how plants adjust their carbon fixation machinery when exposed to excess light energy that could potentially cause photoinhibition. The antibody can be incorporated into co-immunoprecipitation experiments to identify stress-induced protein-protein interactions between PRK and other Calvin cycle enzymes or regulatory proteins, potentially uncovering novel regulatory mechanisms activated during environmental challenges. Additionally, researchers can employ the antibody in immunolocalization studies using confocal microscopy to track changes in PRK subcellular distribution under stress conditions, which may reveal stress-induced relocalization patterns associated with metabolic adaptation. Integration of At1g14686 antibody-based protein data with transcriptomic and metabolomic analyses provides a comprehensive multi-omics approach to understanding how carbon fixation responds to environmental stressors, potentially identifying key regulatory nodes for improving crop resilience.

What approaches enable researchers to study PRK enzyme activity in conjunction with At1g14686 antibody detection?

Advanced research integrating PRK enzyme activity with At1g14686 antibody detection requires complementary experimental strategies that link protein abundance with functional output. Researchers can isolate active PRK enzyme from plant tissues using immunoprecipitation with the At1g14686 antibody coupled to protein A/G magnetic beads or agarose, followed by spectrophotometric activity assays that measure the ATP-dependent conversion of ribulose-5-phosphate to ribulose-1,5-bisphosphate, typically by coupling to NADH oxidation via auxiliary enzymes. For in situ activity visualization, researchers may combine enzyme activity staining in native gels with subsequent Western blotting using the At1g14686 antibody on parallel gel lanes, allowing correlation between activity bands and PRK protein bands. Pulse-chase experiments using radioactive carbon (^14C) can track carbon fixation rates in plant tissues under various conditions, with subsequent immunoprecipitation using the At1g14686 antibody to determine the specific contribution of PRK to observed metabolic fluxes. To investigate post-translational modifications affecting PRK activity, researchers can perform immunoprecipitation with the At1g14686 antibody followed by mass spectrometry analysis to identify phosphorylation, redox modifications, or other regulatory changes that correlate with alterations in enzyme activity under different physiological conditions. Coupling these approaches with reverse genetic techniques, such as CRISPR/Cas9-mediated mutagenesis of specific PRK residues, allows researchers to establish causal relationships between protein structure, abundance, and catalytic function in planta.

How can immunolocalization with the At1g14686 antibody enhance understanding of PRK's role in chloroplast function?

Immunolocalization using the At1g14686 antibody provides valuable spatial information about PRK distribution within chloroplasts, enhancing our understanding of how enzyme compartmentalization contributes to photosynthetic efficiency. Researchers can perform immunogold electron microscopy by fixing plant tissue in paraformaldehyde/glutaraldehyde, embedding in LR White resin, sectioning ultrathin samples, and incubating with the At1g14686 antibody followed by gold-conjugated secondary antibodies, allowing precise nanometer-scale localization of PRK within thylakoid membranes, stroma, or potential microcompartments. For confocal microscopy approaches, researchers should fix plant tissue in 4% paraformaldehyde, permeabilize with a detergent like Triton X-100, block with BSA or normal serum, and incubate with the At1g14686 antibody followed by fluorophore-conjugated secondary antibodies, ideally using co-staining with organelle markers such as chlorophyll autofluorescence for thylakoids or fluorescently-tagged proteins for other chloroplast compartments. Super-resolution microscopy techniques like STORM or PALM can be employed with appropriate fluorophore-conjugated secondary antibodies to achieve resolution beyond the diffraction limit, potentially revealing PRK organization into metabolic microdomains or enzyme complexes not visible by conventional microscopy. Live-cell imaging of PRK dynamics can be achieved by correlating antibody immunolocalization in fixed cells with fluorescent protein fusions in living cells, validating that tagged constructs localize to the same compartments as the endogenous protein detected by the antibody. Integration of immunolocalization data with physiological measurements of carbon fixation rates in wild-type versus PRK mutant plants can establish structure-function relationships between enzyme spatial organization and photosynthetic efficiency, potentially identifying optimization strategies for improved carbon capture.

What are the most common issues when using the At1g14686 antibody and how can they be resolved?

Researchers working with the At1g14686 antibody may encounter several common technical challenges that can be systematically addressed through protocol optimization. Weak or absent signals in Western blotting often result from protein degradation during extraction, which can be mitigated by working quickly on ice, adding additional protease inhibitors to extraction buffers, and avoiding freeze-thaw cycles of samples. High background issues in immunoblotting typically stem from insufficient blocking or washing, and can be resolved by extending blocking time to 2 hours, increasing blocking agent concentration to 5-10%, using alternative blocking agents like casein instead of BSA, and implementing more stringent washing steps (5-6 washes of 10 minutes each) with higher Tween-20 concentrations (0.1-0.2%) in wash buffers. Non-specific bands may appear due to antibody cross-reactivity, which can be addressed by further diluting the primary antibody (1:2000-1:5000), reducing incubation temperature to 4°C, or performing antibody pre-absorption with non-specific plant proteins prior to use. Variable results between experiments often stem from inconsistent sample loading or transfer efficiency, which can be controlled by careful protein quantification, inclusion of multiple housekeeping controls, and implementation of stain-free gel technology or Ponceau S staining to verify transfer efficiency prior to antibody incubation. Temperature sensitivity during shipping or storage may compromise antibody performance, so researchers should aliquot antibodies upon receipt to avoid repeated freeze-thaw cycles, store at -20°C or -80°C for long-term storage, and validate each new lot with positive control samples before use in critical experiments.

What controls should be included when using the At1g14686 antibody in plant research?

Robust experimental design with the At1g14686 antibody requires implementation of appropriate controls to ensure reliable and interpretable results. Positive controls should include wild-type Arabidopsis thaliana leaf extracts known to express PRK protein, ideally alongside recombinant PRK protein when available, which establishes the correct molecular weight band position and confirms antibody functionality. Negative controls should incorporate PRK-knockout or knockdown mutant plant extracts when available, or alternatively, non-photosynthetic plant tissues with minimal PRK expression such as roots, which helps identify potential non-specific binding. Loading controls are essential for Western blotting applications and should include antibodies against stable housekeeping proteins such as actin, tubulin, or GAPDH, or chloroplast-specific proteins like Rubisco for normalization when specifically studying chloroplast proteins. Pre-immune serum controls (using serum collected from the host animal before immunization) can help distinguish between specific binding and inherent serum reactivity to plant proteins, though this may not be available for all commercial antibodies. Peptide competition assays, where the antibody is pre-incubated with excess immunizing peptide prior to application, can confirm binding specificity by demonstrating signal reduction when the antibody's binding sites are occupied by soluble peptide. Technical replicates (multiple lanes of the same sample) and biological replicates (samples from different plants or treatments) should be included to assess reproducibility and natural variation, with a minimum of three biological replicates recommended for statistical validity.

How should researchers properly store and handle the At1g14686 antibody to maintain its activity?

Proper storage and handling practices are crucial for maintaining At1g14686 antibody activity and ensuring consistent experimental results across multiple studies. Upon receipt, researchers should immediately aliquot the antibody into small volumes (10-50 μL) in sterile microcentrifuge tubes to minimize freeze-thaw cycles, as repeated freezing and thawing significantly diminishes antibody binding capacity and specificity. Long-term storage should be at -20°C or -80°C, with antibody aliquots kept in the dark to prevent light-induced degradation of proteins and preservatives. Working dilutions prepared for immediate use should be stored at 4°C and used within 1-2 weeks, with addition of preservatives such as sodium azide (0.02%) to prevent microbial contamination during short-term storage (though researchers should note that the antibody may already contain ProClin 300 as a preservative, which requires careful handling due to its hazardous nature). Researchers should avoid vortexing antibody solutions, as this can lead to protein denaturation and aggregation, instead mixing by gentle inversion or low-speed pulse centrifugation. Prior to each use, antibody aliquots should be thawed completely on ice or at 4°C and centrifuged briefly to collect the solution at the bottom of the tube and remove any potential aggregates. Quality assessments should be performed periodically, especially for antibodies stored for extended periods, by testing activity with positive control samples and validating the expected band pattern in Western blots or signal intensity in ELISA. Proper shipping conditions should be maintained when transferring antibodies between laboratories, ideally on dry ice for frozen antibodies or with ice packs for antibodies shipped at 4°C, with temperature monitoring during transport when possible.

How might the At1g14686 antibody contribute to crop improvement strategies?

The At1g14686 antibody holds significant potential for advancing crop improvement strategies through enhanced understanding of photosynthetic efficiency and carbon fixation mechanisms. Researchers can utilize the antibody in comparative studies across crop varieties with differing photosynthetic efficiencies, quantifying PRK protein levels and correlating them with carbon assimilation rates, biomass production, and yield parameters to identify promising genetic backgrounds for breeding programs. The antibody enables screening of transgenic crops with engineered Calvin cycle modifications, providing a tool to verify altered PRK expression levels, protein stability, and proper subcellular localization in plants modified to enhance photosynthetic capacity through increased carbon fixation enzyme activity. In studies exploring natural variation, the antibody can help characterize PRK protein levels across diverse germplasm collections, potentially identifying naturally occurring variants with optimized enzyme expression that correlate with improved photosynthetic performance under various environmental conditions. Field trials of crops under actual agricultural conditions can incorporate protein-level analysis using the At1g14686 antibody to examine how PRK expression responds to fluctuating light, temperature, and water availability in real-world scenarios, generating data more relevant to farming contexts than controlled laboratory studies. Integration with emerging CRISPR/Cas9 genome editing approaches allows researchers to precisely modify PRK regulatory elements or protein structure, with the antibody serving as a crucial tool to validate the intended protein-level changes in edited plants before proceeding to more extensive phenotypic analysis.

What emerging technologies might enhance the utility of the At1g14686 antibody in plant research?

Emerging technologies are poised to significantly expand the utility of the At1g14686 antibody in advanced plant research applications . Single-cell proteomics approaches, when coupled with the At1g14686 antibody, could enable cell-type-specific analysis of PRK expression across different leaf tissues and developmental stages, revealing previously undetectable spatial heterogeneity in photosynthetic enzyme distribution . Microfluidic antibody arrays may allow simultaneous measurement of PRK alongside dozens of other photosynthetic and metabolic enzymes from minimal sample volumes, providing comprehensive protein interaction networks that regulate carbon fixation under different conditions . CRISPR-based protein tagging systems could be developed to introduce epitope tags into the endogenous PRK locus, enabling more sensitive detection with commercial tag antibodies while validating localization patterns using the original At1g14686 antibody as a reference standard . Combining the antibody with proximity labeling techniques such as BioID or APEX could map the PRK protein interaction network within the chloroplast, potentially revealing previously unknown regulatory partners that influence enzyme activity or stability . Advanced multiplexed imaging techniques using antibody-based detection with metal-tagged secondary antibodies and mass cytometry (CyTOF) could simultaneously visualize dozens of proteins within the same chloroplast sections, contextualizing PRK localization within the broader photosynthetic machinery . Integration with artificial intelligence approaches for image analysis could extract subtle patterns in PRK distribution from immunolocalization experiments that are not apparent to human observers, potentially revealing functional microdomains within chloroplasts that optimize carbon fixation efficiency .

How might antibody engineering improve the specificity and utility of At1g14686 antibody variants?

Antibody engineering approaches offer promising avenues for enhancing the specificity and expanding the utility of At1g14686 antibody variants in plant research applications . Recombinant antibody technology could be employed to convert the polyclonal At1g14686 antibody into monoclonal variants with defined epitope specificity, potentially reducing batch-to-batch variation and enhancing reproducibility across different research groups . Fragment-based approaches could generate smaller antibody derivatives such as Fab or scFv fragments that maintain PRK binding specificity while providing better tissue penetration for immunohistochemistry applications, particularly for densely packed chloroplast samples . Species cross-reactivity could be engineered by modifying the complementarity-determining regions (CDRs) to recognize conserved PRK epitopes across multiple plant species, enabling comparative studies of photosynthetic regulation in crops beyond Arabidopsis . Fusion proteins combining the At1g14686 antibody with fluorescent proteins could enable direct detection without secondary antibodies, simplifying immunofluorescence protocols and reducing background from secondary antibody cross-reactivity . Surface modification strategies could optimize the antibody for specific applications, such as adding polyethylene glycol to improve stability for field-portable diagnostic kits or conjugating to magnetic nanoparticles for more efficient immunoprecipitation of PRK protein complexes . Bispecific antibody formats could be developed to simultaneously target PRK and interacting proteins, enabling visualization or pulldown of complete enzyme complexes rather than individual proteins, potentially revealing dynamic assembly of metabolons during photosynthetic regulation .