At4g31010 Antibody

What is At4g31010 and what is its biological significance?

At4g31010 encodes the CRS2-associated factor 1 mitochondrial protein in Arabidopsis thaliana (Mouse-ear cress), a model plant organism widely used in molecular biology research . This protein consists of 405 amino acids and plays a critical role in mitochondrial function within plant cells . According to database identifiers, this gene/protein is referenced in multiple biological databases including KEGG (ath:AT4G31010), STRING (3702.AT4G31010.1), and UniGene (At.31782), indicating its importance in plant biology research . The protein is involved in mitochondrial RNA processing pathways, making it particularly relevant for studies investigating organellar gene expression and energy metabolism in plants. Understanding this protein's function provides insights into fundamental aspects of plant cellular biology and adaptation mechanisms.

What are the optimal storage conditions for At4g31010 antibodies?

At4g31010 antibodies should be stored according to specific conditions to maintain their functionality and specificity. Upon receipt, store the antibody at -20°C or -80°C to preserve its activity . For ongoing research, the antibody is maintained in a storage buffer containing 50% Glycerol and 0.01M PBS at pH 7.4, with 0.03% Proclin 300 as a preservative . Repeated freeze-thaw cycles should be strictly avoided as they can significantly degrade antibody performance . For working solutions, aliquoting the antibody into smaller volumes is recommended to minimize freeze-thaw cycles. When handling the antibody, always use sterile technique and keep the antibody on ice during experimental procedures. These storage protocols ensure optimal antibody performance in various experimental applications.

What validation methods confirm At4g31010 antibody specificity?

Validation of At4g31010 antibody specificity requires a multi-faceted approach to ensure reliable experimental results. The antibody has been validated through antigen affinity purification methods, which significantly enhances its specificity for the target protein . Western blot analysis should be performed using Arabidopsis thaliana tissue extracts, comparing wild-type samples with At4g31010 knockout/knockdown lines as negative controls. The antibody should recognize a protein band at the expected molecular weight of the At4g31010 protein. Additionally, ELISA testing has been used to validate the antibody's ability to specifically recognize the recombinant Arabidopsis thaliana At4g31010 protein used as the immunogen . Cross-reactivity testing with related plant species can provide further confirmation of specificity. Immunofluorescence studies comparing antibody staining patterns with known mitochondrial markers can validate its ability to detect the native protein in its cellular context.

How can At4g31010 antibody be used to study plant stress responses?

The At4g31010 antibody offers a powerful tool for investigating mitochondrial responses to environmental stressors in plants. To effectively study stress responses, researchers should design experiments comparing At4g31010 protein levels across multiple stress conditions (drought, salt, temperature extremes, and pathogen exposure) using standardized Western blot protocols. Quantitative analysis can be performed by normalizing At4g31010 signal intensity to housekeeping proteins such as actin or tubulin. Time-course experiments are essential to track dynamic changes in protein expression, with sampling at strategic intervals (0, 3, 6, 12, 24, and 48 hours) after stress induction. For subcellular localization studies, combine the At4g31010 antibody with established mitochondrial markers in immunofluorescence microscopy to detect potential stress-induced changes in protein distribution. Co-immunoprecipitation experiments using the At4g31010 antibody can identify stress-specific protein interaction partners. Complementing these approaches with transcriptomic data on At4g31010 gene expression provides a comprehensive understanding of both transcriptional and post-transcriptional regulation under stress conditions.

What are the considerations for optimizing Western blot protocols with At4g31010 antibody?

Optimizing Western blot protocols with At4g31010 antibody requires careful consideration of several technical parameters. The antibody has been specifically purified using antigen affinity methods, which influences its optimal working concentration . Begin optimization with a dilution series (1:500, 1:1000, 1:2000, 1:5000) to determine the optimal antibody concentration that maximizes specific signal while minimizing background. For sample preparation, mitochondrial enrichment protocols significantly improve detection sensitivity, as the target protein is mitochondrially localized . The choice of protein extraction buffer is critical—use buffers containing protease inhibitors to prevent degradation of the target protein. For membrane blocking, compare the effectiveness of 5% non-fat dry milk versus 3-5% BSA in TBST. Detection systems should be chosen based on required sensitivity, with chemiluminescence offering good sensitivity for most applications and fluorescent secondary antibodies providing better quantitative results. Include positive controls (recombinant At4g31010 protein) and negative controls (extracts from At4g31010 knockout lines) to validate specificity in each experiment.

How does antibody selection impact protein interaction studies with At4g31010?

The choice between polyclonal and monoclonal antibodies significantly impacts protein interaction studies involving At4g31010. The available At4g31010 antibody is polyclonal in nature, raised in rabbit against recombinant Arabidopsis thaliana At4g31010 protein . Polyclonal antibodies recognize multiple epitopes, which can be advantageous for capturing a wider range of protein-protein interactions but may increase the risk of non-specific binding. For co-immunoprecipitation (Co-IP) experiments, the antibody's ability to function in native conditions must be validated, as some antibodies only recognize denatured epitopes. Crosslinking methods (such as formaldehyde or DSP) can stabilize transient interactions before immunoprecipitation. Control experiments are essential, including IgG controls, reciprocal Co-IP validation, and validation in At4g31010 knockout lines. For especially challenging interactions, proximity labeling approaches like BioID or APEX2 fused to At4g31010 can complement traditional Co-IP approaches. Mass spectrometry analysis of immunoprecipitated complexes provides unbiased identification of interaction partners, requiring careful experimental design to distinguish genuine interactions from background contaminants.

What methodologies integrate At4g31010 antibody detection with omics approaches?

Integrating At4g31010 antibody detection with multi-omics approaches requires sophisticated experimental design and data analysis strategies. For proteomics integration, immunoprecipitation using the At4g31010 antibody followed by mass spectrometry analysis can identify the complete interactome of this mitochondrial protein under various conditions. Comparative proteomics between wild-type and At4g31010 mutant lines can elucidate downstream effects on the mitochondrial proteome. For transcriptomics integration, correlate RNA-seq or microarray data with At4g31010 protein levels detected by Western blotting across developmental stages or stress conditions to identify post-transcriptional regulation mechanisms. Chromatin immunoprecipitation sequencing (ChIP-seq) can be adapted using transcription factors suspected to regulate At4g31010 expression. Metabolomics data can be correlated with At4g31010 protein levels to establish connections between this mitochondrial factor and metabolic pathways. Computational integration of these multi-omics datasets requires sophisticated bioinformatics approaches, including network analysis, pathway enrichment, and machine learning methods to identify regulatory networks centered around At4g31010 function.

How can non-specific binding be reduced when using At4g31010 antibody?

Non-specific binding is a common challenge when working with plant antibodies that can significantly impact experimental outcomes. For the At4g31010 antibody, several methodological approaches can reduce background and increase specificity. First, optimize blocking conditions by testing different blocking agents—5% non-fat dry milk, 3-5% BSA, or commercial blocking reagents—for their effectiveness in reducing non-specific binding. Implement a pre-adsorption step by incubating the diluted antibody with protein extracts from At4g31010 knockout Arabidopsis lines to remove antibodies that bind to non-target proteins. Adjust antibody concentration through systematic titration experiments, as using excess antibody often increases background. Modify washing conditions by increasing the number of washes, extending wash durations, or adjusting detergent concentration in wash buffers. For Western blots, high-quality membrane blocking and thorough washing with TBST (Tris-buffered saline with 0.1% Tween-20) are essential. If persistent non-specific binding occurs, consider using more stringent washing conditions or adding competing proteins like BSA to the antibody dilution buffer.

What are the best practices for quantifying At4g31010 protein levels across experimental conditions?

Accurate quantification of At4g31010 protein levels requires careful experimental design and standardized methodologies. Western blotting remains the primary method for quantifying relative protein abundance, though it requires rigorous controls and standardization . For reliable quantification, load equal amounts of total protein (validated by Bradford or BCA assay) across all samples, and include a concentration gradient of a reference sample to confirm signal linearity. Always normalize At4g31010 signal intensity to an appropriate loading control—for mitochondrial proteins, porin or cytochrome c oxidase subunits provide better references than whole-cell markers like actin or tubulin. Use digital image acquisition systems rather than film for better dynamic range and linearity. Statistical analysis should include multiple biological replicates (minimum n=3) and appropriate statistical tests for comparing protein levels across conditions. For absolute quantification, consider developing a quantitative ELISA using the available antibody, or employ targeted proteomics approaches like selected reaction monitoring (SRM) with isotopically labeled peptide standards derived from At4g31010.

How do sample preparation methods affect At4g31010 antibody performance?

Sample preparation methodology significantly impacts At4g31010 antibody performance due to the mitochondrial localization of the target protein. For optimal results, subcellular fractionation to enrich mitochondria before immunodetection substantially improves sensitivity. Protein extraction buffers should contain protease inhibitor cocktails to prevent degradation, with EDTA included to inhibit metalloproteases. The choice between denaturing and native extraction conditions depends on the experimental goal—SDS-based buffers are optimal for Western blotting, while non-denaturing conditions are necessary for immunoprecipitation and activity assays. For Western blotting applications, sample heating conditions affect epitope availability; compare standard boiling (95°C for 5 minutes) with more gentle denaturation (70°C for 10 minutes) to determine optimal conditions for At4g31010 detection. Fresh samples generally yield better results than frozen tissues, but if freezing is necessary, rapid freezing in liquid nitrogen followed by storage at -80°C minimizes protein degradation. For plant tissues with high levels of interfering compounds (phenolics, polysaccharides), additional purification steps such as TCA/acetone precipitation or phenol extraction may be necessary to obtain clean protein samples suitable for immunodetection.

What controls are essential when using At4g31010 antibody in advanced applications?

Implementing comprehensive controls is critical when using At4g31010 antibody for advanced research applications. For genetic controls, include samples from At4g31010 knockout or knockdown lines as negative controls to confirm antibody specificity. Complemented lines (knockout plants transformed with functional At4g31010) provide validation that observed phenotypes directly relate to the target protein. Technical controls should include primary antibody omission controls, isotype controls (non-specific rabbit IgG at the same concentration), and antigen pre-adsorption controls (antibody pre-incubated with excess recombinant At4g31010 protein). For subcellular localization studies, co-staining with established mitochondrial markers confirms the expected localization pattern. When studying protein-protein interactions, reciprocal co-immunoprecipitation with antibodies against suspected interaction partners provides stronger evidence than one-directional pull-downs. For developmental or stress response studies, time-course sampling with multiple biological replicates ensures reproducibility of observed changes. Quantitative applications require standard curves using recombinant At4g31010 protein to demonstrate linearity of the detection method within the relevant concentration range.

How can CRISPR-Cas9 gene editing be combined with At4g31010 antibody detection?

CRISPR-Cas9 gene editing technology offers powerful approaches for functional studies of At4g31010 when combined with antibody-based detection methods. Researchers can generate precise mutations in the At4g31010 gene, including knockout lines, domain-specific mutations, and tagged variants for localization studies. After CRISPR editing, the At4g31010 antibody serves as a critical validation tool to confirm successful modification at the protein level through Western blotting . For structure-function studies, researchers can create domain deletion or point mutation variants, then use the antibody to assess how these modifications affect protein stability, localization, and interaction with other mitochondrial factors. Epitope tagging approaches can complement antibody detection—adding small epitope tags (HA, FLAG, etc.) to the native At4g31010 locus allows dual verification with both the specific At4g31010 antibody and commercial tag antibodies. For temporal control, CRISPR interference (CRISPRi) or CRISPR activation (CRISPRa) systems can repress or enhance At4g31010 expression, with antibody detection quantifying the resulting protein level changes.

What approaches enable single-cell analysis of At4g31010 protein expression?

Single-cell analysis of At4g31010 protein expression presents technical challenges that require specialized methodologies, particularly for plant cells with their rigid cell walls. Immunofluorescence microscopy using the At4g31010 antibody combined with confocal imaging and deconvolution techniques can visualize protein distribution in fixed tissue sections or protoplasts . For quantitative analysis, flow cytometry or imaging flow cytometry can be applied to plant protoplasts labeled with fluorescently-conjugated At4g31010 antibody, though protocol optimization is necessary for mitochondrial proteins. More advanced techniques include proximity ligation assays (PLA) that can detect protein-protein interactions at the single-cell level with higher sensitivity than conventional co-immunoprecipitation. Recent developments in single-cell proteomics techniques, such as mass cytometry (CyTOF) or microfluidic platforms like nanovials (as used in research on plasma B cells ), could potentially be adapted for plant cells, though significant protocol modifications would be required. Correlative microscopy approaches combining immunofluorescence with electron microscopy can provide ultrastructural context for At4g31010 localization within mitochondria.

What strategies optimize co-localization studies with At4g31010 antibody?

Co-localization studies with At4g31010 antibody require careful methodological considerations to generate reliable data about protein distribution within plant cells. As At4g31010 is a mitochondrial protein, co-localization with established mitochondrial markers provides important validation . For multi-color immunofluorescence microscopy, select secondary antibodies with well-separated emission spectra to minimize bleed-through (e.g., Alexa Fluor 488 for At4g31010 and Alexa Fluor 647 for co-stained proteins). Sample preparation techniques that preserve mitochondrial morphology are essential—mild fixation with paraformaldehyde (typically 2-4%) maintains structural integrity while allowing antibody penetration. For super-resolution microscopy applications (STED, STORM, or PALM), specialized sample preparation and higher-quality primary antibodies may be required. Quantitative co-localization analysis should employ established metrics such as Pearson's correlation coefficient or Manders' overlap coefficient, calculated using platforms like ImageJ with the JACoP plugin. Z-stack acquisition with appropriate step sizes (typically 0.2-0.5 μm) ensures complete capture of three-dimensional distributions. Live-cell imaging can complement fixed-cell analysis by tracking dynamic changes in protein distribution, though this would require development of fluorescent protein fusions to At4g31010 rather than antibody-based detection.

How does the performance of commercial At4g31010 antibodies compare across vendors?

When selecting an At4g31010 antibody for research, understanding performance variations across commercial sources is crucial for experimental success. Currently, At4g31010 antibodies are available from vendors including Cusabio (product code CSB-PA819810XA01DOA) and Abmart . These antibodies differ in several critical aspects that affect their research applications. The Cusabio antibody is a rabbit polyclonal antibody produced against recombinant Arabidopsis thaliana At4g31010 protein and purified by antigen affinity chromatography . It is specifically validated for ELISA and Western blot applications . The antibody formulation contains 50% glycerol with 0.03% Proclin 300 as a preservative in PBS (pH 7.4) . This information helps researchers evaluate stability and storage requirements. Lead time is an important practical consideration—the Cusabio antibody notes a 14-16 week lead time for made-to-order production , which necessitates advance planning for research projects. For optimal experimental design, researchers should conduct side-by-side comparisons using standardized protocols across multiple antibody sources when possible, evaluating specificity, sensitivity, and lot-to-lot consistency.

What experimental design best validates At4g31010 function in mitochondrial RNA processing?

Validating At4g31010's function in mitochondrial RNA processing requires a comprehensive experimental design combining genetic, biochemical, and cellular approaches. The foundation of this design should be a genetic complementation system using At4g31010 knockout lines rescued with wild-type or mutated versions of the gene. For biochemical validation, RNA immunoprecipitation (RIP) using the At4g31010 antibody followed by RT-PCR or RNA-seq can identify the specific RNA targets bound by this protein in vivo . In vitro RNA binding assays with purified recombinant At4g31010 protein can further confirm direct interactions and identify sequence or structural preferences for binding. For functional analysis of RNA processing, compare mitochondrial transcript profiles between wild-type and At4g31010 mutant plants using Northern blotting or RNA-seq, focusing on splicing efficiency, RNA stability, and post-transcriptional modifications. Microscopy approaches using the At4g31010 antibody can visualize co-localization with mitochondrial nucleoids and RNA processing bodies . For protein interaction networks, co-immunoprecipitation with the At4g31010 antibody followed by mass spectrometry can identify other components of the RNA processing machinery that work in concert with this factor.

How can researchers troubleshoot inconsistent results with At4g31010 antibody across plant developmental stages?

Inconsistent antibody performance across developmental stages is a common challenge in plant research that requires systematic troubleshooting. The expression level of At4g31010 likely varies during development, potentially falling below detection limits in certain stages. To address this, optimize protein extraction by adjusting buffer composition for different tissue types—mature leaves contain different interfering compounds than seedlings or reproductive tissues. Increase the starting material for tissues with lower expression, and consider subcellular fractionation to enrich for mitochondria before immunodetection . Another common source of inconsistency is variable post-translational modifications across developmental stages that may affect epitope accessibility. Testing multiple antibody concentrations for each developmental stage can help compensate for these differences. The choice of detection method also impacts sensitivity—chemiluminescence substrates vary widely in sensitivity, with advanced substrates offering 10-100× higher sensitivity for weakly expressed proteins. Consider whether proteolysis during extraction affects detection, as different tissues contain varying protease activities; adjust protease inhibitor cocktails accordingly. Finally, evaluate whether the inconsistency is biologically meaningful rather than a technical artifact by correlating with RNA expression data across the same developmental stages.

What techniques enable visualization of At4g31010 dynamics in living plant cells?

Visualizing At4g31010 dynamics in living plant cells requires approaches that go beyond traditional antibody-based detection, which is typically limited to fixed samples. The most effective strategy involves creating fluorescent protein fusions to At4g31010, either through stable transformation or transient expression systems. For CRISPR-based tagging, researchers can insert fluorescent protein sequences (GFP, mCherry, etc.) at the endogenous At4g31010 locus, maintaining native expression patterns and regulatory mechanisms. Alternatively, transient expression of fluorescently tagged At4g31010 in protoplasts or through Agrobacterium-mediated transformation provides a faster approach for preliminary studies. For mitochondrial proteins like At4g31010 , researchers must verify that the fusion doesn't disrupt mitochondrial targeting by co-localizing with established mitochondrial markers. Advanced microscopy techniques such as fluorescence recovery after photobleaching (FRAP) can measure protein mobility, while fluorescence resonance energy transfer (FRET) can detect protein-protein interactions in real-time when the interaction partner is also fluorescently labeled. For plant tissues with high autofluorescence, consider using fluorescent proteins with red-shifted emission spectra or advanced techniques like fluorescence lifetime imaging microscopy (FLIM).

What emerging technologies will advance At4g31010 protein research?

Emerging technologies offer exciting new avenues for advancing At4g31010 protein research beyond current methodological limitations. CRISPR-based technologies continue to evolve, with base editing and prime editing enabling precise modifications to the At4g31010 gene without double-strand breaks, allowing subtle alterations to protein function without complete disruption. Spatial transcriptomics and proteomics techniques will provide unprecedented insights into tissue-specific expression patterns of At4g31010, correlating protein presence with functional outcomes at cellular resolution. Cryo-electron microscopy advances may soon enable structural determination of plant mitochondrial protein complexes containing At4g31010, revealing mechanistic details of its function in RNA processing. Single-molecule tracking in living cells, using techniques like lattice light-sheet microscopy combined with photo-convertible fluorescent tags, can reveal dynamic behavior of At4g31010 proteins within mitochondria. Nanobody development against At4g31010 would provide smaller binding molecules with potentially better tissue penetration and less interference with protein function than traditional antibodies. Optogenetic tools adapted for plant mitochondrial proteins could allow temporal control of At4g31010 activity or interactions through light exposure. Integration of machine learning approaches with multi-omics data will help predict functional networks and regulatory mechanisms controlling At4g31010 activity across developmental stages and environmental conditions.