At2g05300 Antibody

What is At2g05300 and why is it significant for Arabidopsis research?

At2g05300 is a gene locus in Arabidopsis thaliana (Mouse-ear cress) that encodes a specific protein of interest to plant researchers. The gene is cataloged in the UniProt database under accession number Q9SJ32, indicating its recognized status in protein databases . Understanding this gene and its protein product is significant because Arabidopsis serves as a model organism for plant molecular biology, with applications ranging from basic protein function studies to investigations of plant development, stress responses, and evolutionary relationships. The antibody against this protein enables researchers to track its expression, localization, and interactions within plant tissues.

What methods can be used to validate At2g05300 antibody specificity?

Antibody validation is a critical first step before conducting extensive experiments. For At2g05300 antibody, researchers should employ multiple complementary approaches to confirm specificity. Western blotting using wild-type and knockout/knockdown plant extracts provides the primary validation method, where a single band of the expected molecular weight should appear only in wild-type samples. Additionally, immunoprecipitation followed by mass spectrometry can identify whether the antibody pulls down primarily the target protein. Peptide competition assays, where pre-incubation with the immunizing peptide blocks antibody binding, offer further confirmation. Pre-immune serum controls and testing across multiple tissue types will strengthen validation. These validation steps prevent misleading results that could arise from antibodies with cross-reactivity or poor specificity.

How should At2g05300 antibody be stored and handled to maintain optimal activity?

Proper storage and handling of At2g05300 antibody is essential to preserve its functionality. The antibody is typically available in 0.1ml or 2ml quantities . For long-term storage, maintain the antibody at -20°C in small aliquots to avoid repeated freeze-thaw cycles, which can cause protein denaturation and loss of binding capacity. When in use, keep the antibody on ice and return to storage promptly. For diluted working solutions, add preservatives such as sodium azide (0.02%) if storing at 4°C for more than a week. Before each use, centrifuge the antibody briefly to collect contents at the bottom of the tube. Record the date of first use and track the number of freeze-thaw cycles to monitor potential degradation. Proper handling ensures consistent experimental results and extends the usable life of the antibody.

What are the optimal fixation and permeabilization methods for immunolocalization of At2g05300 in Arabidopsis tissues?

The choice of fixation and permeabilization methods significantly impacts the success of immunolocalization studies using At2g05300 antibody. For Arabidopsis tissues, a two-step approach is often most effective. Begin with a light fixation using 4% paraformaldehyde in PBS for 20-30 minutes, which preserves cellular architecture while maintaining epitope accessibility. For membranous structures, adding 0.1-0.5% glutaraldehyde may improve structural preservation. The permeabilization step is equally critical - use 0.1-0.3% Triton X-100 for cytoplasmic proteins or 0.05-0.1% for nuclear proteins. For recalcitrant tissues like mature leaves or roots, vacuum infiltration during fixation improves reagent penetration. Notably, overfixation can mask epitopes and reduce antibody binding, while insufficient fixation leads to poor morphological preservation. Test multiple conditions in parallel, as the optimal protocol may vary depending on the specific subcellular localization of the At2g05300 protein and the plant tissue being examined.

How should researchers design proper controls for Western blot experiments using At2g05300 antibody?

Robust experimental design for Western blots with At2g05300 antibody requires multiple controls to ensure valid interpretation of results. Include positive controls such as recombinant At2g05300 protein or extracts from tissues known to express the protein. Negative controls should include knockout/knockdown plant lines or tissues where the protein is not expressed. A loading control using antibodies against housekeeping proteins like actin or tubulin is essential for normalization. For signal specificity validation, include a peptide competition assay where the immunizing peptide is pre-incubated with the antibody before probing the membrane. When conducting comparative expression studies across conditions, maintain identical protein loads, transfer conditions, and exposure times. Additionally, strip and reprobe membranes with secondary antibody alone to identify any non-specific secondary antibody binding. These controls collectively enable confident interpretation of Western blot results and facilitate troubleshooting when unexpected bands appear.

What is the recommended sample preparation protocol for immunoprecipitation using At2g05300 antibody?

Effective immunoprecipitation with At2g05300 antibody requires careful sample preparation to preserve protein-protein interactions while minimizing background. Begin with fresh Arabidopsis tissue (preferably 1-2 grams), flash-frozen in liquid nitrogen and ground to a fine powder. Extract proteins using a mild lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40 or 0.5% Triton X-100) supplemented with protease inhibitors, phosphatase inhibitors, and 1-2 mM DTT or β-mercaptoethanol. For membrane-associated proteins, include 0.5% sodium deoxycholate. Incubate lysates on ice for 30 minutes with gentle mixing, then centrifuge at 14,000g for 15 minutes at 4°C to remove debris. Pre-clear the supernatant with Protein A/G beads for 1 hour to reduce non-specific binding. For the immunoprecipitation step itself, conjugate At2g05300 antibody to fresh Protein A/G beads (typically 2-5 μg antibody per reaction) before adding to the pre-cleared lysate. The critical factors affecting success include maintaining cold temperature throughout, using optimized detergent concentrations, and determining the appropriate antibody-to-lysate ratio through preliminary experiments.

How can researchers use At2g05300 antibody for chromatin immunoprecipitation (ChIP) experiments?

Chromatin immunoprecipitation with At2g05300 antibody requires adaptations specific to plant chromatin structures. If At2g05300 functions as a DNA-binding protein or chromatin-associated factor, begin with 1-3 grams of Arabidopsis tissue crosslinked with 1% formaldehyde for 10-15 minutes under vacuum. The crosslinking time may need optimization, as excessive crosslinking can reduce epitope accessibility. After quenching with glycine, isolate nuclei using a sucrose gradient, then sonicate to generate DNA fragments of 200-500 bp. For plant tissues, sonication parameters typically require more cycles than mammalian samples due to rigid cell walls and different chromatin compaction. Immunoprecipitate with 3-5 μg of At2g05300 antibody overnight, followed by washing with increasing salt concentration buffers to reduce non-specific binding. Include input controls (non-immunoprecipitated chromatin), IgG controls, and positive controls using antibodies against well-characterized chromatin marks. Validate ChIP efficiency using qPCR of known target regions before proceeding to genome-wide sequencing. The inclusion of spike-in chromatin from a different species can aid in normalization across samples and conditions.

What approaches should be used to investigate post-translational modifications of At2g05300 protein using specific antibodies?

Investigating post-translational modifications (PTMs) of At2g05300 requires specialized approaches beyond standard immunoblotting. First, determine potential modification sites through in silico analysis of the protein sequence, looking for consensus motifs for phosphorylation, SUMOylation, ubiquitination, or other relevant PTMs in plants. For phosphorylation studies, use phosphatase inhibitors (10 mM NaF, 1 mM Na3VO4) during extraction. Consider two complementary approaches: (1) immunoprecipitate the total At2g05300 protein with the general antibody, then probe with modification-specific antibodies (anti-phospho, anti-ubiquitin, etc.) or (2) use PTM-specific antibodies for the immunoprecipitation, then detect with the general At2g05300 antibody. For definitive characterization, combine these methods with mass spectrometry analysis of immunoprecipitated proteins to map modification sites precisely. Include appropriate controls such as samples treated with modifying enzymes (phosphatases, deubiquitinases) or from plants exposed to conditions known to induce the modifications (stress treatments, hormone applications). This multi-faceted approach provides robust evidence for specific PTMs and their potential roles in regulating At2g05300 function.

How can researchers quantitatively compare At2g05300 protein levels across different experimental conditions?

Quantitative comparison of At2g05300 protein levels requires rigorous standardization and appropriate normalization strategies. For Western blot analysis, establish a standard curve using recombinant At2g05300 protein to confirm that measurements fall within the linear detection range of the antibody. Implement technical replicates (minimum of three) and biological replicates (from independent plant populations) to account for experimental variation. For normalization, use multiple housekeeping proteins as references, as single reference proteins may vary across developmental stages or stress conditions. Alternative quantitative approaches include ELISA assays developed with the At2g05300 antibody, which offer greater sensitivity and dynamic range than Western blotting. For single-cell or subcellular resolution, consider quantitative immunofluorescence with careful standardization of image acquisition parameters. In all cases, statistical analysis should include tests for normal distribution of data and appropriate parametric or non-parametric methods. Present data with clear indication of variability (standard deviation or standard error) and statistical significance between conditions.

What are common sources of background in immunohistochemistry with At2g05300 antibody and how can they be minimized?

High background in immunohistochemistry with At2g05300 antibody can arise from multiple sources requiring specific mitigation strategies. Non-specific binding of primary antibody often results from inappropriate blocking; optimize with different blocking agents (5% BSA, 5% normal serum, or commercial blocking reagents) and extend blocking time to 2 hours at room temperature. Autofluorescence, particularly pronounced in Arabidopsis tissues due to chlorophyll and phenolic compounds, can be reduced by pre-treating sections with 0.1% sodium borohydride or including 0.1 M NH4Cl in the blocking buffer. Excessive secondary antibody binding can be minimized by using highly cross-adsorbed formulations and diluting appropriately (typically 1:500 to 1:2000). For plant tissues specifically, including 0.1-0.3% Triton X-100 in wash buffers improves signal-to-noise ratio. If background persists despite these measures, consider protocol modifications: reduce primary antibody concentration, perform incubations at 4°C overnight rather than at room temperature, or try alternative fixation methods. For valuable samples, always include a negative control section incubated with non-immune IgG or pre-immune serum at the same concentration as the primary antibody.

How should researchers analyze contradictory results between transcript data and protein detection using At2g05300 antibody?

Discrepancies between transcript levels (e.g., from RNA-seq or qRT-PCR) and protein detection using At2g05300 antibody may reflect important biological phenomena rather than technical artifacts. When facing such contradictions, first verify the technical aspects: confirm primer specificity for transcript analysis and antibody specificity through the validation methods described earlier. If technical issues are ruled out, investigate biological explanations through a systematic approach. Post-transcriptional regulation may be occurring via microRNAs or RNA-binding proteins; examine the transcript sequence for regulatory motifs and consider RNA immunoprecipitation experiments. Post-translational regulation could involve rapid protein turnover; test this hypothesis using proteasome inhibitors like MG132 or by pulse-chase experiments. Temporal delays between transcription and translation are common in plants responding to environmental stimuli; perform time-course experiments sampling at multiple intervals. Subcellular localization changes might make the protein inaccessible to extraction methods; try differential extraction protocols targeting various cellular compartments. These systematic investigations not only resolve contradictions but may uncover novel regulatory mechanisms governing At2g05300 expression and function.

What strategies can overcome weak or inconsistent signal when using At2g05300 antibody in immunoprecipitation experiments?

When facing weak or inconsistent immunoprecipitation results with At2g05300 antibody, implement a stepwise optimization strategy targeting key variables. Begin by examining antibody quality and quantity; increase the amount used (from 2 μg to 5-10 μg per reaction) or consider crosslinking the antibody to beads with dimethyl pimelimidate to prevent antibody leaching during elution. Optimize extraction conditions by testing different buffer compositions – vary detergent types (NP-40, Triton X-100, CHAPS) and concentrations (0.1-1%), salt concentrations (100-500 mM NaCl), and pH values (6.8-8.0). For low-abundance proteins, increase starting material (3-5 grams of tissue) and concentrate the final eluate using TCA precipitation or commercial concentrators. If protein-protein interactions are the research focus, consider gentler extraction using GlycoBio buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 10% glycerol) or try chemical crosslinking (1-2 mM DSP or formaldehyde) before lysis to stabilize transient interactions. For detection of immunoprecipitated proteins, more sensitive methods like silver staining or fluorescent Western blot systems may be necessary. Document all optimization steps systematically to establish a reproducible protocol for your specific experimental system.

How does At2g05300 antibody performance compare with antibodies against related Arabidopsis proteins?

When conducting comparative studies involving multiple Arabidopsis proteins, understanding the relative performance characteristics of different antibodies is essential. At2g05300 antibody (CSB-PA882876XA01DOA) shares similar production platforms with other Arabidopsis antibodies like At3g49040 (CSB-PA882911XA01DOA) and At3g49520 (CSB-PA882820XA01DOA) . These antibodies typically exhibit comparable specificities when properly validated, but performance can vary based on the inherent properties of their target proteins. Factors affecting comparative performance include epitope accessibility, protein abundance, and subcellular localization. For meaningful comparisons, normalize detection protocols across antibodies by determining the optimal working dilution for each through titration experiments. When interpreting experimental outcomes, consider that differences in signal intensity may reflect not only expression levels but also variations in antibody affinity. For multiplex experiments, verify that the antibodies are compatible with simultaneous use by checking species origin and potential cross-reactivity. Understanding these performance differences ensures accurate interpretation of comparative studies investigating related Arabidopsis proteins.

At2g05300 Antibody Technical Information and Sourcing

The following table summarizes key technical information about At2g05300 antibody available for research applications:

What recent research advances have been made using antibodies against Arabidopsis proteins similar to At2g05300?

Recent advances in plant molecular biology have leveraged antibodies against Arabidopsis proteins to elucidate fundamental biological processes. While specific literature on At2g05300 is limited in the provided search results, the research methodologies employed with other Arabidopsis antibodies illustrate applicable approaches. Researchers have increasingly combined antibody-based techniques with advanced imaging methods such as super-resolution microscopy to visualize protein localization at nanometer resolution. Multi-omics approaches integrating immunoprecipitation with mass spectrometry and RNA sequencing have revealed protein interaction networks and their correlation with transcriptional regulation. Studies have also employed antibodies in chromatin immunoprecipitation sequencing (ChIP-seq) to map DNA-binding sites of transcription factors and other chromatin-associated proteins, providing insights into gene regulatory networks. These methodological advances can be applied using At2g05300 antibody to investigate its target protein's function in plant development, stress responses, or metabolic pathways. The continued refinement of antibody-based techniques provides researchers with increasingly powerful tools to explore protein function in Arabidopsis, serving as a model for understanding fundamental plant biology.