At3g50690 Antibody

What applications are At3g50690 antibodies validated for in Arabidopsis research?

At3g50690 antibodies have been primarily validated for Western blotting (WB) applications with an initial recommended dilution of 1:1000 . According to the antibody specifications, each antibody in the package can detect between 0.01-1ng of its corresponding immunogen peptide in dot blot assays. The antibodies are also potentially applicable for immunoprecipitation (IP) procedures, although this would require additional validation by researchers. It's important to note that unlike some other Arabidopsis antibodies (such as anti-AGO1 which has been validated for Chromatin Immunoprecipitation, Immunofluorescence, Immunolocalization, and small-RNA-IP-Seq ), the At3g50690 antibodies have more limited documented applications. Researchers should therefore conduct preliminary validation experiments to confirm antibody performance in their specific experimental systems before proceeding with large-scale studies.

How should At3g50690 antibodies be stored and handled to maintain optimal activity?

The At3g50690 antibodies are typically shipped as lyophilized supernatant at room temperature . Upon delivery, researchers should suspend the antibody according to manufacturer instructions and store at -20°C to maintain reactivity. It is crucial to avoid repeated freeze-thaw cycles, as these can progressively degrade antibody quality and reduce binding affinity. For long-term storage, consider aliquoting the suspended antibody into small volumes that will be used in individual experiments. Before each use, centrifuge the antibody vial briefly to collect the solution at the bottom of the tube. When diluting for applications, use fresh, cold buffer solutions and maintain the antibody on ice during experimental setup. Additionally, inclusion of protease inhibitors in working solutions can help preserve antibody integrity during longer experimental procedures. Proper storage and handling practices significantly impact experimental reproducibility and sensitivity in antibody-based applications.

How can At3g50690 antibodies be optimized for chromatin immunoprecipitation (ChIP) studies?

Optimizing At3g50690 antibodies for ChIP studies requires careful protocol adaptation since these antibodies have not been explicitly validated for ChIP applications . Drawing from established protocols with other nuclear protein antibodies in Arabidopsis, such as SE antibodies , researchers should first perform a fixation optimization. Test different formaldehyde concentrations (typically 1-3%) and fixation times (10-20 minutes) to preserve protein-DNA interactions without overfixing. Sonication conditions require careful calibration - start with 10-15 cycles (30 seconds on/30 seconds off) and verify chromatin fragmentation to 200-500bp by agarose gel electrophoresis. For immunoprecipitation, begin with 10μl antibody per reaction (similar to pol II CTD antibodies in published protocols ) and optimize through titration experiments. Include appropriate controls: IgG negative control, input samples, and if possible, a known ChIP-validated antibody as positive control. For At3g50690 ChIP, validation of enrichment should target gene loci where nuclear phosphoproteins are likely to function, such as actively transcribed regions or sites of RNA processing. After optimization, analyze enrichment through qPCR before proceeding to genome-wide approaches like ChIP-seq.

What evidence exists for At3g50690 interactions with transcriptional machinery in Arabidopsis?

While direct evidence for At3g50690 interactions with transcriptional machinery is not extensively documented in the provided research, insights can be drawn from studies of similar nuclear phosphoproteins in Arabidopsis. For example, the SERRATE (SE) protein, another nuclear-localized factor in Arabidopsis, has been shown to interact with RNA polymerase II complexes phosphorylated at Serine 2 and Serine 5 of the C-Terminal domain (CTD) . This interaction was confirmed through multiple complementary approaches: mass spectrometry analysis of immunoprecipitated complexes, co-immunoprecipitation experiments, and super-resolution microscopy techniques including Airyscan and super-resolution optical fluctuation (SOFI) imaging . These methodologies revealed that SE formed small, dotted speckles in the nucleus, with a subfraction co-localizing with pol II CTD Ser2P and Ser5P speckles. Similar experimental approaches could be employed to investigate potential interactions between At3g50690 and transcriptional machinery. Given that At3g50690 is classified as a nuclear phosphoprotein, it may participate in transcriptional regulation through direct or indirect interactions with polymerase complexes or associated factors.

How does At3g50690 antibody specificity compare with other nuclear protein antibodies used in Arabidopsis research?

Comparing antibody specificities requires evaluation across multiple parameters including epitope recognition, cross-reactivity, and performance in different applications. The At3g50690 antibody targets specifically the Acidic leucine-rich nuclear phosphoprotein 32-related protein in Arabidopsis through recognition of synthetic peptides from the N-terminus of the protein . In contrast, other nuclear protein antibodies like anti-AGO1 are raised against KLH-conjugated N-terminal peptides of Arabidopsis AGO1 (O04379, At1g48410) and show confirmed reactivity in multiple species including Arabidopsis thaliana, Hyacinthus orientalis, and Nicotiana benthamiana . Similarly, SE antibodies used in chromatin studies were raised against specific peptides (QDLDAPEE EVTVIDYRSL) and validated for multiple applications .

The specificity profile can be quantitatively compared as shown in Table 1:

This comparison indicates that At3g50690 antibodies currently have a more limited validation profile compared to some other nuclear protein antibodies used in Arabidopsis research .

What controls should be included when using At3g50690 antibodies for protein detection?

When designing experiments with At3g50690 antibodies, researchers should implement a comprehensive set of controls to ensure reliable interpretation of results. For Western blot applications, include a positive control (if available, recombinant At3g50690 protein or extracts from plants overexpressing the protein) and a negative control (extracts from knockout/knockdown lines for At3g50690 if available). Additionally, competing peptide controls can verify specificity - pre-incubating the antibody with excess immunogenic peptide should abolish specific binding. For loading controls, use antibodies against constitutively expressed proteins such as actin or tubulin. Implementation of technical replicates (minimum three) and biological replicates (from independent plant samples) is essential for statistical validation. When performing immunoprecipitation experiments, inclusion of non-specific IgG controls from the same species as the primary antibody is critical, as exemplified in the SE ChIP-qPCR experiments where mouse IgGs were used as negative controls . For downstream applications like mass spectrometry, consider including isotype controls and "bead-only" controls to identify non-specific binding. These comprehensive controls enable accurate interpretation of results and differentiation between specific signals and experimental artifacts.

How should protein extraction protocols be optimized for At3g50690 detection in different tissues?

Optimization of protein extraction for At3g50690 detection requires consideration of its nuclear localization and phosphoprotein nature. For nuclear-enriched extractions, researchers should modify standard protocols to enhance nuclear protein recovery while preserving phosphorylation states. Begin with tissue-specific considerations: for leaves, grinding in liquid nitrogen followed by immediate extraction in buffer is effective, while for roots or seeds, additional mechanical disruption may be necessary. The extraction buffer should contain: 20mM Tris-HCl (pH 7.5-8.0), 150-300mM NaCl, 5mM MgCl₂, 0.1% NP-40 or Triton X-100, 1-2mM DTT, 10% glycerol, with critical additions of phosphatase inhibitors (10mM NaF, 1mM Na₃VO₄) to preserve phosphorylation states. Proteasome inhibitors (such as MG132 at 1%) should be included to prevent protein degradation, similar to protocols used for AGO1 detection . For nuclear-enriched fractions, after initial homogenization, filter the homogenate through Miracloth, pellet nuclei by centrifugation at 1500g for 10 minutes at 4°C, then resuspend and lyse nuclei in extraction buffer. Performing differential centrifugation (3000g, 10,000g, 100,000g) can provide fractions enriched for different subcellular components. Compare protein yields and At3g50690 signal across different extraction methods through Western blotting to determine optimal conditions for specific experimental questions.

What are the key considerations for using At3g50690 antibodies in co-immunoprecipitation studies?

For successful co-immunoprecipitation (co-IP) studies with At3g50690 antibodies, researchers must address several technical considerations. First, crosslinking optimization is essential - if investigating protein-protein interactions, mild formaldehyde fixation (0.1-0.3%) can preserve transient interactions, but may introduce artifacts; therefore, parallel non-crosslinked samples should be processed. The extraction buffer composition significantly impacts complex preservation; for nuclear protein complexes, use 20mM Tris pH 7.5, 5mM MgCl₂, 2.5mM DTT, 300mM NaCl, 0.1% NP-40 with protease inhibitors as used successfully for SE co-IP studies . Pre-clearing lysates with protein A/G beads (1 hour at 4°C) reduces non-specific binding. For the IP step, optimize antibody amounts (starting with 2-5μg per reaction) and incubation conditions (typically overnight at 4°C with gentle rotation). After immunoprecipitation, implement stringent washing steps (at least 4-5 washes) with progressively lower salt concentrations to reduce background while preserving specific interactions. For elution, gentle conditions (such as competitive elution with immunogenic peptide) may better preserve native protein complexes than boiling in SDS buffer. When analyzing results, compare protein partners identified in At3g50690 IP with those from control IPs to distinguish specific interactions from background. Validation of identified interactions should employ reciprocal co-IPs and independent methods such as yeast two-hybrid or bimolecular fluorescence complementation assays.

What are common issues when using At3g50690 antibodies in Western blotting and how can they be resolved?

Researchers may encounter several technical challenges when using At3g50690 antibodies in Western blotting. One common issue is weak or absent signal, which may result from insufficient protein extraction, particularly given the nuclear localization of At3g50690. To address this, implement nuclear-enriched extraction protocols as described earlier, and ensure complete protein transfer to membranes by using stain-free gels or Ponceau staining to verify transfer efficiency. High background is another frequent problem that can be mitigated through optimization of blocking conditions (test 5% BSA versus 5% non-fat milk) and increasing washing stringency (add 0.1% Triton X-100 to TBS-T washing buffer, as used successfully in AGO1 detection protocols ). Multiple bands may appear due to protein degradation or post-translational modifications; adding protease and phosphatase inhibitors to extraction buffers can reduce degradation, while comparison with knockout/knockdown lines helps identify specific bands. For weak signals, signal enhancement systems or more sensitive detection methods (chemiluminescence versus fluorescence-based detection) can be employed. Quantification challenges can be addressed by using internal loading controls and ensuring linear detection range by performing serial dilutions of samples. Inconsistent results between experiments may stem from variable antibody quality; aliquoting antibodies upon first thawing and maintaining consistent incubation temperatures can improve reproducibility. Document all optimization steps systematically for reproducible and reliable Western blot results.

How can researchers verify At3g50690 antibody specificity in different experimental contexts?

Verifying antibody specificity is critical for experimental validity and requires multiple complementary approaches. For genetic verification, the most definitive approach is comparing signal between wild-type plants and knockout/knockdown mutants of At3g50690; a specific antibody will show reduced or absent signal in mutant lines. If mutant lines are unavailable, RNAi-mediated knockdown or CRISPR-Cas9 edited lines can be generated. Molecular approaches include peptide competition assays, where pre-incubation of the antibody with excess immunogenic peptide should abolish specific binding without affecting non-specific signals. Heterologous expression systems can be valuable - expressing tagged At3g50690 in bacteria, yeast, or plant protoplasts allows parallel detection with both anti-tag antibodies and the At3g50690-specific antibody to confirm co-localization of signals. For immunofluorescence applications, co-localization with known nuclear markers provides additional verification. Mass spectrometry analysis of immunoprecipitated material can confirm capture of the target protein and reveal potential cross-reactivity. When analyzing antibody specificity, it's important to verify under the specific conditions of each experiment, as fixation, extraction methods, and detection systems can all influence specificity profiles. Document all verification steps in research publications to provide comprehensive validation that the observed signals represent the intended target protein.

How does the function of At3g50690 compare with other nuclear phosphoproteins like SERRATE in Arabidopsis?

Comparative analysis between At3g50690 and other nuclear phosphoproteins such as SERRATE (SE) reveals both potential functional similarities and distinct roles in nuclear processes. SE has been extensively characterized as a regulator of gene expression, particularly for intronless genes in Arabidopsis . SE associates with chromatin in a transcription-dependent manner, requiring both RNA production and interaction with the Cap-Binding Complex (CBC) for its recruitment to target loci . SE preferentially binds to intronless genes and associates with Serine 5- and Serine 2-phosphorylated RNA polymerase II complexes during transcriptional pausing and elongation . In contrast, specific chromatin association patterns and transcriptional roles of At3g50690 are less well documented in the current literature. As a nuclear phosphoprotein, At3g50690 may participate in similar nuclear processes, potentially involving RNA metabolism or chromatin regulation, but its precise mechanisms remain to be elucidated. Both proteins likely function within nuclear compartments, as evidenced by SE forming dotted speckles in the nucleus observed through super-resolution microscopy techniques . Further research using complementary approaches such as ChIP-seq, RNA-seq in knockout/knockdown lines, and protein-protein interaction studies would help clarify the functional relationship between these nuclear phosphoproteins and their respective roles in transcriptional regulation and RNA processing pathways.

What techniques can be used to study potential RNA-binding properties of At3g50690?

Investigating potential RNA-binding properties of At3g50690 requires a multi-faceted approach combining in vitro and in vivo techniques. RNA immunoprecipitation (RIP) represents a valuable starting point - using At3g50690 antibodies to immunoprecipitate the protein from plant extracts, followed by RNA extraction and analysis through RT-qPCR or RNA-seq to identify associated transcripts. For higher resolution and to distinguish direct binding from indirect associations, UV crosslinking-based methods such as CLIP (Cross-Linking Immunoprecipitation) or PAR-CLIP (Photoactivatable Ribonucleoside-Enhanced CLIP) can be employed. In vitro binding assays provide complementary evidence - using recombinant At3g50690 protein with synthetic RNA oligonucleotides in electrophoretic mobility shift assays (EMSA) or filter binding assays to determine binding affinities and sequence preferences. Surface plasmon resonance (SPR) or microscale thermophoresis (MST) can provide quantitative binding parameters. Structural approaches such as NMR spectroscopy or X-ray crystallography with RNA-protein complexes could reveal binding mechanisms at the molecular level. For functional validation, tethering assays where At3g50690 is fused to an MS2 coat protein and tethered to reporter transcripts containing MS2 binding sites can reveal effects on RNA processing, stability, or translation. Integrating these approaches would provide comprehensive evidence regarding the RNA-binding capabilities of At3g50690 and its potential roles in RNA metabolism, similar to how SE's RNA-dependent chromatin association was established through multiple complementary techniques .

How might transcriptome and proteome analyses in At3g50690 mutants inform our understanding of its function?

Integrating transcriptome and proteome analyses in At3g50690 mutant lines would provide multi-dimensional insights into its functional roles. For transcriptome analysis, RNA-seq comparing wild-type and At3g50690 knockout/knockdown lines under standard conditions and various stresses would identify differentially expressed genes (DEGs). Analysis of these DEGs for common promoter elements, gene ontology enrichment, and pathway analysis could reveal regulatory networks under At3g50690 control. As nuclear phosphoproteins often affect specific gene subsets, examination of intronless genes versus intron-containing genes (similar to SE target analysis ) would be particularly informative. For greater resolution, techniques like CAGE-seq or GRO-seq could map transcription initiation sites and nascent transcript production to distinguish direct transcriptional effects from secondary consequences. In parallel, proteomic analyses using quantitative mass spectrometry approaches (iTRAQ, TMT, or label-free quantification) would identify proteins with altered abundance in mutant lines. Phosphoproteomic analysis would be especially relevant given At3g50690's classification as a nuclear phosphoprotein, potentially revealing altered phosphorylation cascades in signaling networks. Integration of transcriptome and proteome data through systems biology approaches could identify discordant mRNA-protein pairs, suggesting post-transcriptional regulation. Co-expression network analysis might place At3g50690 within specific functional modules, while comparison with publicly available datasets for other nuclear regulators (such as SE) could reveal overlapping or distinct regulatory roles. This multi-omics approach would provide a comprehensive understanding of At3g50690's function in nuclear processes and plant development.

What are the future research directions for At3g50690 function in Arabidopsis thaliana?

Future research on At3g50690 should focus on comprehensive functional characterization through multiple complementary approaches. Creation of CRISPR-Cas9 knockout and conditional knockdown lines will enable phenotypic analyses across developmental stages and under various stress conditions. Genome-wide binding profiles through ChIP-seq or CUT&RUN would map At3g50690 chromatin association patterns, while RNA-association studies (RIP-seq, CLIP-seq) could reveal potential RNA targets. Protein interactome mapping through immunoprecipitation-mass spectrometry and yeast two-hybrid screening would identify interaction partners and place At3g50690 within nuclear protein networks. Super-resolution microscopy techniques, successful in visualizing SE nuclear distribution , could determine At3g50690's subnuclear localization and potential co-localization with transcriptional machinery or RNA processing factors. Mechanistic studies exploring potential roles in transcriptional regulation could examine effects on RNA polymerase II recruitment and progression, similar to analyses performed for SE . Comparative studies with related nuclear phosphoproteins across plant species would provide evolutionary insights and potential functional conservation. Integration of these approaches through systems biology frameworks would provide a holistic understanding of At3g50690's role in nuclear processes, potentially revealing novel regulatory mechanisms in plant gene expression. Development of improved antibodies and tagged transgenic lines would facilitate these studies by providing validated tools for various applications beyond the current Western blot validation .