At3g18410 Antibody

What is the At3g18410 protein and why is it significant in plant research?

At3g18410 is a protein encoded by the At3g18410 gene in Arabidopsis thaliana (mouse-ear cress), identified in the UniProt database as Q94C12. This protein is significant in plant research due to its role in cellular processes. The antibody against At3g18410 allows researchers to detect, localize, and quantify this protein in various experimental setups, contributing to our understanding of plant cellular mechanisms. The protein's expression patterns across different tissues and under various conditions can provide insights into its functional role in plant development and stress responses .

What are the recommended applications for At3g18410 antibody?

The At3g18410 antibody is primarily validated for Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blot (WB) applications. These techniques allow for the detection and semi-quantitative analysis of the At3g18410 protein in plant tissue samples. For Western Blot applications, the antibody enables identification of the antigen in complex protein mixtures, providing information about protein expression levels and molecular weight . Researchers studying mitochondrial proteins in Arabidopsis may find this antibody particularly useful, as similar antibodies have been effectively employed in studies of respiratory chain complexes .

What are the optimal storage conditions for At3g18410 antibody?

For maximum stability and retention of immunoreactivity, the At3g18410 antibody should be stored at either -20°C or -80°C upon receipt. It is critical to avoid repeated freeze-thaw cycles, as these can compromise antibody performance. The antibody is supplied in a liquid form within a storage buffer containing 0.03% Proclin 300 (as a preservative), 50% Glycerol, and 0.01M PBS at pH 7.4. This formulation helps maintain antibody stability during storage . For long-term studies, it is advisable to prepare small aliquots before freezing to minimize the number of freeze-thaw cycles.

How should sample preparation be optimized for At3g18410 antibody detection?

When preparing Arabidopsis samples for At3g18410 antibody detection, several considerations are important. For protein extraction, a buffer containing protease inhibitors is recommended to prevent degradation. If studying mitochondrial proteins, subcellular fractionation techniques can be employed to separate membrane and soluble fractions, as demonstrated in similar studies of plant mitochondrial proteins . For Western blot applications, samples should be denatured in standard SDS-PAGE sample buffer and separated on an appropriate percentage gel based on the expected molecular weight of At3g18410. Transfer conditions should be optimized for hydrophobic or hydrophilic proteins depending on the protein's properties. For ELISA applications, careful calibration with known standards is essential for quantitative analysis.

How can At3g18410 antibody be used in protein complex studies?

For investigations of protein complexes involving At3g18410, Blue-Native PAGE (BN-PAGE) combined with immunodetection offers a powerful approach. This technique preserves protein-protein interactions during electrophoresis, allowing researchers to identify stable complexes containing the At3g18410 protein. Based on similar studies with mitochondrial proteins, the following methodology is recommended: solubilize protein samples with dodecylmaltoside (1% w/v) in ACA buffer (750 mm aminocaproic acid, 0.5 mm EDTA, 50 mm Tris-HCl, pH 7.0), separate the complexes on a 4.5–16% gradient gel, and then perform immunodetection with the At3g18410 antibody . Two-dimensional electrophoresis combining BN-PAGE with SDS-PAGE can further resolve individual components of identified complexes.

What approaches can resolve contradictory immunodetection results with At3g18410 antibody?

When faced with contradictory immunodetection results using the At3g18410 antibody, a systematic troubleshooting approach is essential. First, verify antibody specificity through parallel experiments with knockout/knockdown lines of At3g18410 as negative controls. Second, optimize antibody dilution ratios systematically; the optimal concentration may vary depending on the specific application and sample preparation method. Third, consider cross-reactivity issues by performing peptide competition assays. Fourth, evaluate different blocking agents (BSA, milk proteins, commercial blockers) to reduce background signal. Fifth, verify the integrity of your protein sample through Coomassie staining of parallel gels. Finally, consider alternative detection systems if horseradish peroxidase-based methods yield inconsistent results .

How can At3g18410 antibody be integrated into studies of protein-protein interactions in plant mitochondria?

To investigate protein-protein interactions involving At3g18410 in plant mitochondria, researchers can employ several complementary approaches. Co-immunoprecipitation (Co-IP) using the At3g18410 antibody can identify direct interacting partners. For this, protein samples should be solubilized under non-denaturing conditions, incubated with At3g18410 antibody, and then precipitated using protein A/G beads. The precipitated complexes can be analyzed by mass spectrometry to identify interacting proteins. Additionally, two-dimensional blue-native/blue-native PAGE as outlined by Sunderhaus et al. can be used to resolve stable protein complexes . This approach has been successfully employed for studying mitochondrial protein complexes, allowing visualization through Coomassie staining, activity staining, or immunoblotting. Selected protein complexes can then be excised and analyzed by mass spectrometry to identify components .

What considerations are important when using At3g18410 antibody in subcellular localization studies?

For subcellular localization studies using the At3g18410 antibody, several methodological considerations are crucial. First, tissue fixation protocols must preserve both antigenicity and cellular architecture; 4% paraformaldehyde is often effective for plant tissues. Second, cell permeabilization conditions should be optimized to allow antibody access while maintaining structure; a combination of low concentration detergents and enzymatic cell wall digestion may be necessary for plant cells. Third, include appropriate controls including pre-immune serum and peptide competition assays to confirm specificity. Fourth, co-localization with established organelle markers (such as mitochondrial markers if studying mitochondrial localization) provides important contextual information. Fifth, for quantitative analysis, establish standardized image acquisition settings and analysis parameters. Finally, consider complementary approaches such as expressing fluorescently-tagged At3g18410 to corroborate immunolocalization findings .

What is the recommended protocol for optimizing Western blot conditions for At3g18410 antibody?

Optimizing Western blot conditions for At3g18410 antibody requires systematic adjustment of multiple parameters. Begin with sample preparation by extracting proteins using a buffer containing detergents suitable for your sample type, supplemented with protease inhibitors. For gel electrophoresis, select an appropriate acrylamide percentage based on the molecular weight of At3g18410. After transfer to nitrocellulose or PVDF membrane, block with 5% non-fat milk or BSA in TBS-T for 1 hour at room temperature. For primary antibody incubation, start with a 1:1000 dilution of the At3g18410 antibody in blocking buffer and incubate overnight at 4°C. After washing, incubate with an appropriate HRP-conjugated secondary antibody (typically anti-rabbit IgG if using the rabbit-raised At3g18410 antibody). For visualization, use a chemiluminescence system with exposure times optimized for signal-to-noise ratio . If non-specific bands appear, systematically adjust antibody concentration, blocking conditions, and washing stringency.

What are the critical parameters for successful immunoprecipitation using At3g18410 antibody?

Successful immunoprecipitation using At3g18410 antibody depends on several critical parameters. First, cell lysis conditions must balance effective protein extraction with preservation of protein complexes; typically, a buffer containing 1% non-ionic detergent (such as NP-40 or Triton X-100), 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), and protease inhibitors works well for plant samples. Second, pre-clearing the lysate with Protein A/G beads removes components that bind non-specifically to the beads. Third, antibody amount requires optimization; typically 2-5 μg of antibody per mg of protein lysate is a good starting point. Fourth, incubation conditions affect complex formation; overnight incubation at 4°C with gentle rotation is often optimal. Fifth, washing stringency must be balanced to remove non-specific interactions while preserving specific ones; typically 4-5 washes with decreasing salt concentrations are effective. Finally, elution conditions should be optimized based on downstream applications; either harsh conditions (SDS buffer with heating) for maximum recovery or gentle conditions (excess competing peptide) to preserve activity .

How can mass spectrometry be integrated with At3g18410 antibody studies for comprehensive protein analysis?

Integrating mass spectrometry with At3g18410 antibody studies enables comprehensive characterization of the protein and its interacting partners. For this integrated approach, begin with immunoprecipitation using the At3g18410 antibody to isolate the protein and its complexes. The immunoprecipitated material can then be prepared for MS analysis through in-gel or in-solution digestion with trypsin. For in-gel digestion, separate the immunoprecipitated proteins by SDS-PAGE, excise bands of interest, destain, reduce with DTT (45 mM, 15 min at 50°C), alkylate with iodoacetamide (0.1 M, 15 min at 25°C), and digest with trypsin (0.1 μg/μl, 16h at 37°C) . For highly sensitive detection, consider using LC-MS/MS analysis with an appropriate chromatography setup. Data analysis should include both identification of peptides/proteins and quantitative comparison between experimental conditions. This approach can reveal post-translational modifications, interaction partners, and structural features of the At3g18410 protein .

What quality control measures should be implemented when using At3g18410 antibody?

Implementing rigorous quality control measures when using At3g18410 antibody is essential for reliable research outcomes. First, validate antibody specificity using positive controls (recombinant At3g18410 protein) and negative controls (knockout/knockdown lines or unrelated proteins). Second, perform lot-to-lot validation when receiving new antibody batches by comparing detection sensitivity and specificity with previous lots. Third, include internal loading controls appropriate for your experimental system in Western blot experiments. Fourth, maintain detailed records of antibody performance across different experiments, including optimal dilutions and detection limits. Fifth, regularly check for antibody stability by monitoring performance over time under standardized conditions. Finally, consider cross-validating critical findings using alternative detection methods or a second antibody targeting a different epitope of At3g18410 .

What are common pitfalls in At3g18410 antibody experiments and how can they be avoided?

Common pitfalls in At3g18410 antibody experiments include several technical issues that can compromise results. First, inadequate sample preparation may lead to protein degradation or incomplete extraction; this can be addressed by using fresh samples, appropriate lysis buffers, and protease inhibitors. Second, non-specific binding often manifests as multiple bands or high background; optimize blocking conditions, antibody dilutions, and washing steps to minimize this issue. Third, inconsistent results between experiments may stem from variable antibody performance; maintain consistent experimental conditions and consider creating a large batch of working dilution stored in small aliquots. Fourth, false negative results might occur if the epitope is masked or modified; try alternative sample preparation methods that may preserve or expose the epitope. Fifth, cross-reactivity with related proteins can lead to misinterpretation; validate specificity using knockout controls or peptide competition assays. Finally, overinterpretation of results without appropriate controls should be avoided by implementing rigorous experimental design including positive, negative, and procedural controls .

How can researchers validate At3g18410 antibody specificity for their specific experimental conditions?

Validating At3g18410 antibody specificity for specific experimental conditions requires a multi-faceted approach. Begin with peptide competition assays, where the antibody is pre-incubated with excess purified antigen (recombinant At3g18410 protein) before application to samples; disappearance of signal confirms specificity. Next, test the antibody on samples from knockout or knockdown plants lacking the At3g18410 gene; absence of signal in these samples strongly supports specificity. For Western blot applications, verify that the detected band appears at the expected molecular weight and that this band disappears in knockout samples or peptide competition assays. In immunohistochemistry, compare staining patterns with those obtained using alternative antibodies or detection methods. Consider using heterologous expression systems where At3g18410 is expressed with a tag that can be detected by an independent method, allowing correlation between tag detection and antibody signal. Finally, mass spectrometry analysis of immunoprecipitated material can provide definitive identification of the proteins recognized by the antibody .

How can computational approaches enhance experimental design when using At3g18410 antibody?

Computational approaches can significantly enhance experimental design when using At3g18410 antibody through several strategies. First, epitope prediction algorithms can identify the likely binding regions of the antibody on the At3g18410 protein, helping researchers anticipate potential cross-reactivity with related proteins or the effects of post-translational modifications on detection. Second, protein structure modeling can predict the accessibility of epitopes under different experimental conditions, guiding sample preparation protocols. Third, sequence analysis across species can inform cross-reactivity expectations when working with non-Arabidopsis samples. Fourth, data mining of published proteomics datasets can reveal expected expression patterns and interactions, helping establish appropriate positive and negative controls. Fifth, statistical power analysis can determine optimal sample sizes for quantitative studies. Finally, machine learning approaches can be applied to optimize image analysis parameters for immunohistochemistry or immunofluorescence experiments, enabling more objective and reproducible quantification of results .

What approaches can be used to study At3g18410 protein dynamics in different cellular compartments?

Studying At3g18410 protein dynamics across cellular compartments requires sophisticated methodological approaches. Cell fractionation coupled with immunoblotting using the At3g18410 antibody can provide a biochemical assessment of protein distribution, as demonstrated in studies of other plant proteins . For this approach, differential centrifugation can separate subcellular fractions (e.g., mitochondria, chloroplasts, cytosol), followed by validation of fraction purity using established marker proteins (such as ANT for mitochondrial membranes or SOD for soluble mitochondrial fractions) . Live-cell imaging can be achieved by creating fluorescent protein fusions with At3g18410, though validation with immunofluorescence using the At3g18410 antibody is crucial to confirm that the tag doesn't disrupt localization. For temporal dynamics, inducible expression systems combined with time-course immunoblotting or imaging can track protein movement. Protein translocation can be studied using selective permeabilization of cellular membranes followed by immunodetection, revealing the topology of the protein within organelles. Finally, super-resolution microscopy combined with immunolabeling using the At3g18410 antibody can achieve nanoscale resolution of protein distribution .

How can At3g18410 antibody be used in comparative studies across different plant species?

Using At3g18410 antibody in comparative studies across different plant species requires careful consideration of evolutionary conservation and epitope preservation. First, perform sequence alignment analysis of the At3g18410 protein across target species to assess homology in the antibody's epitope region; higher conservation predicts better cross-reactivity. Second, conduct preliminary Western blot experiments with protein extracts from different species using gradient gels to accommodate potential size variations in homologs. Third, optimize extraction conditions for each species, as protein solubility may vary due to different cellular compositions. Fourth, validate antibody specificity in each new species using control experiments similar to those in Arabidopsis. Fifth, for immunolocalization studies, adjust fixation protocols to account for differences in tissue structure and cell wall composition across species. Finally, when quantitatively comparing expression levels between species, normalize data to appropriate housekeeping proteins that show consistent expression across the studied species. This approach has been successfully employed in comparative studies of mitochondrial proteins across plant species, where antibodies developed against Arabidopsis proteins were effectively used to study homologs in other plants .

How can chromatin immunoprecipitation be adapted for use with At3g18410 antibody?

Adapting chromatin immunoprecipitation (ChIP) for use with At3g18410 antibody requires several modifications to standard protocols, particularly if investigating potential nuclear functions or DNA interactions of the At3g18410 protein. Begin with tissue crosslinking using 1% formaldehyde for 10-15 minutes to preserve protein-DNA interactions, followed by quenching with glycine. After nuclei isolation, sonicate chromatin to fragments of approximately 200-500 bp, with optimization required for each tissue type. Pre-clear the chromatin with protein A beads to reduce background, then immunoprecipitate using 2-5 μg of At3g18410 antibody per sample, with IgG controls processed in parallel. After washing steps of progressively decreasing stringency, reverse crosslinks at 65°C overnight. Following DNA purification, analyze the immunoprecipitated DNA by qPCR for candidate target sequences or by next-generation sequencing for genome-wide binding profiles. Critical quality control steps include verifying fragment size distribution after sonication, checking enrichment of known control regions, and confirming minimal background in IgG controls .

What considerations are important when combining At3g18410 antibody detection with proteomics approaches?

When combining At3g18410 antibody detection with proteomics approaches, several important considerations ensure optimal results. First, for immunoprecipitation-mass spectrometry (IP-MS), optimize lysis conditions to maintain protein interactions while ensuring efficient extraction; typically, mild non-ionic detergents (0.5-1% NP-40 or Triton X-100) in physiological buffers work well. Second, implement stringent controls including isotype-matched IgG pulldowns and, where possible, samples from At3g18410 knockout lines to distinguish specific from non-specific interactions. Third, consider crosslinking approaches (chemical or ultraviolet) to capture transient interactions before cell lysis. Fourth, for sample preparation for MS analysis, choose between in-gel digestion following SDS-PAGE separation or on-bead digestion based on your specific research questions. Fifth, data analysis should incorporate statistical methods to distinguish true interactors from background proteins, such as significance analysis of interactome (SAINT) or comparison of protein abundance in reciprocal isolations. Finally, validate key interactions using orthogonal methods such as co-immunoprecipitation with antibodies against putative interacting proteins or functional assays .

How can At3g18410 antibody be used effectively in immunohistochemistry studies of plant tissues?

Effective use of At3g18410 antibody in immunohistochemistry studies of plant tissues requires careful adaptation of protocols to overcome the unique challenges of plant material. Begin with fixation using 4% paraformaldehyde in PBS or appropriate buffer, optimizing duration (typically 12-24 hours) based on tissue type and size. For paraffin embedding, use a graded ethanol series followed by xylene and paraffin infiltration; alternatively, cryo-sectioning may better preserve antigenicity. After sectioning (5-10 μm thick), perform antigen retrieval using citrate buffer (pH 6.0) at 95°C for 10-20 minutes to expose epitopes that may be masked during fixation. Block with 5% normal serum (from the species the secondary antibody was raised in) plus 1% BSA in PBS to reduce non-specific binding. Incubate with the At3g18410 antibody at optimized dilution (starting at 1:100-1:500) overnight at 4°C. After washing, apply fluorescent or enzyme-conjugated secondary antibodies. For plant tissues, include controls for autofluorescence (especially from chlorophyll and cell walls) and implement spectral unmixing if using multiple fluorophores. Counter-staining with DAPI for nuclei or cell wall stains provides structural context. Finally, validate specificity using tissue from knockout plants as negative controls .