At1g10110 Antibody

What is At1g10110 and why is it significant for plant research?

At1g10110 (UniProt: O80603) is a protein-coding gene in Arabidopsis thaliana that has become an important research target in plant molecular biology. The protein is involved in cellular processes that make it valuable for understanding plant development and stress responses. Antibodies against this protein allow researchers to conduct fundamental investigations of protein expression, localization, and interactions across different developmental stages and environmental conditions. The significance lies in the ability to track this specific protein's presence and modifications when studying various plant biological processes, enabling correlation between genotype and phenotype in experimental Arabidopsis models .

How is the At1g10110 antibody produced and what are its key specifications?

The At1g10110 antibody is produced using recombinant Arabidopsis thaliana At1g10110 protein as the immunogen. It is raised in rabbits, resulting in a polyclonal IgG antibody that recognizes multiple epitopes of the target protein. The antibody undergoes antigen affinity purification to ensure specificity and is supplied in liquid form with a storage buffer containing 0.03% Proclin 300 as preservative, 50% glycerol, and 0.01M PBS at pH 7.4. This polyclonal nature provides robust detection capabilities across different experimental contexts while maintaining specificity for the target protein .

What are the validated applications for At1g10110 antibody?

The At1g10110 antibody has been validated for enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB) applications. These techniques are fundamental for quantifying protein expression levels and confirming protein identity based on molecular weight, respectively. Similar to other plant protein antibodies, optimal working dilutions must be determined empirically for each specific application and experimental system, as factors such as protein abundance and sample preparation methods can influence detection sensitivity .

What are the optimal protein extraction methods for At1g10110 detection in Western blotting?

For optimal At1g10110 detection in Western blotting, researchers should consider methodologies similar to those used for other Arabidopsis proteins. Based on protocols used for similar plant antibodies, a recommended approach involves extracting proteins using trichloroacetic acid and acetone precipitation methods. Fresh tissue (approximately 7-day-old seedlings) should be collected and flash-frozen in liquid nitrogen before grinding to a fine powder. The powder should be homogenized in extraction buffer containing protease inhibitors to prevent protein degradation. Following extraction, proteins should be denatured with lithium dodecyl sulfate (LDS) sample buffer at 70°C for 10 minutes before loading onto 12% SDS-PAGE gels. For transfer, PVDF membranes with 0.2 μm pore size are recommended using semi-dry transfer systems .

How should researchers optimize blocking and antibody incubation conditions?

For optimal results with At1g10110 antibody, researchers should begin with a blocking solution of 5% non-fat dry milk in TBS-T (Tris-buffered saline with 0.1% Tween-20) for 1-4 hours at room temperature or overnight at 4°C with gentle agitation. The primary antibody dilution should start at 1:500 to 1:1000 in blocking buffer, with incubation for 2 hours at room temperature or overnight at 4°C with gentle agitation. Following primary antibody incubation, membranes should be washed thoroughly (at least three 15-minute washes) with TBS-T before incubation with an appropriate anti-rabbit IgG secondary antibody conjugated to horseradish peroxidase. These conditions should be optimized based on signal-to-noise ratio in preliminary experiments, as protein abundance can vary across different plant tissues and developmental stages .

What controls are essential when using At1g10110 antibody for the first time?

When using At1g10110 antibody for the first time, several controls are essential to validate results. A positive control using wild-type Arabidopsis thaliana tissue known to express the target protein should be included. Negative controls should consist of: (1) samples from knockout or knockdown lines of At1g10110 if available, (2) primary antibody omission control to assess non-specific binding of the secondary antibody, and (3) pre-immune serum control if available. Additionally, a loading control using antibodies against constitutively expressed proteins (such as actin or tubulin) should be performed on the same samples to normalize protein loading. These controls help establish antibody specificity and performance characteristics in your specific experimental system .

How can researchers address weak or absent signal issues when using At1g10110 antibody?

When encountering weak or absent signals with At1g10110 antibody, researchers should systematically troubleshoot by examining several key aspects of their experimental procedure. First, verify protein extraction efficiency using total protein stains (Ponceau S or Coomassie) before immunoblotting. Second, assess whether the target protein might be degrading during extraction by adding fresh protease inhibitors and keeping samples cold throughout processing. Third, try increasing the primary antibody concentration (up to 1:250) or extending incubation time to overnight at 4°C. Fourth, enhance detection sensitivity by using more sensitive detection reagents like enhanced chemiluminescence (ECL) substrates designed for low-abundance proteins. Finally, consider enriching the target protein through immunoprecipitation prior to Western blotting if expression levels are very low. If the target protein is particularly sensitive to denaturation conditions, try alternative buffer systems or gentler denaturation methods .

What strategies can reduce background and non-specific binding when using At1g10110 antibody?

To reduce background and non-specific binding when using At1g10110 antibody, several optimization strategies can be employed. First, increase the stringency of blocking by testing different blocking agents (BSA, casein, or commercial blocking reagents) instead of milk when high background persists. Second, add 0.1-0.5% Tween-20 or 0.1% Triton X-100 to antibody dilution buffers to reduce hydrophobic interactions. Third, increase salt concentration in wash buffers (up to 500 mM NaCl) to disrupt low-affinity non-specific interactions. Fourth, pre-absorb the primary antibody with plant tissue lysate from At1g10110 knockout plants if available. Fifth, use more rigorous washing steps (increasing number and duration of washes) after both primary and secondary antibody incubations. Finally, titrate both primary and secondary antibodies to determine the minimum concentration that provides specific signal while minimizing background .

How should At1g10110 antibody be stored to maintain long-term activity?

For optimal long-term activity of At1g10110 antibody, proper storage is critical. Upon receipt, the antibody should be stored at -20°C or preferably -80°C. After initial thawing, the antibody should be aliquoted into small single-use volumes to avoid repeated freeze-thaw cycles, which can degrade antibody performance. Each aliquot should be sufficient for a single experiment to prevent repeated thawing. Working dilutions should be prepared fresh on the day of use and not stored for future experiments. If extended storage of diluted antibody is necessary, addition of stabilizing proteins (such as 1% BSA) and preservatives compatible with downstream applications may help maintain activity. Always centrifuge antibody vials briefly before opening to collect liquid at the bottom of the tube and prevent loss of material. When handling, use clean pipette tips and avoid contamination with bacteria or fungi .

Can the At1g10110 antibody be adapted for immunohistochemistry or immunofluorescence in plant tissues?

While the At1g10110 antibody is primarily validated for ELISA and Western blotting, adaptation for immunohistochemistry (IHC) or immunofluorescence (IF) in plant tissues may be possible with appropriate optimization. Drawing from protocols for other plant antibodies like Anti-HY5, researchers should begin with fixation using 4% paraformaldehyde, followed by careful permeabilization steps that preserve tissue architecture. Antigen retrieval may be necessary due to fixation-induced epitope masking. Initial antibody concentration should be higher than for Western blotting (starting at 1:50 to 1:200) with overnight incubation at 4°C. Validation should include appropriate negative controls using pre-immune serum and competition with the immunizing peptide if available. Additionally, autofluorescence is a common challenge in plant tissues, so appropriate quenching steps and selection of fluorophores with emission spectra distinct from plant autofluorescence are crucial considerations .

How can At1g10110 antibody be incorporated into chromatin immunoprecipitation (ChIP) protocols?

Adapting At1g10110 antibody for chromatin immunoprecipitation would require extensive optimization if the protein has DNA-binding properties. The polyclonal nature of the antibody makes it potentially suitable for ChIP applications, provided the target protein interacts with DNA either directly or as part of a complex. Researchers should begin with a standard plant ChIP protocol, cross-linking proteins to DNA with 1% formaldehyde for 10-15 minutes at room temperature. After nuclei isolation and chromatin shearing (to fragments of approximately 200-500 bp), immunoprecipitation should be performed using a higher concentration of antibody than for Western blotting (typically 2-5 μg per reaction). Include appropriate controls: input chromatin (pre-immunoprecipitation sample), no-antibody control, and ideally a non-specific IgG control. Optimization of chromatin shearing, antibody concentration, and washing stringency will be critical for success. Validation should include qPCR targeting known or suspected binding regions versus control regions, with results expressed as percent of input or fold enrichment over IgG control .

What considerations are important when using At1g10110 antibody in co-immunoprecipitation experiments?

For co-immunoprecipitation (Co-IP) experiments using At1g10110 antibody, preserving native protein complexes is crucial. Researchers should use non-denaturing lysis buffers (typically containing 0.5-1% non-ionic detergents like NP-40 or Triton X-100) supplemented with protease inhibitors and phosphatase inhibitors if phosphorylation states are important. Pre-clearing lysates with protein A/G beads can reduce non-specific binding. For the immunoprecipitation step, 2-5 μg of antibody per 500 μg of total protein is recommended as a starting point, with overnight incubation at 4°C followed by capture with protein A beads (appropriate for rabbit IgG). Washing steps should balance stringency with preservation of protein-protein interactions. Elution can be performed using antibody-specific peptide competition if available, or by standard SDS elution. Controls should include a pre-immune IgG precipitation and, ideally, samples from knockout/knockdown plants. Validation of interacting partners should include reciprocal Co-IP when possible, along with mass spectrometry identification of novel interaction partners .

How should researchers interpret variability in At1g10110 detection across different plant tissues and developmental stages?

When interpreting variability in At1g10110 detection across different plant tissues and developmental stages, researchers should consider multiple factors. First, expression levels of the target protein naturally vary across tissues and developmental stages, requiring adjustment of protein loading and antibody concentrations accordingly. Second, extraction efficiency may differ between tissue types due to varying compositions of interfering compounds, cell wall components, or proteases. Third, post-translational modifications might affect epitope recognition, potentially resulting in apparent expression differences that actually reflect modification state changes. To address these challenges, researchers should normalize data to multiple housekeeping proteins that remain stable across the conditions tested, use consistent protein extraction protocols optimized for each tissue type, and consider complementary techniques such as RT-qPCR to correlate protein detection with transcript levels. Finally, statistical analysis across multiple biological replicates is essential to distinguish technical variability from genuine biological differences .

What approaches can researchers use to validate At1g10110 antibody specificity in their experimental system?

To rigorously validate At1g10110 antibody specificity in an experimental system, researchers should implement multiple complementary approaches. The gold standard is comparing Western blot results between wild-type plants and genetic knockout/knockdown lines, which should show reduced or absent signal in the latter. If such lines are unavailable, RNAi-mediated silencing or CRISPR-Cas9 targeting of At1g10110 can create validation materials. Another approach is peptide competition assays, where pre-incubation of the antibody with excess immunizing peptide should abolish specific signals. Mass spectrometry analysis of immunoprecipitated material can confirm the identity of the detected protein band. Additionally, correlation between protein levels detected by the antibody and mRNA levels measured by RT-qPCR across different conditions provides supporting evidence for specificity. Finally, detecting the same pattern with a second antibody raised against a different epitope of the same protein (if available) would strongly support specificity claims .

How can researchers quantitatively analyze Western blot data from At1g10110 antibody experiments?

For quantitative analysis of Western blot data using At1g10110 antibody, researchers should follow a structured approach that ensures reliability and reproducibility. First, include a dilution series of a reference sample on each blot to confirm that detection falls within the linear range of signal response. Second, use appropriate normalization controls—typically housekeeping proteins like actin, tubulin, or GAPDH—that remain stable across experimental conditions, blotting these on the same membrane when possible. Third, employ image analysis software (e.g., ImageJ with gel analysis plugins) to quantify band intensities while ensuring background subtraction is consistent across all lanes. Fourth, normalize target protein signals to loading control signals from the same lane. Fifth, analyze data from at least three independent biological replicates using appropriate statistical tests. Finally, report both representative blot images and quantification graphs with error bars and statistical significance indicators. This approach allows for meaningful comparison of At1g10110 protein levels across different experimental conditions or genotypes .

How does the performance of At1g10110 antibody compare with other plant protein antibodies in Arabidopsis research?

The At1g10110 antibody demonstrates performance characteristics that align with other well-established plant protein antibodies used in Arabidopsis research. Like the Anti-HY5 antibody used for detecting the transcription factor ELONGATED HYPOCOTYL 5, the At1g10110 antibody is a rabbit-derived polyclonal antibody that offers robust detection in biochemical applications. Both antibodies require similar optimization steps for Western blotting applications, including empirical determination of dilution factors (typically in the 1:500 to 1:1000 range) and careful blocking steps to minimize background. The species specificity of At1g10110 antibody is currently limited to Arabidopsis thaliana, which is narrower than some plant antibodies that demonstrate cross-reactivity with multiple plant species. As with most plant antibodies, researchers should anticipate the need for protocol optimization when applying the antibody to new experimental conditions or sample types .

How might advances in antibody engineering and production techniques impact future versions of At1g10110 antibody?

Advances in antibody engineering and production techniques are likely to significantly impact future iterations of plant antibodies like the At1g10110 antibody. Recombinant antibody technologies may enable the production of synthetic antibody fragments (such as single-chain variable fragments or nanobodies) with enhanced specificity and reduced background binding. These engineered antibodies could offer advantages including better lot-to-lot consistency, reduced cross-reactivity, and potentially enhanced performance in applications beyond Western blotting and ELISA. Additionally, advances in phage display and yeast display technologies may facilitate the selection of antibodies with optimal characteristics for specific applications such as super-resolution microscopy or in vivo imaging in plant tissues. The incorporation of site-specific conjugation techniques would allow precise control over the attachment of detection molecules (fluorophores, enzymes) to improve sensitivity while maintaining native binding properties. These technological advances may eventually yield At1g10110 detection reagents with superior specificity, sensitivity, and application versatility compared to current polyclonal antibodies .