At4g19070 Antibody

What is the At4g19070 gene and its protein product?

At4g19070 is a gene from Arabidopsis thaliana (Mouse-ear cress) that encodes the Cadmium-induced protein AS8, also described as a putative membrane lipoprotein. The gene is also identified by alternative designations including T18B16.3, F13C5.230, and T18B16.40 . This protein is of particular interest in plant stress biology research due to its induction following cadmium exposure, suggesting a potential role in heavy metal response mechanisms. The protein appears to be membrane-associated based on its characterization as a putative membrane lipoprotein, though further structural and functional studies are needed to fully elucidate its biological role.

What antibody formats are available for At4g19070 detection?

Currently, researchers have access to polyclonal antibodies raised in rabbits against Arabidopsis thaliana At4g19070 protein. These antibodies are generated through antigen-affinity purification and belong to the IgG isotype class . The commercially available antibody products are specifically reactive against Arabidopsis thaliana targets and have been validated for applications including ELISA and Western blot analyses. For researchers requiring specialized formats, it's worth noting that recombinant versions of the At4g19070 protein are also available, which could potentially be used for custom antibody production or as positive controls in experimental procedures.

How should researchers store and handle At4g19070 antibodies?

While specific storage recommendations for At4g19070 antibodies aren't detailed in the available search results, standard antibody handling protocols should be followed. Typically, antibodies should be stored at -20°C for long-term preservation and at 4°C for short-term use. Repeated freeze-thaw cycles should be avoided by aliquoting the antibody into smaller volumes upon receipt. Prior to use, centrifuge the antibody vial briefly to collect the liquid at the bottom of the tube. When handling the antibody, ensure sterile technique is employed to prevent contamination. For optimal performance, always verify the specific storage and handling recommendations provided by the antibody manufacturer, as these may vary slightly depending on specific formulation characteristics.

What is the recommended protocol for Western blot using At4g19070 antibody?

For Western blot applications using the At4g19070 antibody, researchers should follow a standardized protocol adapted for plant protein detection. Begin by extracting total protein from Arabidopsis tissues using an appropriate buffer containing protease inhibitors. After determining protein concentration, load 20-50 μg of protein per lane on an SDS-PAGE gel (10-12% is typically suitable for this protein). Following electrophoresis, transfer proteins to a PVDF or nitrocellulose membrane and block with 5% non-fat dry milk in TBST for 1 hour at room temperature. Incubate the membrane with At4g19070 antibody (typically at 1:1000 to 1:2000 dilution) overnight at 4°C . After washing with TBST, apply an appropriate secondary antibody conjugated with HRP. Develop using ECL substrate and image according to standard protocols. Always include positive and negative controls to validate specificity of detection.

What are the recommended ELISA protocols with At4g19070 antibody?

The At4g19070 antibody has been validated for ELISA applications . A typical indirect ELISA protocol would involve coating microplate wells with protein extract containing At4g19070 or recombinant At4g19070 protein in carbonate buffer (pH 9.6) overnight at 4°C. After washing with PBS-T, block wells with 1-5% BSA for 1-2 hours at room temperature. Apply the primary At4g19070 antibody at an optimized dilution (typically starting at 1:500 to 1:2000) and incubate for 1-2 hours at room temperature. Following washing, add HRP-conjugated secondary antibody and incubate for 1 hour. Develop with TMB substrate and read absorbance at 450 nm after stopping the reaction with sulfuric acid. For quantitative analyses, include a standard curve using purified recombinant At4g19070 protein at known concentrations.

How can immunolocalization of At4g19070 be optimized in plant tissues?

For immunolocalization studies of At4g19070 in plant tissues, fixation and permeabilization steps are critical. Fix tissue samples in 4% paraformaldehyde in PBS for 30-60 minutes, followed by washing in PBS. For membrane-associated proteins like At4g19070, additional permeabilization with 0.1-0.5% Triton X-100 may be necessary to facilitate antibody penetration. Block non-specific binding sites with 3-5% BSA in PBS for 1 hour. Apply the primary At4g19070 antibody at an optimized dilution (1:100 to 1:500 range is typical for immunohistochemistry) and incubate overnight at 4°C. After washing, apply fluorophore-conjugated secondary antibody and incubate for 1-2 hours at room temperature. Include DAPI staining for nuclear visualization and mount slides with anti-fade mounting medium. Critical controls should include omission of primary antibody and ideally, tissue from At4g19070 knockout plants if available.

How can researchers validate the specificity of At4g19070 antibodies?

Validating antibody specificity is crucial for reliable experimental outcomes. For At4g19070 antibodies, several approaches are recommended. First, perform Western blot analysis comparing wild-type Arabidopsis samples with At4g19070 knockout or knockdown lines—the specific band should be absent or significantly reduced in the latter. Second, pre-adsorb the antibody with purified recombinant At4g19070 protein prior to use in applications; this should eliminate specific binding. Third, verify that the antibody detects recombinant At4g19070 protein expressed in a heterologous system. Fourth, demonstrate that the antibody detects increased protein levels in plants exposed to cadmium, as the target is cadmium-induced. Finally, consider peptide competition assays where specific immunogenic peptides used to generate the antibody block binding in a concentration-dependent manner .

What are common issues when using At4g19070 antibodies and their solutions?

How should researchers interpret contradictory results with At4g19070 antibodies?

When faced with contradictory results using At4g19070 antibodies, researchers should systematically evaluate several factors. First, different antibody lots may have varying specificities and sensitivities—check lot numbers and consider testing multiple antibodies targeting different epitopes of At4g19070. Second, experimental conditions significantly impact antibody performance; systematically optimize buffer compositions, incubation times, and temperatures. Third, sample preparation methods affect protein solubilization and epitope accessibility; compare multiple extraction protocols. Fourth, consider biological variables such as plant growth stage, tissue type, and stress conditions, as At4g19070 is cadmium-induced and likely shows variable expression. Finally, consult antibody validation repositories to compare your results with those reported by other researchers . Document all variables meticulously to identify the source of discrepancies.

How does cadmium exposure affect detection of At4g19070 protein?

Cadmium exposure significantly influences At4g19070 protein detection as it is classified as a "Cadmium-induced protein AS8" . Researchers should consider creating a standardized cadmium exposure protocol to achieve consistent protein expression levels. Typically, Arabidopsis plants are treated with cadmium solutions ranging from 10-100 μM CdCl₂ for periods of 24-72 hours before protein extraction. Concentration-dependent and time-course experiments should be performed to determine optimal induction conditions. When comparing samples across experiments, it is critical to maintain identical cadmium exposure parameters. Researchers should note that sensitivity of antibody detection may vary with protein abundance—samples from non-induced plants may require more sensitive detection methods or higher protein loading, while cadmium-treated samples may produce stronger signals even at lower antibody concentrations.

What approaches can be used for quantitative analysis of At4g19070 expression?

For quantitative analysis of At4g19070 protein expression, researchers can employ several antibody-based techniques with appropriate controls and calibration. Quantitative Western blotting can be performed using serial dilutions of recombinant At4g19070 protein to generate a standard curve. Signal intensities should be captured within the linear range of detection and normalized to housekeeping proteins such as actin or tubulin. For more precise quantification, sandwich ELISA provides better quantitative accuracy, again using recombinant At4g19070 for standardization. Advanced techniques such as capillary electrophoresis immunoassay or automated Western platforms (e.g., Jess or Wes systems) offer improved reproducibility and sensitivity for quantification. Regardless of the method chosen, biological replicates (n≥3) and technical replicates are essential for statistical validity.

What are the considerations for using At4g19070 antibodies across different plant species?

When using At4g19070 antibodies across different plant species, sequence homology analysis is essential. Researchers should first perform BLAST searches to identify homologous proteins in target species and determine the degree of conservation within antibody epitope regions. Cross-reactivity testing should be performed empirically using Western blot on protein extracts from multiple species. Higher antibody concentrations may be required for cross-species applications, though this increases the risk of non-specific binding. Positive controls using Arabidopsis thaliana samples should always be included. For species with lower sequence homology, epitope-specific antibodies targeting the most conserved regions may offer better cross-reactivity. If cross-reactivity is weak or absent, researchers might consider custom antibody production against species-specific epitopes or the recombinant protein from the species of interest.

How can researchers find validated At4g19070 antibodies?

Researchers seeking validated At4g19070 antibodies should utilize specialized antibody search engines and data repositories. These platforms allow scientists to compare antibodies from different vendors and access validation data to assess antibody performance . For plant-specific antibodies like At4g19070, researchers should first check general antibody search engines that aggregate products from multiple vendors. Additionally, plant biology resource centers and databases may contain information about antibodies used in published research on Arabidopsis proteins. When selecting an antibody, prioritize those with comprehensive validation data including Western blot images, immunohistochemistry results, and specificity testing (e.g., using knockout lines). Consider reaching out to authors of publications that have successfully used At4g19070 antibodies for recommendations and protocols tailored to specific applications.

What alternative approaches exist when antibodies show limited performance?

When At4g19070 antibodies demonstrate limited performance, researchers have several alternative approaches. First, consider epitope-tagged expression systems where At4g19070 is fused with established tags (FLAG, HA, GFP) that have highly specific commercial antibodies available. This approach requires genetic transformation but provides highly specific detection. Second, targeted proteomics using mass spectrometry can identify and quantify At4g19070 protein without antibodies, though this requires specialized equipment and expertise. Third, transcriptional analysis (qPCR, RNA-Seq) can serve as a proxy for protein expression, acknowledging the limitations of inferring protein levels from transcript abundance. Fourth, CRISPR-based tagging of the endogenous At4g19070 locus with fluorescent proteins enables direct visualization without antibodies. Finally, if studying protein function is the primary goal, phenotypic analysis of knockout/knockdown lines may provide valuable insights without requiring direct protein detection.