At5g35930 Antibody

What is At5g35930 and why is it significant for plant research?

At5g35930 encodes an AMP-dependent synthetase and ligase family protein in Arabidopsis thaliana. This protein functions in phosphopantetheine binding, acyl carrier activity, catalytic activity, AMP binding, and cofactor binding, making it an important component in plant metabolic pathways . The plasma membrane localization (SUBAcon score: 1.000) suggests it may play a role in membrane-associated metabolic processes or signaling . Antibodies against At5g35930 allow researchers to study the protein's expression, localization, and function in plant cellular processes, particularly those involving lipid metabolism or membrane-associated pathways.

What experimental applications are suitable for At5g35930 antibody?

Based on validated testing parameters, At5g35930 antibody is primarily suitable for ELISA and Western blotting (WB) applications . When designing experiments, researchers should consider that this is a polyclonal antibody raised in rabbits against recombinant Arabidopsis thaliana At5g35930 protein . While not explicitly validated for other techniques, experienced researchers might adapt the antibody for immunohistochemistry, immunofluorescence, or immunoprecipitation with proper validation steps. Each application requires specific optimization protocols, as antibody performance can vary significantly between techniques even when the same antibody is used.

What controls should be included when using At5g35930 antibody?

Proper controls are essential for reliable interpretation of results when using At5g35930 antibody. The following table outlines the recommended controls:

The importance of proper controls cannot be overstated, as inadequate antibody characterization significantly contributes to irreproducible research. Studies estimate that approximately 50% of commercial antibodies fail to meet basic standards for characterization, resulting in billions of dollars in research waste annually .

How should At5g35930 antibody be stored and handled?

The At5g35930 antibody should be stored at -20°C or -80°C immediately upon receipt to maintain its functionality . Avoid repeated freeze-thaw cycles, as these can degrade antibody quality and reduce binding efficiency. The antibody is provided in liquid form with a storage buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . When handling the antibody:

• Aliquot upon first thawing to minimize freeze-thaw cycles

• Use sterile technique when handling to prevent contamination

• Allow the antibody to reach room temperature before opening the vial

• Centrifuge briefly before use to collect all liquid at the bottom of the tube

• Return unused portions to -20°C or -80°C immediately after use

Proper storage and handling significantly impact antibody performance and experimental reproducibility.

How can I validate the specificity of At5g35930 antibody?

Antibody validation is a critical step before using At5g35930 antibody in experiments, particularly given the widespread concerns about antibody reproducibility in the scientific community . A comprehensive validation approach includes:

• Western blot analysis: Confirm a single band of expected molecular weight using:

  • Wild-type samples (positive control)

  • At5g35930 knockout/knockdown samples (negative control)

  • Recombinant At5g35930 protein (positive control)

• Immunoprecipitation followed by mass spectrometry: Verify that the antibody captures the intended target protein.

• Cross-reactivity testing: Test against closely related AMP-dependent synthetase and ligase family proteins to ensure specificity.

• Epitope competition assay: Pre-incubate antibody with excess immunizing peptide to confirm signal elimination.

• Orthogonal method verification: Compare protein localization or expression patterns with results from transcript analysis (qPCR or RNA-seq).

What dilution factors are optimal for different applications?

While specific dilution recommendations for At5g35930 antibody are not explicitly provided in the datasheet, the following table provides starting points based on general guidelines for polyclonal antibodies and should be optimized for your specific experimental conditions:

For each application, perform a dilution series experiment to determine the optimal antibody concentration that provides the best signal-to-noise ratio for your specific experimental system.

How can I optimize immunohistochemical protocols for At5g35930 in plant tissues?

Immunohistochemical detection of At5g35930 in plant tissues presents unique challenges due to the presence of cell walls and other structural components. Drawing from immunohistochemical techniques used in other systems , the following optimization steps are recommended:

• Fixation: Test different fixatives:

  • 4% paraformaldehyde (PFA) for 2-4 hours (preserves protein structure)

  • Farmer's fixative (3:1 ethanol:acetic acid) for membrane proteins

  • Cold methanol for 10 minutes (may better preserve certain epitopes)

• Antigen retrieval: Critical for accessing epitopes in fixed tissues:

  • Heat-induced epitope retrieval: 10mM sodium citrate buffer (pH 6.0) at 95°C for 10-20 minutes

  • Enzymatic retrieval: Proteinase K (1-10 μg/ml) or cellulase/pectinase treatment

• Permeabilization: Essential for antibody access:

  • 0.1-0.5% Triton X-100 for 15-30 minutes

  • 1-2% DMSO as an alternative permeabilizing agent

• Blocking: Reduce non-specific binding:

  • 3-5% BSA or normal serum from the same species as the secondary antibody

  • Include 0.1% Tween-20 in blocking solution

• Primary antibody incubation:

  • Extended incubation (overnight at 4°C) often improves signal

  • Consider using a humid chamber to prevent drying

Optimization of these parameters should be done systematically, changing one variable at a time while keeping others constant to determine the optimal conditions for At5g35930 detection in plant tissues.

How can I troubleshoot weak or non-specific signals in Western blotting?

Western blotting with At5g35930 antibody may present challenges that require methodical troubleshooting. The following table outlines common issues and their solutions:

When optimizing Western blot protocols, it's important to remember that approximately 50% of commercial antibodies may not meet basic standards for characterization , so rigorous validation is essential.

How can I use At5g35930 antibody for co-immunoprecipitation studies?

Co-immunoprecipitation (Co-IP) using At5g35930 antibody can reveal protein-protein interactions involving this AMP-dependent synthetase and ligase family protein. The following protocol provides a framework for Co-IP experiments:

• Sample preparation:

  • Harvest fresh plant tissue (preferably young tissue with active metabolism)

  • Grind in liquid nitrogen and lyse in a non-denaturing lysis buffer (50mM Tris-HCl pH 7.5, 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, protease inhibitor cocktail)

  • Centrifuge at 14,000×g for 15 minutes at 4°C to remove debris

• Pre-clearing (reduces non-specific binding):

  • Incubate lysate with Protein A/G beads for 1 hour at 4°C

  • Remove beads by centrifugation

• Immunoprecipitation:

  • Add 2-5 μg of At5g35930 antibody to pre-cleared lysate

  • Incubate overnight at 4°C with gentle rotation

  • Add fresh Protein A/G beads and incubate for 2-4 hours at 4°C

  • Wash beads 4-5 times with lysis buffer

• Elution and analysis:

  • Elute proteins by boiling in SDS sample buffer

  • Analyze by SDS-PAGE followed by Western blotting or mass spectrometry

For identifying novel protein interactions, mass spectrometry analysis of co-immunoprecipitated proteins can reveal potential binding partners of At5g35930, which may provide insights into its functional role in AMP-dependent synthetase and ligase pathways.

How can I design experiments to study At5g35930 expression under different stress conditions?

Investigating At5g35930 expression under different stress conditions requires a well-designed experimental approach. Based on experimental design principles , the following framework is recommended:

• Define variables:

  • Independent variable: Stress condition (e.g., drought, salt, temperature)

  • Dependent variable: At5g35930 protein expression level

  • Control variables: Growth conditions, plant age, tissue type

• Experimental design:

  • Use a between-subjects design with multiple treatment groups

  • Include appropriate controls (untreated plants, positive controls)

  • Define stress treatment parameters (duration, intensity)

• Sample collection and processing:

  • Harvest tissues at defined time points after stress application

  • Process samples consistently using standardized protocols

  • Extract proteins using buffers optimized for membrane proteins

• Quantitative analysis:

  • Use Western blotting with appropriate loading controls (e.g., actin, tubulin)

  • Include standard curves with known amounts of recombinant protein

  • Analyze band intensity using densitometry software

• Data analysis:

  • Apply appropriate statistical tests (ANOVA, t-test)

  • Account for biological and technical replicates

  • Consider time-course analysis for dynamic responses

This experimental design allows for rigorous testing of how At5g35930 expression responds to environmental stresses, potentially revealing its role in stress adaptation mechanisms in plants.

How can I investigate the subcellular localization of At5g35930?

While SUBAcon predicts At5g35930 to be localized to the plasma membrane with high confidence (score: 1.000) , experimental verification is essential. The following approaches can be used to confirm and investigate the subcellular localization:

• Immunofluorescence microscopy:

  • Fix plant tissues with 4% paraformaldehyde

  • Permeabilize with 0.1-0.5% Triton X-100

  • Incubate with At5g35930 antibody followed by fluorescently-labeled secondary antibody

  • Co-stain with markers for different cellular compartments (e.g., plasma membrane, ER, Golgi)

  • Analyze using confocal microscopy

• Subcellular fractionation:

  • Separate cellular components through differential centrifugation

  • Prepare enriched fractions of plasma membrane, cytosol, and other organelles

  • Analyze fractions by Western blotting with At5g35930 antibody

  • Include markers for different subcellular compartments as controls

• Protease protection assay:

  • Treat isolated membrane fractions with proteases with or without detergent

  • Analyze by Western blotting to determine protein topology

• GFP fusion protein approach (complementary method):

  • Generate At5g35930-GFP fusion constructs

  • Express in Arabidopsis or protoplasts

  • Visualize localization by live-cell imaging

  • Compare with antibody-based localization results

These approaches will provide robust evidence for the subcellular localization of At5g35930, confirming or refining the computational prediction from SUBAcon.

What considerations are important when using At5g35930 antibody for cross-species studies?

Using At5g35930 antibody in non-Arabidopsis species requires careful consideration of sequence conservation and epitope accessibility. The following approach is recommended:

Cross-species application of antibodies should always be approached with caution, as even minor sequence variations can significantly affect antibody binding and experimental outcomes.

How can I use At5g35930 antibody in quantitative proteomics studies?

Integrating At5g35930 antibody into quantitative proteomics workflows can provide insights into the regulation and function of this protein. The following approaches are recommended:

• Immunoaffinity enrichment coupled with mass spectrometry:

  • Immunoprecipitate At5g35930 and associated proteins

  • Digest samples with trypsin

  • Analyze by LC-MS/MS

  • Quantify using label-free or isotope labeling approaches (SILAC, TMT, iTRAQ)

• Selected reaction monitoring (SRM)/Multiple reaction monitoring (MRM):

  • Develop specific peptide transitions for At5g35930

  • Use antibody-based enrichment before targeted MS analysis

  • Quantify absolute amounts using isotope-labeled standard peptides

• Reverse phase protein arrays (RPPA):

  • Create lysate dilution series from different experimental conditions

  • Spot onto nitrocellulose slides

  • Probe with At5g35930 antibody

  • Quantify signal intensity relative to standard curves

• Antibody-based absolute quantification:

  • Develop a quantitative ELISA using At5g35930 antibody

  • Generate standard curves with recombinant At5g35930 protein

  • Calculate absolute protein amounts in different tissues/conditions

These approaches allow for precise quantification of At5g35930 across different experimental conditions, developmental stages, or genetic backgrounds, providing insights into its regulation and function in plant metabolism.

How can I use At5g35930 antibody to study protein-protein interactions within the AMP-dependent synthetase pathway?

Understanding protein-protein interactions involving At5g35930 can reveal its functional role within metabolic networks. Several approaches using the At5g35930 antibody can be employed:

• Co-immunoprecipitation followed by targeted Western blotting:

  • Immunoprecipitate using At5g35930 antibody

  • Probe Western blots with antibodies against suspected interaction partners

  • Include appropriate controls (IgG control, input samples)

• Proximity ligation assay (PLA):

  • Use At5g35930 antibody in combination with antibodies against potential partners

  • Apply species-specific secondary antibodies linked to oligonucleotides

  • Amplify signal when proteins are in close proximity (<40 nm)

  • Visualize interaction sites by fluorescence microscopy

• FRET-based immunoassays:

  • Label At5g35930 antibody with donor fluorophore

  • Label partner protein antibody with acceptor fluorophore

  • Measure energy transfer as indicator of protein-protein proximity

• Crosslinking immunoprecipitation (CLIP):

  • Chemically crosslink protein complexes in vivo

  • Immunoprecipitate using At5g35930 antibody

  • Identify interacting proteins by mass spectrometry

  • Verify interactions through reverse immunoprecipitation

These methods can help map the interactome of At5g35930, providing insights into its role in AMP-dependent synthetase and ligase pathways and potentially revealing novel functions in plant metabolism.