At3g15980 Antibody

What is At3g15980 and why would researchers need an antibody against it?

At3g15980 is a gene in Arabidopsis thaliana that is part of a genome cluster including five genes (At3g15940, At3g15950, At3g15960, At3g15970, and At3g15980). The gene At3g15950, known as NAI2, has been identified as necessary for ER body formation. While At3g15980's specific function is less characterized, it appears to be deleted in nai2-1 mutants along with the surrounding genes, suggesting potential related functions in ER body biology . Researchers would need antibodies against At3g15980 to study its protein localization, expression patterns, protein-protein interactions, and potential roles in ER body formation or plant defense mechanisms.

How are antibodies against plant proteins like At3g15980 typically generated?

Antibodies against plant proteins like At3g15980 are typically generated through a process similar to that used for NAI2 antibody development. The methodology typically involves:

• Identification and cloning of partial cDNA fragments of the target gene

• Amplification of these fragments using PCR with gene-specific primers

• Introduction of the amplified fragments into appropriate vectors (such as pCR8/GW/Topo)

• Transfer of the protein-coding region to expression vectors (such as pDEST17)

• Production of His-tagged fusion proteins in bacterial expression systems (commonly E. coli BL21-AI)

• Purification of recombinant proteins using column chromatography (often nickel nitrilotriacetic acid agarose)

• Injection of purified proteins into rabbits or other animals to raise antibodies

This approach allows for the generation of specific antibodies that can then be validated and used in various experimental applications.

What detection methods are suitable for antibodies targeting At3g15980?

For antibodies targeting At3g15980, researchers can employ several detection methods based on established protocols for plant protein antibodies:

• Immunofluorescence analysis: Similar to techniques used for NAI2, this involves using primary antibodies against At3g15980 followed by fluorescently-labeled secondary antibodies for visualization under fluorescence microscopy .

• Western blotting: Using SDS-PAGE to separate proteins followed by transfer to membranes and detection with the At3g15980 antibody.

• Immunoprecipitation: For studying protein-protein interactions, though coimmunoprecipitation experiments should be carefully designed as demonstrated in NAI2 studies which failed to detect direct interaction with PYK10 .

• High-density peptide microarrays: This advanced technique can be employed to assess antibody specificity and cross-reactivity profiles, similar to the approach used in autoantibody studies .

How should I design experiments to evaluate At3g15980 antibody specificity and cross-reactivity?

Designing rigorous experiments to evaluate At3g15980 antibody specificity requires a methodical approach:

• Genetic controls: Include wild-type plants and at3g15980 mutant/knockout lines. The antibody should show positive signal in wild-type and no signal in knockout material if it's specific .

• High-density peptide microarrays: These can be used to systematically test antibody specificity against thousands of peptides:

  • Design overlapping 15-mer peptides from At3g15980 and related proteins with at least 9 amino acids overlap

  • Include peptides from homologous proteins to test cross-reactivity

  • Use dilution series (typically 1:100 for purified antibodies) and appropriate controls

  • Employ fluorescently labeled secondary antibodies (e.g., goat anti-rabbit IgG conjugated to Alexa Fluor 647)

  • Analyze signal intensity patterns across peptides to identify specific binding regions

• Preabsorption controls: Pre-incubate the antibody with purified recombinant At3g15980 protein before immunostaining to confirm specificity.

• Western blot validation: The antibody should detect bands of the predicted molecular weight in wild-type samples but not in knockout lines .

What factors should be considered when optimizing immunohistochemistry protocols for At3g15980 antibody?

When optimizing immunohistochemistry protocols for At3g15980 antibody, consider these critical factors:

• Fixation method: The choice between paraformaldehyde, glutaraldehyde, or other fixatives affects epitope accessibility. For ER body proteins like NAI2, paraformaldehyde fixation has proven effective .

• Antigen retrieval: May be necessary if the fixation process masks epitopes.

• Blocking conditions: To reduce non-specific binding, optimize:

  • Blocking agent (BSA, serum, commercial blockers)

  • Concentration (typically 0.1-5%)

  • Duration (1-2 hours at room temperature)

• Antibody dilution: Determine optimal primary antibody concentration through titration experiments. For plant antibodies similar to NAI2, typical dilutions range from 1:100 to 1:1000 .

• Incubation conditions:

  • Temperature (4°C, room temperature)

  • Duration (2-4 hours or overnight)

  • Buffer composition (PBS, TBS, with appropriate detergents)

• Signal detection system: Choose appropriate secondary antibodies and visualization methods based on your imaging system.

• Controls: Include wild-type and knockout tissues, as well as secondary-only controls to assess background.

How can I troubleshoot inconsistent results when using At3g15980 antibody in different plant tissues?

Troubleshooting inconsistent results with At3g15980 antibody across different plant tissues requires systematic investigation:

• Tissue-specific expression analysis:

  • Verify if At3g15980 expression varies across tissues using RT-qPCR

  • Check public expression databases for tissue-specific expression patterns

  • Consider that NAI2 (At3g15950) shows specific expression patterns that may also apply to neighboring genes

• Sample preparation optimization:

  • Adjust fixation time for different tissues (denser tissues may require longer fixation)

  • Modify permeabilization protocols based on tissue type

  • Consider tissue-specific clearing methods to improve antibody penetration

• Extraction buffer optimization:

  • Different tissues may require different extraction buffers

  • For ER body proteins like NAI2, specialized fractionation methods have been developed to preserve structure and function

• Antibody concentration adjustment:

  • Perform titration curves for each tissue type

  • Consider using higher antibody concentrations for tissues with lower target expression

• Positive and negative controls:

  • Include tissues known to express or not express At3g15980

  • Use genetic knockouts as definitive negative controls

How can Design of Experiments (DOE) methodology be applied to optimize At3g15980 antibody-based assays?

Design of Experiments (DOE) methodology can significantly improve the optimization process for At3g15980 antibody-based assays:

• Parameter identification: Identify key variables affecting assay performance:

  • Antibody concentration (typically ranging from 1:50 to 1:1000)

  • Incubation temperature (4°C to 37°C)

  • Incubation time (1-24 hours)

  • Buffer pH (typically 6.8-7.8)

  • Blocking agent concentration

  • Washing steps duration and number

• Statistical design selection:

  • For early-phase optimization, use factorial design (full or fractional)

  • Include center points to assess variability and detect curvature

  • Define response variables (signal-to-noise ratio, background, specificity)

• Execution and analysis:

  • Run experiments according to the design matrix

  • Analyze data using appropriate statistical software

  • Generate response surface models to identify optimal conditions

  • Validate findings with confirmation runs

• Design space definition:

  • Establish boundaries where all critical quality attributes are met

  • Define robust setpoints within this space

  • Document parameters that significantly affect assay performance

This approach allows for systematic optimization while minimizing the number of experiments required, leading to more robust and reproducible antibody-based assays.

What are the recommended protocols for antibody purification from polyclonal antisera against At3g15980?

For purifying antibodies against At3g15980 from polyclonal antisera, the following protocol is recommended based on established methods:

• Antigen preparation:

  • Express recombinant At3g15980 protein fragments in E. coli using systems like BL21-AI

  • Include a His-tag or other purification tag

  • Purify using affinity chromatography (nickel columns for His-tagged proteins)

• Affinity purification:

  • Couple purified recombinant At3g15980 protein to an appropriate matrix (CNBr-activated Sepharose or similar)

  • Pass polyclonal serum through the column

  • Wash extensively to remove non-specific antibodies

  • Elute specific antibodies using low pH buffer (typically glycine-HCl, pH 2.5-3.0)

  • Immediately neutralize with Tris buffer

  • Dialyze against PBS or appropriate storage buffer

• Quality control:

  • Test specificity using Western blotting against plant extracts

  • Confirm activity in relevant applications (immunofluorescence, immunoprecipitation)

  • Determine protein concentration using Bradford or BCA assay

  • Assess purity using SDS-PAGE

• Storage:

  • Add preservatives (0.02% sodium azide, if compatible with intended applications)

  • Aliquot to avoid freeze-thaw cycles

  • Store at -20°C or -80°C for long-term storage

How can I develop a quantitative assay to measure At3g15980 protein levels in plant extracts?

Developing a quantitative assay for At3g15980 protein levels requires systematic approach:

• ELISA development:

  • Coat plates with purified anti-At3g15980 antibody (capture antibody)

  • Optimize blocking conditions (typically 1-5% BSA or similar)

  • Create standard curve using purified recombinant At3g15980 protein

  • Develop detection system using either:

    • Direct detection with enzyme-conjugated anti-At3g15980 antibody

    • Sandwich ELISA with a second antibody recognizing a different epitope

  • Optimize incubation times, temperatures, and washing steps

  • Validate assay for linearity, precision, accuracy, and detection limits

• Western blot quantification:

  • Use purified recombinant At3g15980 protein to generate standard curves

  • Ensure equal loading using housekeeping protein controls

  • Optimize transfer conditions for At3g15980's molecular weight

  • Use fluorescent secondary antibodies for wider dynamic range

  • Analyze using appropriate imaging software with correction for background

• Mass spectrometry-based quantification:

  • Develop Selected Reaction Monitoring (SRM) or Parallel Reaction Monitoring (PRM) assays

  • Identify suitable proteotypic peptides from At3g15980

  • Use stable isotope-labeled peptide standards for absolute quantification

  • Optimize extraction procedures to maximize protein recovery

• Validation across tissue types:

  • Confirm assay performance in different plant tissues

  • Assess matrix effects and modify protocols accordingly

  • Use genetic controls (knockout lines) to confirm specificity

What advanced imaging techniques are most suitable for studying At3g15980 localization in relation to ER bodies?

For studying At3g15980 localization in relation to ER bodies, several advanced imaging techniques are particularly valuable:

• Confocal laser scanning microscopy:

  • Use immunofluorescence with anti-At3g15980 antibodies

  • Combine with ER markers (e.g., anti-BiP antibodies) for colocalization studies

  • Apply appropriate resolution settings (typically 10 μm resolution with 16-bit depth for adequate detection of fluorescent signals)

  • Consider spectral unmixing for multiple fluorophores

• Super-resolution microscopy:

  • Structured Illumination Microscopy (SIM) to achieve resolution beyond diffraction limit

  • Stochastic Optical Reconstruction Microscopy (STORM) for nanoscale resolution

  • Stimulated Emission Depletion (STED) microscopy for detailed structural analysis of ER bodies

• Correlative Light and Electron Microscopy (CLEM):

  • Combine fluorescence microscopy with electron microscopy

  • Use immunogold labeling for electron microscopy detection

  • Enables ultrastructural context while preserving specific labeling

• Live-cell imaging approaches:

  • Generate fluorescent protein fusions (if antibodies cannot be used in living cells)

  • Use spinning disk confocal microscopy for rapid acquisition

  • Apply FRAP (Fluorescence Recovery After Photobleaching) to study protein dynamics

• Image analysis considerations:

  • Use appropriate software for colocalization analysis (Pearson's correlation, Manders' overlap)

  • Apply deconvolution algorithms to improve signal-to-noise ratio

  • Consider 3D reconstruction for complete spatial understanding

  • Quantify signal intensities relative to ER body structures

What strategies can be employed to study At3g15980 protein-protein interactions within the ER body context?

To study At3g15980 protein-protein interactions within ER bodies, consider these methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Use anti-At3g15980 antibodies for pulldown experiments

  • Analyze precipitated proteins by mass spectrometry or Western blotting

  • Consider crosslinking to capture transient interactions

  • Be aware that even when interactions exist, they may be difficult to detect by Co-IP, as observed with NAI2 and PYK10

• Proximity-dependent labeling:

  • Fuse At3g15980 to BioID, TurboID, or APEX2

  • These enzymes biotinylate proteins in close proximity

  • Analyze biotinylated proteins to identify interaction partners

  • Particularly useful for detecting weak or transient interactions

• Förster Resonance Energy Transfer (FRET):

  • Generate fluorescent protein fusions to At3g15980 and potential partners

  • Measure energy transfer between fluorophores

  • Provides spatial information about interactions in living cells

• Yeast two-hybrid screening:

  • Use At3g15980 as bait to screen for interacting proteins

  • Validate positive hits in planta

  • Consider membrane-based yeast two-hybrid systems if transmembrane domains are present

• Split fluorescent/luminescent reporter assays:

  • Fuse complementary fragments to At3g15980 and potential partners

  • Reconstitution of fluorescence/luminescence indicates interaction

  • Can be performed in planta for native context

• Protein complex analysis:

  • Use Blue Native PAGE to preserve protein complexes

  • Combine with antibody-based detection or mass spectrometry

  • Consider size exclusion chromatography to isolate complexes

• Bioinformatic prediction tools:

  • Use resources like ATTED-II to identify co-expressed genes

  • These may indicate functional relationships, as demonstrated for NAI1 and NAI2