At3g18320 Antibody

What is the At3g18320 gene and what does the corresponding antibody detect?

The At3g18320 gene (UniProt Number: Q9LS58) encodes an F-box family protein in Arabidopsis thaliana. F-box proteins are components of SCF ubiquitin-ligase complexes that play crucial roles in protein degradation and various cellular processes including development and stress responses. The At3g18320 antibody is a rabbit polyclonal antibody that specifically recognizes this protein in plant samples . The antibody has been purified using Protein A/G affinity chromatography to ensure specificity and reduced background .

What are the recommended applications for the At3g18320 antibody?

The At3g18320 antibody has been validated for use in multiple experimental applications:

• Western Blot (WB): For detecting the native or denatured protein in cell/tissue lysates

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of the protein in solution

For Western blot applications, researchers typically use dilutions ranging from 1:1000 to 1:5000, while ELISA applications generally require dilutions between 1:5000 and 1:20000, though optimal concentrations should be determined empirically for each experimental setup .

How should the At3g18320 antibody be stored and handled?

For optimal antibody performance:

• Store the antibody at -20°C or -80°C for long-term storage (>1 month)

• For short-term storage (<1 month), the antibody can be kept at 4°C

• Avoid repeated freeze-thaw cycles, which can lead to protein denaturation and loss of activity

• When shipping, maintain cold chain using blue ice or similar cooling methods

• The antibody should be handled using standard laboratory safety procedures for biological materials

How can I validate the specificity of the At3g18320 antibody for my research?

Antibody validation is critical for ensuring experimental reliability. For At3g18320 antibody, consider implementing these validation strategies:

A. Orthogonal Validation:
Compare protein levels determined by the antibody-dependent method (e.g., Western blot) with levels determined by an antibody-independent method (e.g., mass spectrometry or RNA-seq) across a panel of samples . This approach reveals whether the antibody signal correlates with actual protein abundance.

B. Genetic Validation:
Use RNA interference (RNAi) or CRISPR-Cas9 to knockdown At3g18320 expression in your plant system, then perform Western blot analysis. A specific antibody will show reduced signal intensity proportional to the knockdown efficiency .

C. Recombinant Expression Validation:
Express the At3g18320 protein recombinantly in a system that normally doesn't express it, then compare antibody reactivity between samples with and without expression .

D. Independent Antibody Validation:
Use two different antibodies that recognize distinct epitopes of the At3g18320 protein. Similar staining patterns would support specificity .

What controls should I include when using the At3g18320 antibody in immunoprecipitation experiments?

When performing immunoprecipitation (IP) with At3g18320 antibody, proper controls are essential:

• Non-specific IgG control: Include a sample with the same amount of non-specific IgG from the same species (rabbit) to identify non-specific binding . This control helps distinguish specific precipitation from background.

• Input sample: Reserve a portion (typically 5-10%) of your lysate before IP to confirm the presence of your target protein and enable quantitative assessment of enrichment .

• No-antibody control: Process a sample without antibody to identify proteins that bind non-specifically to the beads.

• Blocking peptide control: If available, pre-incubate the antibody with the immunizing peptide to block specific binding sites.

For example, in a typical At3g18320 ChIP experiment, you would prepare at least two samples for each condition - one for the non-specific antibody (IgG) and one for the anti-At3g18320 antibody (specific). Add 3-4 μg of antibody to each sample and proceed with the protocol .

How can I optimize Western blot conditions for the At3g18320 antibody?

Optimizing Western blot conditions for the At3g18320 antibody requires methodical testing of several parameters:

• Sample preparation: For plant tissues containing At3g18320, use a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and protease inhibitors. Grind tissue in liquid nitrogen before adding buffer to prevent protein degradation.

• Blocking conditions: Test different blocking reagents (2-2.5% RPN2125 or 5% non-fat dry milk) in TBS-T (20 mM Tris, 137 mM sodium chloride, pH 7.6 with 0.1% Tween-20) .

• Antibody dilution: Start with a 1:1000 to 1:10000 dilution range. For example, a successful protocol used an anti-PR-1 antibody at 1:10000 in blocking reagent for 1 hour at room temperature .

• Secondary antibody: Use anti-rabbit IgG conjugated to horseradish peroxidase at 1:25000 dilution for optimal signal-to-noise ratio .

• Detection method: Chemiluminescent detection reagents in the extreme low femtogram range provide excellent sensitivity. Exposure time should be optimized (typically 1-5 minutes) depending on your imaging system .

How can I use the At3g18320 antibody for chromatin immunoprecipitation (ChIP) studies?

Chromatin immunoprecipitation with the At3g18320 antibody requires careful optimization for plant tissues:

Detailed ChIP Protocol for At3g18320:

• Crosslinking: Crosslink plant material with 1% formaldehyde for 10 minutes at room temperature under vacuum. Quench with 0.125 M glycine for 5 minutes.

• Chromatin preparation: Grind tissue in liquid nitrogen, extract nuclei with extraction buffer (0.4 M sucrose, 10 mM Tris-HCl pH 8.0, 10 mM MgCl2, 5 mM β-mercaptoethanol, 0.1 mM PMSF, protease inhibitors), and sonicate to shear chromatin to 200-500 bp fragments.

• Immunoprecipitation: Pre-clear chromatin with protein A/G beads, then incubate with At3g18320 antibody (4 μg) overnight at 4°C. Include an IgG control in parallel.

• Washes and elution: Wash beads with low-salt, high-salt, LiCl, and TE buffers. Elute DNA-protein complexes with elution buffer (1% SDS, 0.1 M NaHCO3).

• Reverse crosslinking and DNA purification: Reverse crosslinks by incubating at 65°C overnight, treat with RNase A and Proteinase K, then purify DNA .

• qPCR analysis: Design primers for potential At3g18320 binding sites based on known F-box protein binding motifs. Calculate enrichment using the percent input method: 100 × 2^(Ct[input] − Ct[IP]) .

What approaches can be used to determine if the At3g18320 protein undergoes persulfidation and how does this affect its function?

Recent research suggests that persulfidation (addition of -SSH group) is an important post-translational modification in plants, especially under endoplasmic reticulum (ER) stress conditions:

Methodology to Detect At3g18320 Persulfidation:

• Biotin-switch technique: Modify the protocol used for ATG18a . Block free thiols with methylmethanethiosulfonate (MMTS), treat with sodium sulfide (Na2S) to reduce persulfide groups, then label with biotin-HPDP. Detect by Western blot with the At3g18320 antibody.

• Mass spectrometry analysis: Purify recombinant At3g18320 protein, digest with trypsin under non-reducing conditions, and perform LC-MS/MS analysis looking for a 32-Da mass increase plus carbamidomethylation (SS-CAM) in the fragmentation spectrum .

• Tag-switch assay: Use methylsulfonyl benzothiazole (MSBT) to block free thiols followed by CN-biotin to label persulfide groups. Visualize with fluorescent streptavidin.

• Functional assays: Compare the activity of wild-type At3g18320 with that of a mutant where potential persulfidation sites (cysteine residues) are replaced with serine. Assess protein-protein interactions and protein localization.

Given that other F-box proteins are known to be regulated by post-translational modifications, persulfidation may influence At3g18320's ability to form SCF complexes and target substrate proteins for ubiquitination.

Why might I see multiple bands when using the At3g18320 antibody in Western blot?

Multiple bands in Western blot can occur for several legitimate biological reasons as well as technical issues:

Potential Biological Explanations:

• Post-translational modifications: F-box proteins like At3g18320 can undergo modifications such as phosphorylation, ubiquitination, or persulfidation

• Alternative splicing: Check if At3g18320 has known splice variants

• Protein complexes: Incomplete denaturation may show complexes with other proteins

• Proteolytic processing: F-box proteins may be cleaved as part of their regulation

Technical Troubleshooting:

• Improve sample preparation: Add additional protease inhibitors, maintain samples at 4°C, and avoid repeated freeze-thaw cycles

• Optimize blocking: Test different blocking reagents; 2-2.5% RPN2125 in TBS-T has shown good results in plant protein detection

• Antibody dilution: Test a dilution series (e.g., 1:500, 1:1000, 1:5000) to find optimal specificity

• Enhanced validation: Apply orthogonal validation using mass spectrometry to identify the molecular weight of authentic At3g18320

If background persists, consider increased washing steps (e.g., one 15-minute wash followed by three 5-minute washes in TBS-T) as used successfully for other plant proteins .

How can I resolve weak or absent signals when using At3g18320 antibody in immunofluorescence microscopy?

Immunofluorescence with plant tissues presents unique challenges:

Methodical Troubleshooting Approach:

• Fixation optimization: Test different fixation methods:

  • 2% formaldehyde in MTSB buffer for 30 minutes at 37°C

  • Methanol fixation for 10 minutes at -20°C

  • Combined formaldehyde/methanol fixation

• Cell wall digestion: For plant tissues, enzymatic digestion is critical:

  • Use 0.25% Dricelaze, 0.1% Macerozyme in 5 mM MES buffer, pH 5.2 for 5-7 minutes

  • Alternatively, try 1% cellulase, 0.3% macerozyme, 0.5% pectinase for 30 minutes

• Permeabilization: Ensure adequate permeabilization with 10% DMSO/3% NP40 in MTSB buffer

• Antibody concentration: Use higher antibody concentrations for immunofluorescence than for Western blot (start with 1:200 dilution)

• Signal amplification: Consider tyramide signal amplification (TSA) or quantum dot-conjugated secondary antibodies

• Antigen retrieval: Heat-mediated antigen retrieval may expose masked epitopes (citrate buffer, pH 6.0, 95°C for 10-20 minutes)

Remember that some proteins may be present in very low amounts in non-induced plant material, similar to observations with PR-1 protein . If using Arabidopsis, consider inducing expression with appropriate treatments based on the gene's known regulation pattern.

How can I implement a strategy to systematically generate additional antibodies against At3g18320 using novel approaches?

For researchers seeking to develop new antibodies against At3g18320 with improved properties:

Comprehensive Antibody Generation Strategy:

• Antigen design:

  • Express full-length recombinant At3g18320 protein with His-tag for purification

  • Alternatively, design synthetic peptides from unique regions of At3g18320

  • Create MBT (molecular bacterial toxin) fusion constructs for enhanced immunogenicity

• Immunization approach:

  • Traditional hybridoma technology using Arabidopsis inflorescence total proteins following the flowchart from reference

  • Phage display methods using human or synthetic antibody libraries

  • Single B-cell isolation from immunized animals

• Screening methodology:

  • Primary screen using Western blot against Arabidopsis total proteins

  • Secondary screen testing tissue specificity (leaves, stems, flowers)

  • Tertiary screen by immunofluorescence on floral histological sections

• Verification procedure:

  • Immunoprecipitation followed by mass spectrometry analysis to confirm target identity

  • Orthogonal validation using transcriptomics/proteomics correlation

  • Testing in At3g18320 knockout/knockdown lines

• Bispecific approach:

  • Consider developing a common light chain (CLC) bispecific antibody targeting At3g18320 and a second protein of interest using Hybridoma-to-Phage-to-Yeast platform

This systematic approach, inspired by successful antibody generation against other Arabidopsis proteins , can yield multiple antibodies with different characteristics suitable for various applications.

How can I use the At3g18320 antibody to investigate the role of this F-box protein in plant stress responses?

F-box proteins play crucial roles in plant stress responses through selective protein degradation via the ubiquitin-proteasome system. To investigate At3g18320:

Experimental Design for Stress Response Studies:

• Stress treatments: Subject Arabidopsis plants to various stresses (drought, salt, heat, pathogen infection, ER stress)

• Protein expression analysis: Use the At3g18320 antibody in Western blot to quantify changes in protein levels across stress conditions

• Immunoprecipitation-based interactome analysis:

  • Perform IP with At3g18320 antibody under different stress conditions

  • Identify interacting proteins by mass spectrometry

  • Compare interactome shifts between normal and stress conditions

  • Validate key interactions with reciprocal co-IP

• Subcellular localization: Use the antibody for immunofluorescence to track changes in At3g18320 localization during stress responses, similar to approaches used for PR-1 protein visualization

• Chromatin immunoprecipitation (if At3g18320 has nuclear functions): Follow protocols similar to those outlined in reference

Since ER stress induces autophagy in plants dependent on ATG18a through a persulfidation mechanism , it would be interesting to investigate whether At3g18320 is involved in similar regulatory pathways during stress conditions.

What is the broader significance of using the People Also Ask methodology for enhancing antibody research in plant sciences?

Analyzing Google's "People Also Ask" data provides valuable insights for antibody researchers:

Strategic Applications in Plant Antibody Research:

• Research question formulation: PAA data helps identify knowledge gaps and common challenges faced by researchers using plant antibodies1.

• Methodology optimization: By analyzing frequently asked questions about experimental protocols, researchers can address common pain points in plant antibody applications1.

• Content development: Research institutions can create better educational resources and protocols by addressing the most common questions about plant antibodies110.

• Collaborative research opportunities: Identifying trending questions can reveal emerging research areas where antibody development is needed.

• Troubleshooting resources: PAA data can help create more comprehensive troubleshooting guides specific to plant antibody applications.