At2g21870 Antibody

What is the At2g21870 gene product and why would researchers develop antibodies against it?

At2g21870 is an Arabidopsis thaliana gene locus. While specific information about this particular gene is limited in the provided search results, generating antibodies against plant proteins serves critical research purposes. Antibodies enable researchers to detect, quantify, and localize proteins of interest within plant tissues and cells. In the context of plant immunity and stress responses, antibodies against specific proteins allow researchers to track expression levels under various conditions, similar to how antibodies against mitochondrial proteins were used to study ATP synthase components . Developing antibodies against At2g21870 would enable researchers to understand its expression patterns, potential involvement in protein complexes, and responses to environmental conditions.

What are the main considerations when designing an antibody against a plant protein like At2g21870?

When designing antibodies against plant proteins, researchers should consider several key factors. First, they must determine which region of the protein to target. As demonstrated in the search results, researchers often select specific domains or regions of a protein, such as "the C-terminal part" of a subunit . For example, selecting a unique epitope that distinguishes At2g21870 from related proteins is crucial for specificity.

Second, researchers must decide whether to produce antibodies against the full-length protein or a fragment. The search results show that researchers often use partial protein sequences, such as specific domains (e.g., "Met 1–Asp 81") , which can improve antibody specificity and simplify protein production for immunization.

Third, post-translational modifications must be considered, as these can affect antibody recognition. Finally, researchers should evaluate the protein's hydrophobicity, as highly hydrophobic regions can present challenges for antibody production and specificity.

How can I validate the specificity of an At2g21870 antibody?

Validating antibody specificity is critical for reliable experimental results. Based on the immunodetection approaches described in the search results, several methods can be employed:

• Western blot analysis using wild-type plant tissue alongside knockout/knockdown lines (such as RNAi lines) to confirm the absence or reduction of the signal in the mutant samples, similar to how RNAi lines were used to validate antibodies against ATPd .

• Comparison of observed protein size with predicted molecular mass. The search results show that researchers typically compare observed band sizes with calculated molecular masses, as demonstrated in Table 1 of the third search result .

• Testing for cross-reactivity with similar proteins through recombinant protein controls.

• Immunoprecipitation followed by mass spectrometry to confirm that the antibody pulls down the intended target.

• Immunolocalization studies to verify that the detected protein localizes as expected based on predicted or known subcellular localization.

How can I use an At2g21870 antibody to study protein-protein interactions and protein complexes?

Antibodies are powerful tools for studying protein-protein interactions. Based on the approaches described in the search results, researchers can use At2g21870 antibodies in several advanced applications:

• Co-immunoprecipitation (Co-IP): After immunoprecipitating At2g21870 with its specific antibody, mass spectrometry can identify co-precipitating proteins, revealing potential interaction partners.

• Blue-Native PAGE combined with immunodetection: This technique, described in the third search result, allows the identification of stable subcomplexes and can reveal whether At2g21870 participates in larger protein assemblies. As noted: "Mitochondrial proteins (100 μg) were solubilized with dodecylmaltoside (1% (w/v) final) in 75 μl of ACA buffer... and loaded onto a 4.5–16% (w/v) gradient gel" . Similar approaches could be applied to study At2g21870-containing complexes.

• Sequential immunoprecipitation: Using antibodies against At2g21870 and its potential partners sequentially can confirm direct interactions versus membership in the same complex.

• Crosslinking coupled with immunoprecipitation: This can capture transient interactions before immunoprecipitation with the At2g21870 antibody.

How does environmental stress affect At2g21870 protein levels, and how can antibodies help measure these changes?

Environmental stresses significantly impact plant protein expression. While specific information about At2g21870's response to stress is not provided in the search results, the approach used to study R-gene expression under different environmental conditions offers a valuable framework .

Researchers can use At2g21870 antibodies to:

• Perform quantitative immunoblotting on plant tissues subjected to different stresses (temperature, drought, pathogen exposure), similar to how researchers analyzed protein levels in RNAi lines under different conditions .

• Compare protein levels across different Arabidopsis accessions (natural variants) from different geographic origins to understand environmental adaptation patterns. The first search result describes analyzing expression across "12 Arabidopsis thaliana accessions selected from a broad geographical distribution across Europe and a latitudinal cline in the Midwest USA" . Similar approaches could be applied using antibodies instead of or in addition to qRT-PCR.

• Correlate protein levels with transcript levels (measured by qRT-PCR) to identify post-transcriptional regulation mechanisms under stress conditions.

• Examine protein localization changes under stress using immunolocalization techniques.

How can I use an At2g21870 antibody to study protein modifications and turnover?

Antibodies can be valuable tools for studying post-translational modifications (PTMs) and protein stability. Advanced applications include:

• Using phospho-specific antibodies: If At2g21870 undergoes phosphorylation, researchers can develop phospho-specific antibodies against predicted phosphorylation sites.

• Immunoprecipitation followed by PTM-specific detection: After immunoprecipitating with the At2g21870 antibody, samples can be probed with antibodies against specific modifications (phosphorylation, ubiquitination, etc.).

• Pulse-chase experiments combined with immunoprecipitation: This approach can measure protein turnover rates under different conditions.

• Comparing modified versus unmodified protein ratios in different tissues or under different conditions using quantitative immunoblotting.

• Immunoprecipitation followed by mass spectrometry: This can identify the precise sites and types of modifications, as indicated by the mass spectrometry approach described in search result 3: "Samples were analyzed on a 6510 Q-TOF mass spectrometer (Agilent Technologies) with an HPLC Chip Cube source" .

What is the optimal protocol for generating antibodies against At2g21870?

Based on the antibody production approach described in search result 3, the following protocol would be appropriate for generating antibodies against At2g21870:

• Identify suitable antigenic regions: Select unique, hydrophilic, surface-exposed regions of the At2g21870 protein that are likely to be immunogenic.

• Express the selected protein fragment as a fusion protein: "cDNAs encoding for the C-terminal part... were cloned in the Gateway entry vector pDONR207 (Invitrogen) and subsequently transferred to the destination vector, pDEST15 (Invitrogen), which allows the N-terminal fusion of GST" .

• Purify the fusion protein: "These chimeric proteins were overexpressed in E. coli BL21 pLysS cells and purified on GST-Sepharose" .

• Immunize rabbits with the purified protein: "The purified proteins were injected into independent rabbits (4 doses of 250 μg per protein)" .

• Collect antisera: "The rabbits were bled after the third and fourth injections, and the sera were used unpurified in immunodetection experiments" .

• Validate the antibody specificity using the approaches discussed in FAQ 1.3.

What are the optimal conditions for using At2g21870 antibodies in Western blotting?

Optimal conditions for Western blotting with plant protein antibodies can be derived from the protocols described in the search results:

• Sample preparation: Extract proteins from plant tissue using an appropriate buffer. For membrane proteins, consider solubilization with detergents like dodecylmaltoside as described: "Mitochondrial proteins (100 μg) were solubilized with dodecylmaltoside (1% (w/v) final)" .

• Protein quantification: Ensure equal loading of samples, typically 50-100 μg of total protein per lane as suggested in the search results .

• Gel separation: Choose an appropriate percentage acrylamide gel based on the predicted molecular weight of At2g21870.

• Transfer conditions: Optimize transfer time and voltage based on protein size.

• Blocking: Use 3-5% non-fat dry milk or BSA in TBST.

• Primary antibody incubation: The optimal dilution would need to be determined empirically, but based on the table in search result 3, plant protein antibodies are typically used at dilutions ranging from 1:1000 to 1:20000 .

• Secondary antibody: "Secondary antibodies linked to horseradish peroxidase were used, and the signals were detected by chemiluminescence (GE Healthcare)" .

• Detection: "The images were recorded using a Luminescent Image Analyzer (LAS 100, Fuji, Japan)" or equivalent imaging system.

How can I optimize immunoprecipitation protocols for At2g21870?

Based on the experimental approaches in the search results, an optimized immunoprecipitation protocol for At2g21870 would include:

• Tissue selection: Choose appropriate plant tissues where At2g21870 is expected to be expressed. Consider using information from expression databases or qRT-PCR data to guide tissue selection.

• Protein extraction: Use a buffer that maintains protein-protein interactions if studying complexes. The search results describe using ACA buffer (750 mM aminocaproic acid, 0.5 mM EDTA, 50 mM Tris-HCl, pH 7.0) for protein complex studies .

• Pre-clearing: Incubate the lysate with protein A/G beads alone to reduce non-specific binding.

• Antibody binding: Incubate the pre-cleared lysate with the At2g21870 antibody at 4°C (typically overnight).

• Immunoprecipitation: Add protein A/G beads to capture the antibody-protein complexes.

• Washing: Perform stringent washes to remove non-specifically bound proteins.

• Elution: Elute bound proteins for subsequent analysis by SDS-PAGE, Western blotting, or mass spectrometry.

• Controls: Include a negative control using pre-immune serum or IgG from the same species as the primary antibody.

Why might I detect multiple bands or non-specific signals when using an At2g21870 antibody?

Multiple bands or non-specific signals can occur for several reasons. The search results mention facing similar issues: "Immunoblot of ATPd with total seedling protein extracts showed a weak signal and a non-specific band right above subunit d preventing accurate quantification" . Based on this and standard troubleshooting approaches, consider:

• Cross-reactivity: The antibody may recognize similar epitopes in related proteins. Consider using more purified samples or optimizing antibody dilution.

• Protein degradation: Multiple bands at lower molecular weights may indicate degradation products. Add protease inhibitors to extraction buffers.

• Post-translational modifications: Different bands may represent modified forms of the protein.

• Non-specific binding of the secondary antibody: Perform a control blot with only the secondary antibody.

• Sample purity: As demonstrated in the search results, using purified subcellular fractions can improve specificity: "To increase sensitivity of immunoblot signals, we repeated the experiment with purified mitochondria... Immunoblots with mitochondrial proteins resulted in a clear signal without the non-specific band" .

How can I determine the appropriate sample preparation method to maximize At2g21870 detection?

Sample preparation significantly impacts protein detection. Based on the approaches described in the search results:

• Test different extraction buffers: Compare buffers with different detergents (e.g., dodecylmaltoside as mentioned in search result 3) and salt concentrations.

• Consider subcellular fractionation: The search results demonstrate that using purified organelles (like mitochondria) can improve detection specificity . Determine At2g21870's predicted subcellular localization and consider enriching for that fraction.

• Optimize protein amount: Test different protein concentrations (50-100 μg is typical as shown in the search results) .

• Sample treatment: Consider treatments that may expose epitopes, such as denaturation conditions.

• Protein precipitation: Methods like acetone precipitation can concentrate proteins and remove interfering compounds: "50 μg of each fraction was acetone-precipitated for 2 h at −80 °C, centrifuged at 20,000 × g for 20 min at 4 °C, and the pellets were air-dried" .