At3g21620 Antibody

What is At3g21620 and why is it significant in Arabidopsis research?

At3g21620 is classified as an acid phosphatase/vanadium-dependent haloperoxidase-related protein in Arabidopsis thaliana. It is a putative uncharacterized protein with 174 amino acids . Despite being annotated as "putative uncharacterized," this protein belongs to a functionally significant class of enzymes that may play important roles in plant metabolism and stress responses. Studying At3g21620 contributes to our understanding of plant biochemical pathways and potential regulatory mechanisms in Arabidopsis.

The protein's relatively small size and specific structural motifs make it an interesting target for functional studies using antibody-based approaches. Researchers typically investigate this protein to understand its subcellular localization, expression patterns during development, and possible roles in plant stress responses or metabolism.

What validation methods should be employed before using At3g21620 antibodies?

Antibody validation is critical for ensuring experimental reliability. For At3g21620 antibodies, multiple validation approaches should be employed:

• Western blot analysis in wild-type and mutant backgrounds: The most definitive validation comes from demonstrating that the antibody detects the expected band in wild-type samples but shows reduced or absent signal in knockout/knockdown mutants. Several Arabidopsis antibodies have been validated using this approach, including AXR4, ACO2, AtBAP31, and ARF19 .

• ELISA titration: Commercial antibodies against At3g21620 typically undergo ELISA testing to determine binding affinity. Look for antibodies with high ELISA titers (e.g., 10,000), which approximately correspond to 1 ng detection of target protein on Western blots .

• Immunocytochemistry controls: If using the antibody for localization studies, include appropriate negative controls (primary antibody omission, pre-immune serum) and positive controls (co-localization with known markers).

• Cross-reactivity assessment: Bioinformatic analysis should confirm that the antigenic region used to generate the antibody has limited similarity to other Arabidopsis proteins. A threshold of less than 40% sequence similarity is typically used as a cutoff to minimize cross-reactivity .

Which regions of At3g21620 make the most effective antibody targets?

When selecting antibodies against At3g21620, researchers should consider which protein region will provide optimal results for their specific application:

• N-terminal antibodies: Target the amino terminus of At3g21620, useful for detecting the full-length protein. Commercial antibodies targeting this region are typically generated against 3 synthetic peptides representing the N-terminus sequence .

• C-terminal antibodies: Target the carboxyl terminus and are valuable for confirming full-length protein expression or detecting specific isoforms. These are also typically generated using 3 synthetic peptides representing the C-terminus sequence .

• Middle-region antibodies: Target non-terminal sequences and may provide higher specificity in some applications. Similar to other regions, these are typically generated using 3 synthetic peptides representing internal sequences .

The selection of the optimal target region depends on protein structure, post-translational modifications, and the specific experimental application. For novel investigations, using antibodies targeting different regions can provide complementary data and higher confidence in results.

What is the recommended Western blot protocol for At3g21620 antibody?

For optimal Western blot results with At3g21620 antibody, follow this research-validated protocol:

Sample preparation:

• Harvest Arabidopsis tissue (preferably roots, as many Arabidopsis antibodies are validated in root tissue)

• Homogenize in extraction buffer containing protease inhibitors

• Clarify lysate by centrifugation (14,000 × g, 15 min, 4°C)

• Quantify protein concentration using Bradford assay

SDS-PAGE and transfer:

• Load 20-30 μg protein per lane alongside molecular weight markers

• Separate proteins on 12-15% polyacrylamide gel (appropriate for the 174 amino acid At3g21620 protein)

• Transfer to PVDF membrane (recommended over nitrocellulose for small proteins)

Antibody incubation:

• Block membrane with 5% non-fat milk in TBST for 1 hour at room temperature

• Incubate with primary At3g21620 antibody at 1:1000 dilution overnight at 4°C

• Wash 3× with TBST, 10 minutes each

• Incubate with HRP-conjugated secondary antibody at 1:5000 for 1 hour at room temperature

• Wash 3× with TBST, 10 minutes each

• Develop using ECL reagent and image

Critical controls:

• Include a positive control sample with known At3g21620 expression

• Include a negative control (ideally, At3g21620 knockout/knockdown line)

• Reserve 50 μl of each sample for Western blot confirmation when performing other antibody-based experiments

How should immunolocalization experiments with At3g21620 antibody be designed?

For successful immunolocalization of At3g21620 in Arabidopsis tissues:

Tissue preparation:

• Fix tissue in 4% paraformaldehyde in PBS for 2 hours at room temperature

• Wash with PBS (3× for 10 minutes each)

• Dehydrate through ethanol series and embed in paraffin or prepare for whole-mount immunostaining

• Section paraffin-embedded tissues at 8-10 μm thickness

Immunostaining procedure:

• Dewax sections or permeabilize whole-mount samples (0.1% Triton X-100 in PBS, 15 minutes)

• Block with 3% BSA in PBS for 1 hour at room temperature

• Incubate with affinity-purified At3g21620 antibody (1:100 to 1:500 dilution) overnight at 4°C

• Wash 3× with PBS, 10 minutes each

• Incubate with fluorescently-labeled secondary antibody (1:500) for 2 hours at room temperature

• Wash 3× with PBS, 10 minutes each

• Counterstain nuclei with DAPI (1 μg/ml, 10 minutes)

• Mount and image using confocal microscopy

Essential controls:

• Negative control: omit primary antibody

• Peptide competition: pre-incubate antibody with immunizing peptide

• Positive control: co-stain with established subcellular marker proteins, such as BiP (ER), γ-cop (Golgi), PM-ATPase (plasma membrane), or MDH (mitochondria)

The success rate of peptide antibodies for immunocytochemistry is often low, but affinity purification significantly improves detection rates. Of 70 protein antibodies tested in one Arabidopsis study, only 22 (31.4%) were suitable for immunocytochemistry applications .

What are the recommended protocols for immunoprecipitation with At3g21620 antibody?

For immunoprecipitation of At3g21620 and associated proteins:

Lysate preparation:

• Harvest 5-10 g of Arabidopsis tissue

• Grind in liquid nitrogen to fine powder

• Resuspend in IP buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, protease inhibitor cocktail)

• Clarify by centrifugation (14,000 × g, 15 minutes, 4°C)

• Pre-clear with Protein A/G beads (1 hour, 4°C with rotation)

Immunoprecipitation:

• Reserve 10% of lysate as input control

• Add 5 μg of At3g21620 antibody to remaining lysate

• Incubate overnight at 4°C with gentle rotation

• Add 50 μl Protein A/G beads, incubate 2 hours at 4°C

• Wash beads 4× with IP buffer

• Elute proteins with SDS sample buffer (95°C, 5 minutes)

Analysis methods:

• SDS-PAGE followed by Western blotting to detect At3g21620

• Mass spectrometry to identify co-immunoprecipitated proteins

Standard IP experiments typically use 250 μl of lysate for antibody pulldown and 50 μl for Western blot confirmation .

How can At3g21620 antibody be used for chromatin immunoprecipitation (ChIP)?

If At3g21620 is suspected to interact with chromatin or be part of a chromatin-modifying complex, ChIP can be performed:

Sample preparation:

• Crosslink plant tissue with 1% formaldehyde for 10 minutes

• Quench with 0.125 M glycine for 5 minutes

• Wash tissues with ice-cold PBS

• Extract and sonicate chromatin to 200-500 bp fragments

Immunoprecipitation:

• Pre-clear chromatin with Protein A/G beads (1 hour, 4°C)

• Incubate pre-cleared chromatin with At3g21620 antibody (5 μl) overnight at 4°C

• Add Protein A/G beads and incubate for 2 hours

• Wash beads thoroughly with progressively stringent buffers

• Reverse crosslinks (65°C overnight)

• Purify DNA

Data analysis:

• Perform qPCR targeting specific genomic regions of interest

• For genome-wide analysis, prepare ChIP-seq libraries and sequence

• Analyze enrichment relative to input and IgG control samples

For Arabidopsis proteins, ChIP protocols often require optimization due to the relatively low abundance of many regulatory proteins and the presence of cell wall components that can interfere with chromatin extraction.

How can contradictory results with At3g21620 antibody be resolved?

When facing contradictory results using At3g21620 antibody:

Systematic troubleshooting approach:

• Verify antibody specificity:

  • Test antibody in At3g21620 mutant/knockout line

  • Perform peptide competition assay

  • Try antibodies targeting different epitopes of At3g21620

• Validate experimental conditions:

  • Test multiple protein extraction methods

  • Adjust antibody concentration and incubation conditions

  • Ensure proper sample handling to prevent protein degradation

• Confirm protein expression:

  • Verify At3g21620 expression in your experimental tissue/condition by RT-qPCR

  • Consider that protein expression may be condition-dependent or tissue-specific

  • Analyze transcript data from public repositories to predict expression patterns

• Cross-validate with complementary approaches:

  • Express tagged version of At3g21620 (e.g., GFP fusion) and detect with tag-specific antibody

  • Perform transient expression in heterologous system

  • Use alternative detection methods (mass spectrometry, RNA-seq)

What are the considerations for using At3g21620 antibody in protein-protein interaction studies?

For studying protein-protein interactions involving At3g21620:

Co-immunoprecipitation (Co-IP):

• Perform standard IP as described in section 2.3

• Probe Western blots with antibodies against suspected interaction partners

• Compare pull-down in experimental vs. control conditions

Proximity Ligation Assay (PLA):

• Perform standard immunofluorescence up to primary antibody incubation

• Incubate with primary antibodies against At3g21620 and potential interactor

• Use PLA probes and follow manufacturer's protocol for detection

• Analyze PLA signals using confocal microscopy

Bimolecular Fluorescence Complementation (BiFC) as validation:
While not directly using the antibody, BiFC can validate antibody-based interaction studies:

• Clone At3g21620 and potential interactor into BiFC vectors

• Express in Arabidopsis protoplasts or via agroinfiltration

• Visualize fluorescence if proteins interact

Considerations for plant-specific challenges:

• Plant tissues contain compounds that can interfere with antibody binding

• Cell wall can impede antibody penetration in intact tissues

• Protein abundance may vary significantly across tissues and conditions

• Use of antibodies from the same host species requires special considerations

What strategies can improve signal detection with At3g21620 antibody?

To enhance detection sensitivity with At3g21620 antibody:

Antibody optimization:

• Affinity purification: This significantly improves detection rates. Studies show that affinity purification of antibodies massively improved detection rates in Arabidopsis antibodies, increasing success from a very low rate to 55% for protein antibodies .

• Titration: Test multiple antibody dilutions (1:100 to 1:5000) to identify optimal concentration.

• Incubation conditions: Extend primary antibody incubation (overnight at 4°C) and optimize secondary antibody parameters.

Sample preparation optimization:

• Extraction buffers: Test different extraction buffers to improve protein solubilization.

• Protease inhibitors: Use fresh, complete protease inhibitor cocktail.

• Sample concentration: Consider using protein concentration methods for low-abundance targets.

Signal amplification methods:

• Enhanced chemiluminescence: Use high-sensitivity ECL reagents for Western blots.

• Tyramide signal amplification: For immunohistochemistry applications.

• Biotin-streptavidin systems: For additional signal enhancement.

What are the common causes of non-specific binding with At3g21620 antibody?

Non-specific binding is a common challenge with plant antibodies. For At3g21620 antibody:

Common sources of non-specificity:

• Cross-reactivity with related proteins: At3g21610 shares sequence similarity with At3g21620 , potentially causing cross-reactivity. Bioinformatic analysis using a 40% sequence similarity cutoff helps identify potential cross-reacting proteins .

• Inappropriate blocking: Insufficient blocking or wrong blocking agent may increase background. Test alternative blocking agents (BSA, casein, commercial blockers).

• Secondary antibody issues: Non-specific binding of secondary antibody can occur. Include controls without primary antibody.

• Plant-specific interference: Phenolic compounds, abundant RuBisCO, and endogenous peroxidases can interfere with antibody experiments.

Solutions to reduce non-specificity:

• Blocking optimization: Increase blocking agent concentration or try different blocking agents.

• Stringent washing: Increase number or duration of washes with higher detergent concentration.

• Antibody pre-adsorption: Pre-incubate antibody with plant extract from knockout lines.

• Peptide competition: Pre-incubate antibody with immunizing peptide as specificity control.

What controls are essential for At3g21620 antibody experiments?

Proper controls are critical for reliable results with At3g21620 antibody:

Essential controls for all applications:

• Negative genetic controls:

  • At3g21620 knockout/knockdown mutant (gold standard control)

  • RNAi lines with reduced At3g21620 expression

• Technical controls:

  • No primary antibody control

  • Non-immune IgG control (same concentration as primary antibody)

  • Peptide competition/neutralization (pre-incubate antibody with immunizing peptide)

• Positive controls:

  • Recombinant At3g21620 protein (if available)

  • Overexpression line with tagged At3g21620

Application-specific controls:

• For Western blots:

  • Molecular weight markers

  • Loading control (anti-tubulin, anti-actin, or anti-GAPDH)

• For immunolocalization:

  • Co-staining with known subcellular markers (e.g., BiP for ER, γ-cop for Golgi)

  • Competing peptide control

  • Signal from overexpression line

• For immunoprecipitation:

  • Input sample (pre-IP lysate)

  • IgG control precipitation

  • Beads-only control

How can At3g21620 antibody contribute to understanding protein function in stress responses?

At3g21620 antibody can be instrumental in elucidating protein function during stress responses:

Experimental approaches:

• Expression profiling: Use At3g21620 antibody to monitor protein levels across stress treatments (drought, salt, pathogens, temperature) and compare with transcriptional changes.

• Post-translational modifications: Combine At3g21620 antibody with phospho-specific or other PTM antibodies to detect stress-induced modifications.

• Protein-protein interaction dynamics: Perform co-IP with At3g21620 antibody under different stress conditions to identify condition-specific interactors.

• Subcellular relocalization: Use immunolocalization to track potential stress-induced changes in At3g21620 localization.

This research direction is particularly relevant given that many acid phosphatases and haloperoxidase-related proteins in plants are involved in stress responses, though the specific function of At3g21620 remains to be fully characterized.

What approaches integrate At3g21620 antibody data with other omics techniques?

Multi-omics integration with At3g21620 antibody data:

Integrative approaches:

• Proteogenomics: Correlate At3g21620 antibody-derived protein abundance data with:

  • Transcriptomics (RNA-seq)

  • Epigenomics (ChIP-seq for histone modifications)

  • Genomics (natural variation, QTL analysis)

• Protein interaction networks: Combine At3g21620 antibody Co-IP with:

  • Mass spectrometry to identify interactors

  • Network analysis tools to position At3g21620 in functional networks

  • Validation of interactions using BiFC or FRET

• Metabolomics integration: Correlate At3g21620 levels/activity with:

  • Metabolite profiles to identify affected pathways

  • Flux analysis to determine metabolic impact

• Phenomics correlation: Connect At3g21620 abundance with:

  • Growth phenotypes

  • Stress tolerance metrics

  • Developmental transitions

This integrated approach can reveal functional roles of At3g21620 that might be missed by single-technique studies, particularly important for putative uncharacterized proteins.