At5g18180 Antibody

What is At5g18180 and what organism is it found in?

At5g18180 is a protein found in Arabidopsis thaliana (Mouse-ear cress), a model organism widely used in plant molecular biology research. The protein has been identified in studies examining protein-protein interactions, particularly in relation to iron-sulfur cluster assembly pathways . According to UniProt annotation (Q9FK53), this protein is part of the Arabidopsis proteome and may play roles in cellular processes that are still being characterized through ongoing research .

What applications can At5g18180 antibody be used for?

At5g18180 antibody has been validated for the following applications:

For Western blot applications, it is essential to include proper controls to ensure specificity. When using this antibody for the first time in Western blot analysis, run a positive control sample (Arabidopsis thaliana leaf extract) alongside experimental samples. The antibody should detect a band at the expected molecular weight of the At5g18180 protein .

For ELISA applications, a standard curve using recombinant At5g18180 protein is recommended to determine optimal antibody concentration and assay sensitivity limits.

What are the optimal storage conditions for At5g18180 antibody?

For long-term storage, keep the antibody at -20°C or -80°C in small aliquots to avoid repeated freeze-thaw cycles, which can degrade antibody quality . The antibody is supplied in a storage buffer containing:

• 50% Glycerol

• 0.01M PBS, pH 7.4

• 0.03% Proclin 300 (preservative)

When working with the antibody, keep it on ice and return to -20°C promptly after use. For short-term storage (less than 2 weeks), the antibody can be kept at 4°C. Always centrifuge the antibody vial briefly before opening to ensure all liquid is at the bottom of the tube .

How should I validate the specificity of At5g18180 antibody?

Validating antibody specificity is crucial for reliable experimental results. Implement the following validation protocols:

• Positive and negative controls: Use wild-type Arabidopsis samples as positive controls and, if available, At5g18180 knockout/knockdown lines as negative controls.

• Western blot analysis: Perform Western blot analysis with recombinant At5g18180 protein alongside plant extracts to confirm specific binding.

• Pre-absorption test: Pre-incubate the antibody with excess purified antigen before using in your application. Loss of signal indicates specificity.

• Multiple detection methods: Compare results across different techniques (e.g., Western blot and immunoprecipitation) to ensure consistent detection.

• Cross-species reactivity testing: Test the antibody against proteins from related plant species to determine cross-reactivity profiles.

Remember that validation is an ongoing process, and antibody performance may vary between batches and experimental conditions .

How can I optimize Western blot protocols for At5g18180 antibody detection?

Optimizing Western blot protocols for At5g18180 detection requires attention to several parameters:

Sample preparation:

• Use fresh plant tissue and include protease inhibitors in extraction buffer

• Extract proteins in buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA

• Heat samples at 95°C for 5 minutes in Laemmli buffer with β-mercaptoethanol

Gel electrophoresis and transfer:

• Use 10-12% SDS-PAGE gels for optimal resolution

• Transfer to PVDF membrane at 100V for 1 hour in cold transfer buffer

Blocking and antibody incubation:

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

• Dilute primary antibody 1:1000 in 5% BSA in TBST

• Incubate overnight at 4°C with gentle rocking

• Wash 4× with TBST, 5 minutes each

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

Detection and troubleshooting:

• Use ECL substrate for detection

• If background is high, increase washing steps or dilute antibody further

• If signal is weak, increase antibody concentration or protein load

For challenging samples, consider using gradient gels (4-20%) to improve resolution and signal clarity .

What are the known interactions between At5g18180 and iron-sulfur cluster assembly pathways?

Research has shown potential associations between At5g18180 and iron-sulfur cluster assembly pathways in plants:

At5g18180 was identified in mass spectrometry analysis of proteins that copurified with GRXS17 (glutaredoxin S17), a protein involved in cytosolic iron-sulfur cluster assembly in Arabidopsis . This suggests At5g18180 may be part of the protein interaction network associated with iron-sulfur protein biogenesis.

In the study by Core et al., At5g18180 showed differential expression with a log2 fold change of -0.6141 in GRXS17-deficient plants, suggesting its expression may be influenced by GRXS17 function . Iron-sulfur clusters are essential cofactors for proteins involved in diverse cellular processes including respiration, photosynthesis, and DNA metabolism.

To investigate this interaction further, researchers can:

• Perform co-immunoprecipitation using At5g18180 antibody followed by mass spectrometry to identify interacting partners

• Use yeast two-hybrid assays to confirm direct protein-protein interactions

• Conduct bimolecular fluorescence complementation (BiFC) to visualize interactions in vivo

• Analyze phenotypes of plants with mutations in both At5g18180 and known CIA pathway components

How does At5g18180 expression change under different stress conditions?

While comprehensive stress-response data specifically for At5g18180 is limited in the provided search results, several approaches can be employed to investigate its expression pattern under various stress conditions:

Methodological approach:

• RT-qPCR analysis: Design specific primers for At5g18180 and measure transcript levels in plants exposed to various stresses (drought, salt, cold, heat, pathogen infection). Use appropriate reference genes such as ACTIN2 or UBQ10 for normalization.

• Western blot analysis: Use At5g18180 antibody to detect protein levels in stress-treated samples compared to controls. This approach can reveal post-transcriptional regulation that might not be apparent at the RNA level.

• Promoter-reporter fusion: Generate transgenic plants with At5g18180 promoter fused to GUS or fluorescent reporters to visualize spatial and temporal expression patterns under different stress conditions.

• Transcriptome data mining: Analyze existing RNA-Seq datasets from stress-treated Arabidopsis to extract At5g18180 expression profiles. Public repositories like TAIR, GEO, and ArrayExpress contain valuable resources.

Based on information from related research, iron-sulfur cluster proteins and their interacting partners often show altered expression under oxidative stress and DNA damage conditions . The observation that At5g18180 interacts with GRXS17 suggests it might show similar expression patterns to other proteins in this pathway, which are known to respond to stress conditions .

What are the best control samples to use when studying At5g18180 in Arabidopsis?

Selecting appropriate controls is crucial for meaningful interpretation of results when studying At5g18180:

Positive controls:

• Wild-type Arabidopsis thaliana (Col-0 ecotype) tissue expressing At5g18180

• Arabidopsis plants overexpressing At5g18180 (if available)

• Recombinant At5g18180 protein (for antibody validation)

Negative controls:

• T-DNA insertion mutants or CRISPR-generated knockout lines of At5g18180

• RNAi knockdown lines with reduced At5g18180 expression

• Non-plant tissue for testing antibody cross-reactivity

Experimental controls:

• Single-stain controls if using fluorescence microscopy to ensure proper compensation and avoid spectral overlap issues

• FMO (Fluorescence Minus One) controls rather than isotype controls for flow cytometry experiments, as FMOs account for spreading error from other fluorophores

• Untreated wild-type samples as baseline for stress response studies

Tissue-specific controls:

• Different tissue types (roots, leaves, flowers) to determine tissue-specific expression patterns

• Developmental stage series to assess temporal expression changes

Remember that when using antibody-based detection methods, proper controls are essential for accurate data interpretation, as emphasized in flow cytometry research which highlights the importance of appropriate controls for all immunodetection methods .

How can I use At5g18180 antibody for immunolocalization in plant tissues?

Immunolocalization is a powerful technique to determine the subcellular localization of At5g18180. Here is a methodological approach:

Tissue preparation:

• Fix fresh Arabidopsis tissue in 4% paraformaldehyde in PBS (pH 7.4) for 2 hours at room temperature

• Wash 3× in PBS, 10 minutes each

• Either:

  • For sections: Embed in paraffin or OCT compound, section at 5-10 μm thickness

  • For whole-mount: Proceed with cell wall digestion using 1% cellulase, 0.5% macerozyme in PBS for 15-30 minutes

Immunostaining protocol:

• Permeabilize with 0.1% Triton X-100 in PBS for 15 minutes

• Block with 3% BSA in PBS for 1 hour

• Incubate with At5g18180 antibody (1:100 to 1:500 dilution) overnight at 4°C

• Wash 3× with PBS, 10 minutes each

• Incubate with fluorophore-conjugated secondary antibody (e.g., Alexa Fluor 488 anti-rabbit, 1:500) for 1 hour at room temperature

• Wash 3× with PBS, 10 minutes each

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

• Mount in anti-fade mounting medium

Co-localization studies:
To determine precise subcellular localization, co-stain with organelle markers:

• Anti-BiP (ER marker)

• Anti-H+-ATPase (plasma membrane)

• Mitotracker (mitochondria)

• Anti-PsbA (chloroplast)

• Anti-Sec21 (Golgi)

Confocal microscopy settings:

• Use sequential scanning to avoid bleed-through

• Include single-labeled controls to confirm absence of spectral overlap

• Acquire z-stacks to visualize the complete 3D distribution

Always include appropriate negative controls, such as omitting the primary antibody or using pre-immune serum, to confirm staining specificity .

What potential factors can affect the reproducibility of results when using At5g18180 antibody?

Several factors can influence the reproducibility of experiments using At5g18180 antibody:

Antibody-related factors:

• Lot-to-lot variations in antibody production

• Storage conditions and antibody stability

• Freeze-thaw cycles that may degrade antibody quality

• Contaminants in antibody preparations

Experimental design factors:

• Lack of proper controls (positive, negative, isotype, FMO)

• Variations in sample preparation protocols

• Inconsistent blocking conditions

• Variations in incubation times and temperatures

Sample-related factors:

• Plant growth conditions affecting protein expression

• Developmental stage of the plant material

• Stress conditions altering protein levels

• Presence of proteases in sample preparations

Technical factors:

• Instrument calibration and settings for detection methods

• Data analysis parameters and software

• Buffer composition and pH variations

• Transfer efficiency in Western blotting

To improve reproducibility:

• Use the same antibody lot when possible for an entire study

• Document detailed protocols, including all reagents and their concentrations

• Include appropriate controls in every experiment

• Validate new antibody batches against previously used lots

• Standardize plant growth and sampling conditions

How can At5g18180 antibody be used to investigate protein interactions in the cytosolic iron-sulfur cluster assembly pathway?

At5g18180 antibody can be a valuable tool for studying protein-protein interactions within the cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) pathway:

Co-immunoprecipitation (Co-IP):

• Lyse plant tissue in buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and protease inhibitors

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

• Pre-clear with Protein A/G beads for 1 hour at 4°C

• Incubate with At5g18180 antibody overnight at 4°C

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

• Wash 4× with lysis buffer

• Elute with SDS sample buffer

• Analyze by Western blotting using antibodies against suspected interaction partners

Research has shown that At5g18180 may interact with components of the CIA pathway through its association with GRXS17 . Based on this, potential interaction partners to investigate include:

Proximity ligation assay (PLA):
This technique allows visualization of protein-protein interactions in situ:

• Fix and permeabilize plant tissue as for immunolocalization

• Incubate with At5g18180 antibody and antibody against suspected interaction partner

• Apply PLA probes (secondary antibodies with DNA oligonucleotides)

• Add circularization and ligation oligos

• Perform rolling circle amplification with fluorescent nucleotides

• Visualize using fluorescence microscopy

These approaches can help elucidate the role of At5g18180 in the CIA pathway and identify its function in iron-sulfur cluster protein biogenesis .

What are the best approaches for troubleshooting non-specific binding or weak signals when using At5g18180 antibody?

When encountering issues with At5g18180 antibody performance, consider the following troubleshooting strategies:

For non-specific binding:

• Optimize blocking conditions:

  • Try different blocking agents (5% BSA, 5% non-fat milk, commercial blocking buffers)

  • Increase blocking time (2-3 hours at room temperature or overnight at 4°C)

  • Add 0.1-0.3% Tween-20 to reduce background

• Optimize antibody concentration:

  • Perform a dilution series (1:500, 1:1000, 1:2000, 1:5000) to determine optimal concentration

  • Consider using antibody diluent with background reducing components

• Increase washing stringency:

  • Add additional washing steps (5-6 washes instead of 3-4)

  • Extend washing times to 10-15 minutes per wash

  • Use higher salt concentration in wash buffer (up to 500 mM NaCl)

• Pre-absorb the antibody:

  • Incubate with non-specific proteins (e.g., extract from unrelated species)

  • Pre-clear with Protein A/G beads

For weak signals:

• Sample preparation improvements:

  • Use fresh tissue and avoid repeated freeze-thaw cycles

  • Add protease inhibitors to prevent degradation

  • Optimize protein extraction buffer composition

• Detection system enhancements:

  • Use more sensitive detection systems (e.g., chemiluminescent substrates with enhanced sensitivity)

  • Try signal amplification methods like tyramide signal amplification

  • Increase exposure time for Western blots

• Antigen retrieval for fixed samples:

  • For immunohistochemistry, try heat-induced or enzymatic antigen retrieval methods

  • Optimize fixation conditions to preserve epitope accessibility

• Antibody handling:

  • Avoid repeated freeze-thaw cycles of antibody aliquots

  • Store according to manufacturer recommendations

  • Check for antibody precipitation before use

Similar to flow cytometry experiments where compensation and control issues can cause difficulties in data interpretation , antibody-based detection methods require careful optimization of multiple parameters to achieve reliable results.