ATL35 Antibody

What is ATL35 and why is it significant in plant research?

ATL35 refers to a specific protein in Arabidopsis thaliana (Uniprot No. Q9M0R6) that has become an important research target in plant molecular biology . The corresponding ATL35 antibody enables detection and characterization of this protein across various experimental platforms. Understanding ATL35 functionality contributes to our knowledge of plant cellular mechanisms, though it should not be confused with the human disease marker ATL (Adult T-cell Leukemia), which represents an entirely different biological entity .

When designing studies around ATL35, researchers should consider its biological context within plant systems. While relatively understudied compared to some plant proteins, ATL35 antibodies provide researchers with tools to explore protein-protein interactions, expression patterns across development, and responses to various environmental stimuli. The significance lies in its potential role in broader plant molecular pathways that remain to be fully characterized.

Methodologically, researchers should approach ATL35 studies with careful consideration of tissue-specific expression patterns, appropriate extraction methods for plant tissues, and validation strategies specific to plant antibodies, which often face different challenges than mammalian systems.

What are the optimal storage conditions for ATL35 antibody?

For maximum stability and maintained reactivity, ATL35 antibody should be stored at -20°C or -80°C upon receipt . Repeated freeze-thaw cycles should be carefully avoided as they significantly degrade antibody quality and experimental performance. Creating single-use aliquots immediately upon reconstitution represents best practice for long-term research planning.

For lyophilized formats, reconstitution should be performed using sterile water, and researchers should briefly centrifuge tubes before opening to ensure no product is lost due to adhesion to caps or tube walls . Once reconstituted, the antibody solution should be maintained at -20°C in appropriate working aliquots.

Short-term storage (1-2 weeks) at 4°C may be acceptable for actively used aliquots, but extended storage at this temperature is not recommended. The antibody is typically provided in a buffer containing 50% glycerol and 0.01M PBS at pH 7.4 with 0.03% Proclin 300 as a preservative, which helps maintain stability . Researchers should monitor for any visible precipitates before use, as these may indicate compromised antibody quality.

What applications is the ATL35 antibody validated for?

The ATL35 antibody has been specifically validated for ELISA and Western blot applications, making these the primary recommended techniques for researchers . When applying this antibody in Western blotting, recommended dilution ranges typically fall between 1:1000 and 1:3000, though optimal conditions may require laboratory-specific optimization .

For immunocytochemistry applications, researchers have successfully used similar plant antibodies at dilutions around 1:20, though this should be systematically optimized for ATL35 specifically . The antibody preparation workflow for immunocytochemistry typically involves embedding samples in Lowicryl HM20, sectioning to 100-nm thickness, placing on Formvar-coated gold slot grids, and blocking with 5% nonfat milk solution in TBST before antibody application .

It's important to note that while ATL35 antibody has confirmed reactivity with Arabidopsis thaliana, extrapolation to other plant species should be experimentally verified rather than assumed. Cross-reactivity testing is essential when applying this antibody to new model systems not previously documented in the literature.

How can I verify ATL35 antibody specificity in my experimental system?

Validating antibody specificity represents a crucial step before conducting definitive experiments with ATL35 antibody. The most rigorous approach follows the multiple-pillar validation strategy described in contemporary literature . For ATL35 specifically, consider implementing:

• Knockout/knockdown validation: Generate or obtain Arabidopsis thaliana lines with confirmed ATL35 knockout or knockdown modifications. These serve as negative controls - any signal detected in these samples suggests non-specific binding. RNAi or CRISPR-based approaches for creating these controls are increasingly accessible for plant systems .

• Multiple antibody validation: Where available, utilize different antibodies targeting distinct epitopes of ATL35. Concordant results between different antibodies significantly strengthen specificity claims .

• Recombinant protein controls: Express recombinant ATL35 protein as a positive control for Western blot analysis. The appearance of a band at the expected molecular weight (~35 kDa) supports antibody specificity claims .

• Biological validation: Leverage known biological properties of ATL35, such as its expected subcellular localization or expression patterns under specific conditions. Results contradicting established knowledge suggest potential specificity issues .

For plant-specific considerations, researchers should be particularly vigilant about potentially confounding factors such as high levels of endogenous peroxidases, polyphenols, and other compounds that may interfere with antibody-based detection methods.

What are the major cross-reactivity concerns with ATL35 antibody?

Cross-reactivity represents a significant concern in plant antibody applications due to the presence of numerous protein families with conserved domains. For ATL35 antibody specifically, researchers should be aware of several potential sources of cross-reactivity:

• Related plant proteins: The antibody was raised against recombinant Arabidopsis thaliana ATL35 protein , which may share epitopes with related proteins. Preliminary data suggests limited cross-reactivity with proteins from certain plant species, though comprehensive cross-reactivity profiles remain to be established.

• Non-specific binding in high-polysaccharide tissues: Plant tissues with high polysaccharide content may exhibit non-specific binding. This can be mitigated through extended blocking steps (increasing from standard 20 minutes to 60 minutes) and using higher concentrations of blocking reagents (5-10% nonfat milk solution in TBST) .

• Potential for non-specific binding in protein-dense regions: Some plant organelles exhibit especially high protein density, which may result in non-specific signal. Controls lacking primary antibody are essential for distinguishing true signal from background.

It's worth noting that some plant antibodies show reliable reactivity across multiple plant species including Arabidopsis thaliana, Citrus reticulata, and Gossypium hirsutum, while showing no reactivity with Chlamydomonas reinhardtii, Pheodactylum tricornutum, or Pisum sativum . This pattern suggests conservative epitope recognition that may be present in ATL35 antibody as well, though empirical testing remains necessary.

How can I optimize ATL35 antibody performance in challenging plant tissues?

Plant tissues present unique challenges for antibody applications due to their cell wall structures, secondary metabolites, and high levels of proteases. For optimizing ATL35 antibody performance in difficult samples:

• Enhanced extraction protocols: For recalcitrant tissues, consider supplementing standard extraction buffers with higher concentrations of protease inhibitors, reducing agents (DTT or β-mercaptoethanol at 5-10 mM), and detergents like Triton X-100 (0.5-1%) or SDS (0.1-0.5%) to improve protein accessibility.

• Extended blocking parameters: Increase blocking time to 45-60 minutes using 5% nonfat milk in TBST for tissues known to exhibit high background . Some researchers report success substituting standard milk blocking with 2-5% BSA or 5% normal serum from the species of secondary antibody origin.

• Optimized washing regimen: For tissues showing persistent background, implement more stringent washing steps using higher concentrations of Tween-20 (0.5% instead of standard 0.1%) in TBS . Extending wash durations and increasing wash repetitions can significantly reduce non-specific signal.

• Antigen retrieval considerations: Though less common in plant applications than mammalian IHC, heat-induced or enzymatic antigen retrieval methods may improve antibody accessibility to certain epitopes in fixed tissues. For ATL35, pilot experiments with citrate buffer (pH 6.0) heat retrieval or enzymatic retrieval with proteases like proteinase K may improve signal detection in some applications.

Through systematic optimization of these parameters, researchers can substantially improve signal-to-noise ratios when working with ATL35 antibody across diverse plant tissue types.

What techniques should I use to validate ATL35 antibody specificity?

Comprehensive antibody validation requires multiple complementary approaches. For ATL35 antibody, implement this systematic validation workflow:

• Western blot verification: The most fundamental validation step involves confirming a single band of appropriate molecular weight (~35 kDa) in Western blot analysis of plant extracts expressing ATL35 . Multiple bands or bands of unexpected sizes suggest potential specificity issues.

• Peptide competition assay: Pre-incubate the ATL35 antibody with excess purified antigen (the immunogen sequence) before application in your experimental system. Disappearance of signal confirms epitope-specific binding rather than non-specific interactions.

• Genetic knockout controls: As previously mentioned, utilize genetic approaches to generate or obtain ATL35 knockout or knockdown lines as negative controls. The absence of signal in these samples strongly supports antibody specificity .

• Orthogonal validation: Compare antibody-based detection results with non-antibody methods for detecting the same target, such as mass spectrometry or RNA-seq data for transcript expression . Correlation between methods significantly strengthens confidence in antibody specificity.

• Recombinant expression system: Express recombinant ATL35 protein with an orthogonal tag (e.g., GFP, FLAG, or His-tag) and confirm co-localization of anti-ATL35 signal with the tagged protein. This approach is particularly valuable for confirming subcellular localization patterns.

Documentation of these validation steps should accompany all publications utilizing ATL35 antibody to ensure reproducibility and reliability of reported findings.

How should I establish appropriate dilution parameters for ATL35 antibody?

Determining optimal dilution conditions for ATL35 antibody requires systematic titration experiments tailored to each application. Follow this methodical approach:

• Western blot optimization:

  • Begin with a broad dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000)

  • Analyze signal-to-noise ratio, background levels, and specific band intensity

  • Refine with a narrower range around promising dilutions

  • Published recommendations suggest starting with 1:1000-1:3000 range

• ELISA optimization:

  • Conduct checkerboard titration using both coating antigen concentration and antibody dilution variables

  • Plot signal-to-noise ratios to identify optimal working parameters

  • Confirm with replicates before finalizing protocol conditions

• Immunocytochemistry optimization:

  • Begin with manufacturer's recommended dilution (typically more concentrated than for Western blot)

  • For plant antibodies similar to ATL35, a starting dilution of 1:20 has shown success

  • Evaluate using positive and negative control samples

  • Adjust secondary antibody dilutions proportionally

The optimal dilution will be the one that provides maximum specific signal with minimal background across multiple experimental replicates. Document all optimization parameters including incubation time, temperature, blocking conditions, and washing protocols to ensure reproducibility.

How can I differentiate between true ATL35 signal and experimental artifacts?

Distinguishing genuine ATL35 signal from artifacts requires implementing rigorous controls and analytical approaches:

• Essential controls for each experiment:

  • No primary antibody control (secondary antibody only)

  • Isotype control (irrelevant primary antibody of same isotype/host species)

  • Biological negative control (tissue known not to express target)

  • Competition control (antibody pre-incubated with immunizing peptide)

• Signal validation strategies:

  • Consistency across multiple detection methods (fluorescence, colorimetric, chemiluminescence)

  • Reproducibility across biological replicates

  • Appropriate subcellular localization pattern consistent with known biology

  • Expected molecular weight in Western blot applications

• Artifact identification and elimination:

  • Edge effects in tissue sections (typically non-specific)

  • Trapping in plant cell walls (verify with high-magnification imaging)

  • Autofluorescence (use appropriate filters and controls)

  • Non-specific binding to vascular tissues (common in plant samples)

When analyzing contradictory results between experiments, systematically evaluate all variables including sample preparation methods, antibody lot numbers, detection reagents, and imaging parameters. Maintaining detailed laboratory records of all experimental conditions facilitates troubleshooting inconsistent results.

What are the critical steps for successful Western blot with ATL35 antibody?

Western blotting with ATL35 antibody requires attention to plant-specific protein extraction considerations and optimization of several key parameters:

• Sample preparation optimization:

  • Use extraction buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, supplemented with protease inhibitors

  • For plant tissues, add 10 mM DTT and 1% polyvinylpolypyrrolidone (PVPP) to minimize interference from phenolic compounds

  • Maintain cold temperatures throughout extraction to prevent proteolysis

  • Centrifuge at 15,000g for 15 minutes at 4°C to remove cell debris

• Electrophoresis considerations:

  • Load 20-50 μg total protein per lane

  • Use 12% SDS-PAGE for optimal resolution around the expected 35 kDa molecular weight

  • Include molecular weight markers that clearly demarcate the 25-40 kDa range

• Transfer and detection parameters:

  • Transfer to PVDF membrane at 100V for 60 minutes in cold transfer buffer

  • Block membrane with 5% nonfat milk in TBST for 1 hour at room temperature

  • Incubate with ATL35 antibody at 1:1000-1:3000 dilution overnight at 4°C

  • Wash extensively with TBST (3 x 10 minutes)

  • Incubate with HRP-conjugated secondary antibody at 1:5000-1:10000 for 1 hour

  • Develop using enhanced chemiluminescence substrate

For troubleshooting weak signals, consider extending primary antibody incubation time, reducing washing stringency, or using signal enhancement systems like biotin-streptavidin amplification. For high background, increase blocking time, adjust antibody dilutions, or implement more stringent washing conditions.

How can I optimize immunolocalization studies using ATL35 antibody?

For successful immunolocalization of ATL35 in plant tissues, researchers should follow these optimized protocols based on experience with similar plant antibodies:

• Sample preparation protocol:

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

  • For electron microscopy applications, embed samples in Lowicryl HM20 resin

  • Section to appropriate thickness (100 nm for EM, 5-10 μm for light microscopy)

  • Mount on appropriate slides or grids (Formvar-coated gold slot grids for EM)

• Blocking and antibody application:

  • Block with 5% nonfat milk in TBS containing 0.1% Tween-20 for 20 minutes

  • Dilute primary ATL35 antibody in 2.5% nonfat milk in TBST

  • Apply at 1:20 dilution for 1 hour at room temperature

  • Rinse thoroughly with TBS containing 0.5% Tween-20

  • Apply appropriate secondary antibody (anti-rabbit IgG conjugated to fluorophore or gold particles) at 1:20 dilution

• Visualization optimization:

  • For fluorescence imaging, use appropriate filter sets based on secondary antibody

  • For confocal microscopy, establish z-stack parameters to capture full cellular distribution

  • For electron microscopy, examine multiple fields to quantify gold particle distribution

To enhance specific signal, some researchers report success with tyramide signal amplification systems, which can increase detection sensitivity 10-50 fold. When comparing localization patterns between different conditions or genotypes, maintain identical acquisition parameters to allow valid comparisons of signal intensity and distribution.

What approaches should I use to quantify ATL35 expression levels?

Accurate quantification of ATL35 expression requires calibrated approaches that account for technical variability:

• Western blot quantification methodology:

  • Include concentration gradient of recombinant ATL35 protein as standard curve

  • Utilize housekeeping protein (such as actin or GAPDH) as internal loading control

  • Capture images within linear dynamic range of detection system

  • Apply appropriate background subtraction methods

  • Use densitometry software for quantification (ImageJ with gel analysis plugins)

  • Express results as normalized intensity relative to loading controls

• ELISA quantification approach:

  • Develop standard curve using recombinant ATL35 protein

  • Ensure samples fall within linear range of standard curve

  • Perform technical triplicates for each biological sample

  • Calculate concentration based on 4-parameter logistic regression of standard curve

  • Report results as absolute quantity (ng/ml or ng/mg total protein)

• Statistical analysis for expression studies:

  • Conduct minimum of three biological replicates

  • Apply appropriate statistical tests based on data distribution

  • Calculate variation coefficients to assess method reliability

  • Consider bootstrapping approaches for robust confidence intervals

For temporal expression studies, maintain consistent harvest times and developmental stages across samples. When comparing expression across tissue types, normalize to total protein rather than using single housekeeping proteins, which may vary in expression across different tissues.

How can ATL35 antibody be utilized in plant stress response studies?

ATL35 antibody presents valuable opportunities for investigating plant stress response mechanisms through several advanced applications:

• Stress-induced protein modification analysis:

  • Monitor post-translational modifications of ATL35 under various stress conditions

  • Combine with phospho-specific antibodies to assess stress-induced phosphorylation

  • Implement 2D gel electrophoresis followed by Western blotting to resolve modified forms

  • Correlate modifications with specific stress responses (drought, salinity, pathogens)

• Protein interaction dynamics during stress:

  • Utilize ATL35 antibody for co-immunoprecipitation studies before and during stress

  • Identify stress-specific interaction partners through mass spectrometry

  • Validate interactions through reverse co-IP and proximity ligation assays

  • Map interaction networks across stress response timeline

• Subcellular relocalization studies:

  • Track potential redistribution of ATL35 during stress responses

  • Employ subcellular fractionation followed by Western blot analysis

  • Complement with immunofluorescence microscopy for spatial resolution

  • Correlate relocalization with functional outcomes in stress adaptation

These approaches can provide mechanistic insights into plant stress responses, potentially revealing novel aspects of ATL35 function in environmental adaptation. When designing such studies, researchers should implement appropriate controls for each stress condition and validate findings across multiple plant accessions to establish biological relevance.

What considerations apply when integrating ATL35 antibody with advanced imaging technologies?

Integrating ATL35 antibody with cutting-edge imaging technologies requires specific methodological adaptations:

• Super-resolution microscopy applications:

  • For STORM/PALM: Consider directly conjugated primary antibodies to minimize localization error

  • For SIM: Optimize fixation protocols to preserve native protein distribution while enabling antibody access

  • For expansion microscopy: Validate epitope preservation throughout expansion process

  • Expected resolution improvement from ~200nm (confocal) to ~20-50nm (super-resolution)

• Live cell imaging considerations:

  • Explore cell-permeable antibody derivatives or nanobody alternatives with smaller size

  • Validate function-preserving ATL35 fluorescent protein fusions as alternatives

  • Implement FRAP (Fluorescence Recovery After Photobleaching) to assess protein dynamics

  • Compare live imaging with fixed-cell antibody labeling to identify potential fixation artifacts

• Correlative light and electron microscopy (CLEM):

  • Optimize ATL35 antibody for compatible fixation protocols (e.g., light glutaraldehyde fixation)

  • Consider gold-conjugated secondary antibodies for direct EM visualization

  • Implement fiducial markers for precise alignment between light and EM images

  • Validate localization pattern across both imaging modalities

These advanced imaging approaches can significantly enhance our understanding of ATL35 spatial organization and dynamics within plant cells. Researchers should conduct appropriate control experiments specific to each imaging modality and carefully account for the potential influence of sample preparation on antibody accessibility and epitope preservation.