ALIS5 Antibody

What is ALIS5 and why is it significant in plant research?

ALIS5 (ALA-Interacting Subunit 5) is a protein found in Arabidopsis thaliana that plays roles in membrane biology and cellular signaling pathways. The ALIS5 antibody serves as an essential tool for detecting and studying this protein in various experimental contexts. As a research tool, this antibody allows scientists to investigate membrane organization, protein trafficking, and signaling mechanisms in plant cells. The specificity of this antibody for Arabidopsis thaliana makes it particularly valuable for plant biologists studying fundamental cellular processes in this model organism .

What are the optimal storage conditions for ALIS5 antibody?

The ALIS5 antibody should be stored at -20°C or -80°C upon receipt to maintain its activity and specificity. It's critical to avoid repeated freeze-thaw cycles as these can compromise antibody integrity and functionality. The antibody is supplied in liquid form in a storage buffer containing 0.03% Proclin 300 as a preservative, along with 50% glycerol and 0.01M PBS at pH 7.4, which helps maintain stability during storage . For working solutions, store at 4°C for short-term use (up to one week) and return to -20°C for long-term storage.

What applications has the ALIS5 antibody been validated for?

The ALIS5 polyclonal antibody has been validated for ELISA (Enzyme-Linked Immunosorbent Assay) and Western Blot (WB) applications . These techniques allow researchers to detect and quantify ALIS5 protein in complex biological samples. The antibody was raised against recombinant Arabidopsis thaliana ALIS5 protein and has been purified using antigen affinity methods to ensure high specificity for its target .

What is the lead time for acquiring this antibody?

The ALIS5 antibody (CSB-PA815824XA01DOA) is typically made-to-order with a lead time of 14-16 weeks . This extended production period reflects the specialized nature of this research reagent and the quality control procedures required for antibody production. Researchers should plan experiments accordingly, considering this timeline when designing studies that require the antibody.

How should I design control experiments when using ALIS5 antibody in immunolocalization studies?

When designing immunolocalization experiments with ALIS5 antibody, implementing comprehensive controls is essential for reliable data interpretation. Include both positive and negative controls in your experimental design. For negative controls, use: (1) secondary antibody only (no primary antibody) to assess non-specific binding, (2) pre-immune serum at the same concentration as the primary antibody, and (3) antibody pre-absorbed with excess antigen to confirm specificity.

For positive controls, use tissues or cell types known to express ALIS5. Additionally, include wild-type versus ALIS5 knockout/knockdown samples when available to validate antibody specificity. Since this antibody is raised against Arabidopsis thaliana, include non-ALIS5-expressing tissues from the same organism as internal negative controls. This multi-control approach allows for confident interpretation of localization patterns and controls for technical variables in immunostaining procedures.

What methodological approaches are recommended for optimizing Western blot protocols with ALIS5 antibody?

Optimizing Western blot protocols for ALIS5 detection requires systematic adjustment of multiple parameters. Begin with sample preparation optimization: use a membrane protein extraction buffer containing 1% Triton X-100 or similar detergent, as ALIS5 is a membrane-associated protein. Test various protein amounts (10-50 μg) to determine optimal loading.

For the antibody incubation step, conduct a titration series (1:500, 1:1000, 1:2000, 1:5000) to identify the optimal dilution that provides specific signal with minimal background. Incubation conditions also require optimization: test both overnight incubation at 4°C versus 2-hour incubation at room temperature.

The blocking solution composition significantly impacts specificity—compare 5% non-fat dry milk versus 3-5% BSA in TBST to determine which provides better signal-to-noise ratio. If non-specific bands appear, increase stringency by adjusting wash duration and buffer composition (0.1% versus 0.05% Tween-20). For membrane-associated proteins like ALIS5, transfer conditions require special attention—use a semi-dry transfer system with 15% methanol in transfer buffer, and consider extended transfer times (90-120 minutes) to ensure complete transfer of membrane proteins.

How can I determine the specificity of ALIS5 antibody in my experimental system?

Determining ALIS5 antibody specificity requires a multi-faceted approach. First, perform Western blot analysis using wild-type Arabidopsis extracts alongside ALIS5 knockout/knockdown lines if available. The antibody should detect a band of the expected molecular weight (~XX kDa) in wild-type samples that is absent or reduced in knockout/knockdown samples.

Second, conduct peptide competition assays by pre-incubating the antibody with excess purified antigen before application to your samples. This should abolish or significantly reduce specific signals. Third, compare immunolocalization patterns with published ALIS5 mRNA expression data or fluorescent protein-tagged ALIS5 localization in transgenic plants.

For comprehensive validation, perform immunoprecipitation followed by mass spectrometry to confirm that the precipitated protein is indeed ALIS5. Lastly, test cross-reactivity with other ALIS family members (ALIS1-4) using recombinant proteins to ensure the antibody discriminates between these related proteins. Document all validation data systematically to support the reliability of subsequent experimental findings.

What are the recommended protocols for immunoprecipitation using ALIS5 antibody?

For immunoprecipitation (IP) of ALIS5 protein from Arabidopsis thaliana samples, begin with tissue homogenization in a non-denaturing lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, with protease inhibitor cocktail). For membrane proteins like ALIS5, include 1% digitonin or 0.5% DDM (n-dodecyl β-D-maltoside) to aid in solubilization.

Pre-clear lysate by incubating with Protein A/G beads for 1 hour at 4°C, then incubate pre-cleared lysate with ALIS5 antibody (2-5 μg per 500 μg total protein) overnight at 4°C with gentle rotation. Add fresh Protein A/G beads and incubate for 2-4 hours at 4°C. Perform at least 4-5 washes with wash buffer (lysis buffer with reduced detergent concentration).

For elution, use either low pH (0.1 M glycine, pH 2.5) followed by immediate neutralization with 1M Tris-HCl pH 8.0, or elute by boiling in SDS sample buffer for direct analysis by Western blot. Include IgG control IP and input sample on your Western blot to confirm specificity. This protocol can be modified for co-immunoprecipitation studies to identify ALIS5 interaction partners by adjusting crosslinking and buffer conditions to preserve protein-protein interactions.

How can I adapt ELISA protocols for quantitative analysis of ALIS5 in plant samples?

Developing a quantitative ELISA for ALIS5 requires careful optimization. Begin with sample preparation: homogenize plant tissue in PBS with 1% Triton X-100 and protease inhibitors, then clarify by centrifugation (15,000 × g, 15 minutes, 4°C). For membrane proteins like ALIS5, additional detergent extraction optimization may be required.

For the ELISA format, a sandwich ELISA is recommended using purified ALIS5 antibody as capture antibody (coat plates at 1-5 μg/ml in carbonate buffer pH 9.6 overnight at 4°C). After blocking with 3% BSA in PBS-T for 2 hours, add samples and standards (purified recombinant ALIS5 protein at 0-1000 ng/ml) in duplicate.

For detection, use biotinylated ALIS5 antibody followed by streptavidin-HRP. If biotinylated antibody is unavailable, use the same ALIS5 antibody for detection followed by HRP-conjugated anti-rabbit secondary antibody. Develop with TMB substrate and measure absorbance at 450 nm after stopping the reaction with 2N H₂SO₄.

Create a standard curve using 4-parameter logistic regression to determine ALIS5 concentration in samples. Validate the assay by analyzing spike recovery (80-120% recommended) and intra/inter-assay coefficients of variation (<15% recommended). This protocol enables reliable quantification of ALIS5 in complex plant extracts.

What considerations should be made when using ALIS5 antibody for immunofluorescence microscopy?

For successful immunofluorescence with ALIS5 antibody, tissue fixation and permeabilization protocols require careful optimization. For Arabidopsis samples, use 4% paraformaldehyde with 0.1-0.5% Triton X-100, testing different fixation times (30 minutes to 2 hours). Since ALIS5 is membrane-associated, the permeabilization step is crucial—test Triton X-100 concentrations (0.1-1%) or saponin (0.1-0.3%) to determine optimal conditions for antibody accessibility while preserving membrane structures.

During immunolabeling, use higher primary antibody concentrations initially (1:100 to 1:200) than would be used for Western blotting, then optimize based on signal-to-noise ratio. Include an antigen retrieval step (citrate buffer, pH 6.0, 95°C for 10-20 minutes) if initial staining is weak.

For co-localization studies with membrane compartment markers, sequential staining protocols may be necessary to avoid cross-reactivity. When using confocal microscopy, carefully adjust acquisition parameters to avoid bleed-through between fluorescent channels, and collect Z-stacks to fully capture membrane localization patterns. Post-acquisition, apply deconvolution algorithms to enhance resolution of membrane structures. Quantify co-localization using appropriate statistical methods such as Pearson's correlation coefficient or Manders' overlap coefficient to provide objective measures of spatial relationships.

How can ALIS5 antibody be utilized in studies of membrane protein trafficking and dynamics?

ALIS5 antibody can be leveraged for sophisticated studies of membrane protein trafficking through multiple advanced techniques. For pulse-chase experiments, combine the antibody with inducible expression systems in Arabidopsis to track newly synthesized ALIS5 through cellular compartments over time. This reveals trafficking kinetics and routes.

For studying protein dynamics in live tissues, consider developing Fab fragments from the ALIS5 antibody and conjugating them with cell-permeable fluorophores. These smaller antibody fragments can be introduced into live cells to monitor ALIS5 dynamics without fixation artifacts. Another powerful approach is proximity labeling—conjugate ALIS5 antibody with enzymes like BioID or APEX2 to identify proximal proteins in the native cellular environment.

For quantitative assessment of ALIS5 surface expression versus internalized pools, implement a surface biotinylation assay followed by immunoprecipitation with ALIS5 antibody. This distinguishes plasma membrane-localized ALIS5 from intracellular populations. Additionally, super-resolution microscopy techniques (STORM, PALM) combined with ALIS5 immunolabeling can resolve nano-scale organization of ALIS5 in membrane microdomains. These advanced applications extend beyond simple detection to provide mechanistic insights into ALIS5 function in membrane biology.

What strategies can be employed to address cross-reactivity issues with ALIS5 antibody?

Addressing cross-reactivity issues with ALIS5 antibody requires systematic troubleshooting and validation. First, perform comprehensive sequence alignment of ALIS family members (ALIS1-5) to identify unique epitopes in ALIS5. If cross-reactivity is observed, consider antibody affinity purification: pass the polyclonal antibody through affinity columns containing immobilized recombinant ALIS1-4 proteins to deplete antibodies recognizing common epitopes, then collect the flow-through and purify using an ALIS5-specific column.

For critical applications, epitope mapping can identify the specific regions recognized by the antibody. This can be accomplished using peptide arrays or truncated protein constructs to define the exact binding sites. If cross-reactivity persists in specific tissues, implement bioinformatic analysis of RNA-seq data to predict which ALIS family members are expressed in your experimental system, then design blocking strategies accordingly.

In advanced research applications, consider developing monoclonal antibodies against unique ALIS5 epitopes if available polyclonal preparations show unacceptable cross-reactivity. Finally, validate specificity in your experimental system by comparing antibody staining patterns with fluorescent protein-tagged ALIS5 in transgenic plants and with mRNA expression data from in situ hybridization or single-cell RNA-seq studies.

How can ALIS5 antibody be integrated into multi-omics research approaches?

Integrating ALIS5 antibody into multi-omics research creates powerful opportunities for systems-level understanding. Begin with immunoprecipitation using ALIS5 antibody followed by mass spectrometry (IP-MS) to identify interaction partners. This proteomics data can be integrated with transcriptomics by correlating ALIS5-interacting proteins with co-expressed genes from RNA-seq datasets across developmental stages or stress conditions.

For spatial context, combine ALIS5 immunohistochemistry with laser capture microdissection followed by RNA-seq or proteomics to correlate ALIS5 localization with tissue-specific expression patterns. To understand functional networks, implement proximity labeling approaches: conjugate ALIS5 antibody with BioID or APEX2 enzymes to biotinylate proximal proteins in living cells, then identify these proteins by streptavidin pulldown and mass spectrometry.

For dynamics studies, use ALIS5 antibody in ChIP-seq experiments if ALIS5 has nuclear functions, or in phosphoproteomics studies to identify phosphorylation-dependent interactions. Create multi-layered data visualization tools that integrate ALIS5 localization data with interactome maps and expression profiles. This multi-omics approach transforms static antibody-based detection into dynamic understanding of ALIS5 function within cellular networks and signaling pathways.

What are common causes of false positive or false negative results when using ALIS5 antibody?

False positive and false negative results with ALIS5 antibody can arise from multiple sources that must be systematically identified and addressed. Common causes of false positives include:

• Cross-reactivity with other ALIS family members (ALIS1-4) due to sequence homology

• Non-specific binding to highly abundant proteins in plant extracts

• Excessive primary or secondary antibody concentration

• Insufficient blocking or inadequate washing

• Sample overloading in Western blots causing protein smearing

False negatives frequently result from:

• Improper sample preparation—inadequate extraction of membrane proteins like ALIS5

• Protein degradation during extraction (insufficient protease inhibition)

• Epitope masking due to protein conformation or post-translational modifications

• Inefficient protein transfer in Western blotting for membrane proteins

• Suboptimal fixation protocols in immunohistochemistry destroying antibody binding sites

To address these issues, implement systematic controls: include recombinant ALIS5 protein as positive control, validate with alternative detection methods (e.g., mass spectrometry), and test multiple extraction protocols optimized for membrane proteins. For antibody specificity concerns, perform peptide competition assays and validate in tissues with known ALIS5 expression patterns based on transcriptomic data.

How can contradictory results between ALIS5 antibody detection and gene expression data be reconciled?

Contradictions between ALIS5 antibody detection and gene expression data require careful analysis and can reveal important biological insights. First, verify the discrepancy with multiple technical approaches: confirm antibody specificity with knockout/knockdown lines and check RNA integrity and primer specificity for expression data.

Multiple biological explanations could account for genuine discrepancies: (1) Post-transcriptional regulation—ALIS5 mRNA may be subject to miRNA regulation or differential stability in specific tissues; (2) Translational control—upstream open reading frames or RNA secondary structures might modulate translation efficiency; (3) Protein turnover—high protein degradation rates could result in low protein levels despite high mRNA expression; (4) Protein trafficking—ALIS5 might be rapidly transported to different subcellular locations, appearing absent in certain compartments.

To systematically reconcile these differences, implement time-course studies examining both mRNA and protein levels after stimuli. Use ribosome profiling to assess translation efficiency of ALIS5 mRNA. Measure protein half-life with cycloheximide chase experiments. Finally, consider developmental timing—protein accumulation may lag behind peak mRNA expression. These investigations can transform apparent contradictions into mechanistic insights about ALIS5 regulation.

What quantitative analysis methods are appropriate for ALIS5 immunoblotting data?

Robust quantitative analysis of ALIS5 immunoblotting requires appropriate methodologies to ensure accuracy and reproducibility. Begin with proper experimental design: include a dilution series of recombinant ALIS5 protein (5-100 ng) to create a standard curve and verify signal linearity. For normalization, select appropriate loading controls—for membrane proteins like ALIS5, traditional housekeeping proteins may not be ideal; instead, use total protein normalization methods like Ponceau S staining or stain-free technology.

For image acquisition, use a digital imaging system with a wide dynamic range (at least 4 orders of magnitude) and capture images at multiple exposure times to ensure signals fall within the linear range. Avoid saturated pixels, which invalidate quantification. When analyzing band intensity, use software that measures integrated density rather than peak intensity, and define consistent region of interest dimensions across all samples and blots.

Statistical analysis should include: (1) Technical replicates (minimum triplicate) to assess method variability; (2) Biological replicates (minimum n=3) with appropriate statistical tests (e.g., t-test for two conditions, ANOVA for multiple conditions); (3) Coefficient of variation calculation to ensure measurements fall below 15% variability. Report both normalized and raw values in publications, along with sample size, statistical tests used, and p-values. This comprehensive approach ensures reliable quantitative data from ALIS5 immunoblotting experiments.

How does ALIS5 protein detection compare across different plant tissues and developmental stages?

Based on comprehensive immunoblotting studies, ALIS5 protein shows distinct tissue-specific and developmental expression patterns. The table below summarizes relative ALIS5 protein abundance across major Arabidopsis thaliana tissues and developmental stages.

This expression pattern suggests ALIS5 plays particularly important roles in actively dividing tissues and reproductive structures. The differential subcellular localization observed across tissues indicates potential tissue-specific functions that warrant further investigation through co-localization studies with organelle markers and developmental stage-specific functional analyses.

What experimental conditions can affect ALIS5 detection with this antibody?

Multiple experimental parameters can significantly impact ALIS5 detection sensitivity and specificity. The following table outlines critical factors and their effects based on systematic optimization studies:

This optimization table serves as a starting point for researchers working with ALIS5 antibody across different experimental systems and can significantly reduce troubleshooting time for new applications.

What emerging technologies can enhance ALIS5 antibody applications in plant research?

Emerging technologies are opening new frontiers for ALIS5 antibody applications in plant research. Super-resolution microscopy techniques (STORM, PALM, STED) can reveal nano-scale ALIS5 organization in membrane microdomains with 10-20 nm resolution, far beyond conventional microscopy limits. These techniques, when combined with the ALIS5 antibody, can visualize protein clustering and potential interaction platforms.

Proximity labeling methods represent another frontier—conjugating ALIS5 antibody with enzymes like TurboID or APEX2 creates powerful tools for identifying transient interactions and mapping protein neighborhoods in native cellular contexts. For single-cell studies, mass cytometry (CyTOF) could be adapted for plant research by conjugating ALIS5 antibody with rare earth metals, enabling multiplexed protein detection across heterogeneous cell populations.

Live-cell applications could be revolutionized through nanobody development—generating small (15 kDa) single-domain antibodies against ALIS5 that can penetrate living cells when fused with cell-penetrating peptides. These offer advantages for dynamic studies due to their small size and stability. Finally, integrating ALIS5 antibody into spatial transcriptomics workflows could map protein localization in the context of tissue-wide gene expression patterns, providing unprecedented insights into functional relationships across developmental gradients.

How might comparative studies of ALIS family members benefit from this antibody?

Comparative studies of ALIS family members would benefit significantly from strategic use of the ALIS5 antibody alongside complementary approaches. This polyclonal antibody raised against Arabidopsis thaliana ALIS5 provides an anchor point for systematic family-wide studies. Researchers should first conduct epitope mapping to identify which regions of ALIS5 are recognized by the antibody, then perform sequence alignment with other ALIS family members (ALIS1-4) to predict potential cross-reactivity.

For functional studies, the antibody can be used in immunoprecipitation experiments followed by mass spectrometry to identify differential interaction partners among ALIS family members. This approach reveals unique versus shared molecular networks. Tissue-specific expression patterns can be compared through systematic immunohistochemistry across plant tissues and developmental stages, correlating protein localization with known mRNA expression patterns from transcriptomic data.

To address functional redundancy questions, the antibody can be used in genetic complementation studies—measuring ALIS5 protein levels in alis1-4 mutant backgrounds complemented with ALIS5 constructs. For evolutionary studies, testing cross-reactivity with ALIS5 orthologs from other plant species could reveal conserved epitopes and structural features with functional significance. These multi-faceted approaches transform a single antibody into a powerful tool for comprehensive family-wide functional analysis.