EXPA25 Antibody

What is EXPA25 and why is it important in plant research?

EXPA25 (Expansin A25) is a member of the expansin protein family in plants that plays a crucial role in cell wall loosening and cellular expansion processes. Encoded by gene ID 833926 in Arabidopsis thaliana, EXPA25 (UniProt Number: Q9FL77) functions within the complex network of proteins that regulate plant growth and development through cell wall modifications . Expansins like EXPA25 are particularly important during rapid growth phases, stress responses, and developmental transitions in plants. The EXPA25 antibody allows researchers to detect, quantify, and localize this protein in various experimental contexts, providing valuable insights into cellular processes that would otherwise be difficult to observe directly.

What types of EXPA25 antibodies are available for research applications?

Currently, the primary type of EXPA25 antibody available for research is rabbit polyclonal antibody purified by antigen affinity methods. These antibodies are typically generated using recombinant Arabidopsis thaliana EXPA25 protein as the immunogen . Commercial preparations often include:

• Purified rabbit polyclonal antibodies (typically 2mg concentration)

• Positive control materials (200μg antigens)

• Negative control materials (1ml pre-immune serum)

Unlike some therapeutic antibodies that have been developed in multiple formats (such as the well-characterized EA5 antibody against EphA2) , EXPA25 antibodies remain primarily available as polyclonal reagents. This reflects their current research applications rather than therapeutic development.

What are the validated applications for EXPA25 antibodies?

Based on validation studies, EXPA25 antibodies have been confirmed for use in:

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Western Blotting (WB)

While these represent the formally validated applications, researchers studying plant proteins have successfully adapted similar antibodies for additional techniques including:

• Immunohistochemistry (IHC) in plant tissue sections

• Immunoprecipitation when studying protein complexes

• Subcellular localization studies through cell fractionation followed by western blotting

This approach has been demonstrated with other plant antibodies such as those against ACBP6, where western blotting of subcellular fractions revealed cytosolic localization patterns .

How should researchers design proper controls when using EXPA25 antibodies?

Proper experimental design with EXPA25 antibodies requires multiple control types:

• Positive controls: Use the supplied antigen (200μg) to confirm antibody activity and establish detection sensitivity . A concentration series (10-100ng) can help determine the linear detection range.

• Negative controls: The pre-immune serum provided with commercial antibodies serves as an excellent negative control to identify non-specific binding . Additionally, samples from EXPA25 knockout plants (similar to the acbp6 T-DNA insertional mutants described in plant antibody research) would serve as biological negative controls .

• Loading controls: For western blots, researchers should include detection of housekeeping proteins (e.g., actin, tubulin) to normalize between samples.

• Cross-reactivity controls: When working with species other than Arabidopsis, testing against known expansin-deficient tissues or heterologously expressed EXPA proteins can confirm specificity.

The experimental approach used for validating ACBP6 antibodies provides an excellent model, where researchers combined PCR genotyping with western blot analysis to confirm the absence of protein in knockout lines .

What are the optimal storage and handling conditions for maintaining EXPA25 antibody activity?

For maximum stability and reproducibility in experiments, EXPA25 antibodies should be:

• Stored at either -20°C or -80°C for long-term preservation

• Aliquoted to minimize freeze-thaw cycles (ideally ≤5 cycles)

• Kept on ice during experimental procedures

• Stored with appropriate preservatives (typically 0.02-0.05% sodium azide)

• Handled using low-protein binding tubes to prevent loss through adsorption

When diluting the antibody for specific applications, researchers should use freshly prepared buffer systems that maintain protein stability. For western blotting, 5% non-fat dry milk or 1-3% BSA in TBST (Tris-buffered saline with 0.1% Tween-20) is typically effective, similar to protocols used with other plant antibodies .

What is the recommended protocol for detecting EXPA25 using western blotting?

Based on established protocols for plant antibodies and the specific properties of EXPA25, the following western blotting approach is recommended:

Sample Preparation:

• Extract total protein from plant tissue using a buffer containing:

  • 50mM Tris-HCl (pH 7.5)

  • 150mM NaCl

  • 1% Triton X-100

  • 0.5% sodium deoxycholate

  • Protease inhibitor cocktail

• Determine protein concentration using Bradford or BCA assay

• Mix samples with Laemmli buffer and heat at 95°C for 5 minutes

SDS-PAGE and Transfer:

• Load 20-30μg total protein per lane on 12% SDS-PAGE gel

• Include molecular weight markers for reference

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

Immunodetection:

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

• Incubate with EXPA25 antibody (1:1000 dilution) overnight at 4°C

• Wash 3× with TBST (10 minutes each)

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

• Wash 3× with TBST (10 minutes each)

• Develop using ECL substrate and image

This protocol is based on successful approaches used with other plant antibodies in western blot analysis .

How can researchers optimize EXPA25 antibody specificity for challenging applications?

For applications requiring enhanced specificity, researchers can employ several optimization strategies:

• Affinity purification: Further purify commercial EXPA25 antibodies against the immunizing antigen to reduce non-specific binding

• Cross-adsorption: Pre-incubate antibodies with plant extracts from EXPA25 knockout lines to remove antibodies that recognize non-target epitopes

• Optimized blocking: Test different blocking agents (BSA, casein, commercial blockers) to identify those that minimize background while preserving specific signal

• Dilution optimization: Perform titration experiments to identify the minimum effective antibody concentration that provides specific signal with minimal background

• Alternative detection systems: For weak signals, consider amplification methods like tyramide signal amplification or more sensitive detection reagents

Recent advances in antibody engineering have demonstrated how targeted modifications can dramatically improve specificity. For example, studies with therapeutic antibodies have shown that strategic modifications to complementarity-determining regions (CDRs) and framework regions can reduce non-specific binding while maintaining or improving target affinity . Though these approaches were developed for therapeutic antibodies, the principles can guide optimization strategies for research antibodies like those against EXPA25.

How can researchers address non-specific binding issues with EXPA25 antibodies?

Non-specific binding is a common challenge when working with antibodies in plant systems. To address this issue with EXPA25 antibodies:

• Optimize antibody concentration: Test serial dilutions to find the minimal concentration that provides specific signal

• Modify blocking conditions: Increase blocking agent concentration (5-10%) and/or duration (overnight at 4°C)

• Add competitive blocking agents: Include 0.1-0.2% Tween-20 or 0.05% Triton X-100 in antibody dilution buffer

• Increase wash stringency: Use higher salt concentration (up to 500mM NaCl) in wash buffers

• Pre-adsorb antibody: Incubate diluted antibody with membrane prepared from knockout/negative control tissue

• Use quantitative metrics: Employ specificity assessment methods similar to the polyspecificity reagent (PSR) approach, which measures non-specific interactions using protein A-coated magnetic beads

Recent research has established correlations between antibody properties and non-specific binding tendencies. For example, the presence of positively charged patches in antibody complementarity-determining regions (CDRs) has been linked to increased non-specific interactions . This knowledge can guide the selection or development of EXPA25 antibodies with improved specificity profiles.

What approaches can be used for studying EXPA25 subcellular localization?

To investigate the subcellular localization of EXPA25, researchers can employ several complementary approaches:

• Subcellular fractionation with western blotting:

  • Perform differential centrifugation to separate cellular compartments

  • Analyze fractions by western blotting with EXPA25 antibodies

  • Include markers for different cellular compartments (e.g., cytosol, membrane, nuclear fractions)

  This approach was successfully used to demonstrate the cytosolic localization of ACBP6 in Arabidopsis .

• Immunofluorescence microscopy:

  • Fix and permeabilize plant cells or tissue sections

  • Incubate with EXPA25 antibody followed by fluorescent secondary antibody

  • Co-stain with organelle markers for colocalization analysis

• Transgenic approach with fluorescent protein fusions:

  • Generate plants expressing EXPA25-GFP fusion proteins

  • Validate fusion protein functionality

  • Visualize localization in living cells

  Similar approaches with ACBP6-GFP confirmed cytosolic localization of ACBP6 .

How can researchers evaluate EXPA25 expression changes under stress conditions?

To investigate EXPA25 expression responses to environmental stresses (similar to cold induction of ACBP6) , researchers should implement a multi-level analysis approach:

• Transcript level analysis:

  • Perform RT-qPCR to quantify EXPA25 mRNA levels

  • Include multiple reference genes for normalization

  • Sample at multiple time points (0, 6, 12, 24, 48 hours) after stress application

• Protein level analysis:

  • Use western blotting with EXPA25 antibodies to quantify protein abundance

  • Include loading controls (constitutively expressed proteins)

  • Perform densitometric analysis for quantification

• Spatial expression analysis:

  • Use immunohistochemistry to identify tissue-specific expression changes

  • Consider reporter gene fusions (EXPA25 promoter driving GUS or luciferase)

• Functional correlation:

  • Measure relevant physiological parameters (e.g., cell elongation, wall extensibility)

  • Compare wild-type and EXPA25 mutant/overexpression lines

Studies with ACBP6 demonstrated that both northern blot and western blot analyses were essential to document cold-induced expression, with peak induction observed at 48 hours post-treatment . Similar comprehensive approaches would be valuable for EXPA25 stress response studies.

What methods are available for generating knockout or knockdown EXPA25 Arabidopsis lines?

To create EXPA25-deficient plants for functional studies, researchers can employ several genetic approaches:

• T-DNA insertion lines:

  • Screen public repositories (TAIR, NASC) for existing T-DNA insertions in EXPA25

  • Confirm homozygosity using PCR with gene-specific and T-DNA border primers

  • Validate knockout status using RT-PCR and western blotting with EXPA25 antibodies

  This approach was successfully used to generate and characterize acbp6 knockout mutants .

• CRISPR/Cas9 genome editing:

  • Design sgRNAs targeting EXPA25 coding sequences

  • Transform plants and screen for mutations

  • Confirm mutations by sequencing and protein loss by western blotting

• RNAi or artificial microRNA approaches:

  • Design constructs targeting EXPA25 mRNA

  • Generate transgenic lines with variable knockdown efficiency

  • Quantify knockdown levels by RT-qPCR and western blotting

• Inducible silencing systems:

  • Use estrogen or dexamethasone-inducible promoters to drive RNAi constructs

  • Allow temporal control of EXPA25 suppression

  • Monitor phenotypes before and after induction

Validation of knockout/knockdown status should include both genomic PCR, transcript analysis (RT-PCR), and protein detection using EXPA25-specific antibodies, similar to the comprehensive validation performed for acbp6 mutants .

How can new antibody engineering technologies be applied to improve EXPA25 antibodies?

Recent advances in antibody engineering offer opportunities to develop next-generation EXPA25 research tools:

• Machine learning-guided optimization: Computational approaches can predict mutations that enhance both affinity and specificity, as demonstrated in therapeutic antibody development . These methods analyze sequence-function relationships to suggest targeted modifications.

• Recombinant antibody technology: Moving from traditional polyclonal antibodies to recombinant formats provides several advantages, including:

  • Increased sensitivity through affinity maturation

  • Confirmed specificity via negative selection strategies

  • High reproducibility between batches

  • Sustainable supply through defined expression systems

  • Animal-free production methods

• Fragment-based approaches: Smaller antibody fragments (Fab, scFv) can offer advantages for certain applications, especially where tissue penetration is challenging

• Bi-specific formats: Developing antibodies that simultaneously recognize EXPA25 and a second target could enable novel co-localization studies

The Hi-AffiTM recombinant antibody approach demonstrates how next-generation antibody technologies can provide numerous advantages over traditional antibody production methods .

What cross-disciplinary approaches might enhance EXPA25 research?

Integrating techniques from multiple disciplines can significantly advance EXPA25 research:

• Structural biology integration:

  • Determine EXPA25 protein structure through X-ray crystallography or cryo-EM

  • Map epitopes recognized by EXPA25 antibodies

  • Guide rational antibody improvement and experimental design

• Systems biology approaches:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Identify networks and pathways involving EXPA25

  • Build predictive models of EXPA25 function during development and stress

• Advanced imaging technologies:

  • Super-resolution microscopy for precise subcellular localization

  • Live-cell imaging to track EXPA25 dynamics

  • Correlative light and electron microscopy to combine functional and structural insights

• Synthetic biology implementations:

  • Create plants with engineered EXPA25 variants

  • Develop optogenetic control of EXPA25 expression

  • Design synthetic regulatory circuits involving EXPA25

These interdisciplinary approaches build upon fundamental antibody-based detection methods while extending research capabilities into new dimensions of understanding plant cell wall dynamics.