ABCI21 Antibody

What is ABCI21 and what cellular functions does it perform?

ABCI21 is a member of the ABC transporter family in plants, specifically classified as part of the ABCI subfamily. It functions as a plasma membrane-localized ABC transporter involved in IAA (indole-3-acetic acid) transport in an outward direction. Studies have demonstrated that ABCI21 transports auxin less efficiently than naphthalene acetic acid (NAA), indicating substrate specificity . More significantly, ABCI21 interacts with ABCI19 and ABCI20 to form a protein complex at the endoplasmic reticulum membrane, where they collectively function to modulate cytokinin response during early seedling development. Light regulates the expression of these proteins through the transcription factor ELONGATED HYPOCOTYL 5 (HY5) .

What is the specificity profile of commercial ABCI21 antibodies?

Commercial ABCI21 antibodies show varying specificity profiles depending on the immunogen used for production. Currently available antibodies demonstrate reactivity with Arabidopsis thaliana, while some cross-react with related species like Brassica rapa and Brassica napus . This cross-reactivity reflects the conservation of ABCI21 protein structure across these plant families. When selecting an antibody for your research, consider whether you need species-specific detection or cross-reactivity with multiple plant species, as this will impact experimental design and interpretation.

What are the recommended storage conditions for ABCI21 antibodies?

ABCI21 antibodies are typically supplied in lyophilized form and require careful storage to maintain functionality. Storage recommendations include:

• Using a manual defrost freezer to avoid temperature fluctuations

• Avoiding repeated freeze-thaw cycles which can degrade antibody performance

• Immediate storage at recommended temperatures upon receipt

• The product is typically shipped at 4°C but should be stored according to manufacturer specifications

Following these guidelines ensures optimal antibody performance and extends shelf-life for research applications.

How should ABCI21 antibodies be used in Western blot applications?

When using ABCI21 antibodies for Western blot applications, follow this optimized protocol:

• Extract total protein using a buffer containing 50 mM Tris-HCl pH 8, 10 mM KCl, 10 mM MgCl₂, 1 mM EDTA, 20% glycerol (v/v), 0.25% Triton X-100 (v/v), with protease inhibitors

• Separate proteins by SDS-PAGE using 10-12% gels for optimal resolution

• Transfer to PVDF or nitrocellulose membranes using standard transfer conditions

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

• Incubate with ABCI21 antibody at 1:1000-1:5000 dilution (optimize for your specific antibody) overnight at 4°C

• Wash 3-5 times with TBST

• Incubate with appropriate HRP-conjugated secondary antibody

• Visualize using chemiluminescence detection

This methodology has been adapted from similar approaches used for ABCI20 antibodies in plant research . When troubleshooting Western blot issues, consider adjusting antibody concentrations, incubation times, or washing stringency.

What methodologies are effective for immunoprecipitation with ABCI21 antibodies?

For effective immunoprecipitation of ABCI21 and associated proteins:

• Extract total proteins from plant tissues using extraction buffer (50 mM Tris-HCl pH 8, 10 mM KCl, 10 mM MgCl₂, 1 mM EDTA, 20% Glycerol, 0.25% Triton X-100 with protease inhibitors)

• Incubate total protein extracts with anti-ABCI21 antibody overnight at 4°C

• Add affinity resin (such as Affi-Gel 15) and incubate overnight at 4°C

• Place the mixture in chromatography columns

• Rinse sequentially with increasing NaCl concentration buffers (100 mM, 200 mM, 500 mM, and 1 M NaCl/Tris-HCl buffers)

• Elute ABCI21 and associated proteins using an elution buffer containing 8 M Urea, 0.1 M NaH₂PO₄ pH 8, 50 mM Tris-HCl pH 8

This protocol has been adapted from successful approaches used with related ABCI proteins and allows for isolation of ABCI21 complexes for downstream analysis .

How can I generate custom antibodies against ABCI21 for specialized applications?

Custom antibody generation against ABCI21 can be approached through peptide antigen design:

• Select a unique, accessible region of the ABCI21 protein (regions between 310-330 amino acids have proven successful for related proteins)

• Synthesize peptide antigens representing this region

• Conjugate peptides to carrier proteins (KLH or BSA)

• Immunize rabbits with multiple boosts (typically 4 immunizations)

• Collect serum and purify antibodies using affinity chromatography with Affi-Gel 15 resin

• Validate antibody specificity using both wild-type and knockout mutant samples

This approach mirrors the successful generation of anti-ABCI20 antibodies, which used the peptide antigen RTEESRVTGDPARMLN (amino acids 311-326) . For ABCI21, identify unique epitopes using sequence alignment and structural prediction tools to ensure specificity.

How can ABCI21 antibodies be used to study protein-protein interactions in the ABC transporter complex?

To investigate protein-protein interactions involving ABCI21:

• Co-immunoprecipitation (Co-IP):

  • Use anti-ABCI21 antibodies to pull down the protein complex

  • Analyze co-precipitated proteins by mass spectrometry or Western blotting with antibodies against suspected interaction partners (especially ABCI19 and ABCI20)

• Proximity labeling:

  • Generate fusion constructs of ABCI21 with biotin ligase (BioID or TurboID)

  • Express in plant cells to biotinylate proteins in close proximity

  • Purify biotinylated proteins and identify by mass spectrometry

• Fluorescence microscopy:

  • Use fluorescently-tagged anti-ABCI21 antibodies in combination with antibodies against other proteins

  • Analyze co-localization using confocal microscopy

Research has demonstrated that ABCI19, ABCI20, and ABCI21 interact with each other to form a large protein complex at the ER membrane, regulating cytokinin response . These techniques can further elucidate the composition and dynamics of this complex.

What are the methodological approaches for studying ABCI21 subcellular localization?

For investigating ABCI21 subcellular localization:

• Immunofluorescence microscopy:

  • Fix plant cells with 4% paraformaldehyde

  • Permeabilize with 0.1% Triton X-100

  • Block with 3% BSA

  • Incubate with anti-ABCI21 primary antibody and fluorescently-labeled secondary antibody

  • Co-stain with organelle markers (BiP:RFP for ER)

  • Analyze using confocal microscopy

• Subcellular fractionation:

  • Fractionate plant cells into membrane and soluble components

  • Further separate membrane fractions (plasma membrane, ER, Golgi, etc.)

  • Analyze fractions by Western blotting with anti-ABCI21 antibody

  • Compare with known organelle marker proteins

• Transient expression systems:

  • Clone ABCI21 in fusion with fluorescent proteins (similar to approaches used for ABCI21:RFP)

  • Express in tobacco leaves via Agrobacterium infiltration

  • Observe localization 48 hours post-infiltration

  • Co-express with organelle markers for co-localization studies

Research has established that ABCI21 forms a complex with ABCI19 and ABCI20 at the ER membrane, challenging earlier assumptions about exclusive plasma membrane localization .

How can ABCI21 antibodies help investigate the role of these transporters in cytokinin response?

To investigate ABCI21's role in cytokinin response:

• Immunoblotting analysis of ABCI21 protein levels:

  • Treat plants with various concentrations of cytokinins (trans-zeatin)

  • Extract proteins at different time points

  • Quantify ABCI21 protein levels via Western blotting

  • Compare with cytokinin response markers

• Chromatin immunoprecipitation (ChIP):

  • Using antibodies against HY5 (transcription factor)

  • Analyze binding to ABCI21 promoter regions containing HY5 binding motifs

  • Correlate with light conditions and cytokinin treatment

• Immunohistochemistry:

  • Use anti-ABCI21 antibodies on tissue sections

  • Compare expression patterns in wild-type vs. cytokinin signaling mutants

  • Analyze developmental regulation in seedlings

Research has established that abci19 abci20 abci21 triple knockout and abci20 abci21 double knockout seedlings exhibit hypersensitive growth retardation in response to exogenous cytokinin, indicating these proteins negatively regulate cytokinin responses . Antibody-based approaches can help elucidate the molecular mechanisms behind this phenotype.

What are common issues when using ABCI21 antibodies and how can they be resolved?

When troubleshooting ABCI21 antibody applications, remember that expression levels vary throughout development, with higher expression in young seedlings and becoming more root-selective at 14 days after sowing .

How should researchers interpret data from ABCI21 knockout studies in relation to antibody specificity?

When interpreting antibody specificity using knockout mutants:

• Western blot analysis:

  • Compare protein extracts from wild-type and abci21 knockout plants

  • A specific antibody should show a band at the expected molecular weight in wild-type samples that is absent in knockout samples

  • Any remaining bands in knockout samples represent non-specific binding

• Immunolocalization validation:

  • Perform parallel immunostaining on wild-type and knockout tissues

  • Specific signals should be absent in the knockout

  • Remaining signals indicate non-specific binding or cross-reactivity

• Quantitative considerations:

  • Evaluate background-to-signal ratio in both genotypes

  • Calculate specificity index (ratio of wild-type to knockout signal intensity)

  • Values >10 generally indicate good specificity

When working with ABCI21, consider using established knockout lines like SALK_064144, which has been verified as a null mutant by RT-PCR . This approach is particularly important because ABCI21 functions redundantly with ABCI19 and ABCI20, and antibody cross-reactivity between these related proteins must be carefully assessed.

What comparative analyses should be performed when studying ABCI21 in relation to ABCI19 and ABCI20?

When analyzing the ABCI19/20/21 protein family:

• Expression pattern analysis:

  • Compare tissue-specific expression using qRT-PCR data

  • Note that ABCI19 transcript levels are much lower than ABCI20 and ABCI21

  • All three genes show primarily root-selective expression at 14 days after sowing

  • Expression data table (relative expression levels):

  Gene	Shoot (3-8 DAS)	Root (3-8 DAS)	Shoot (14 DAS)	Root (14 DAS)
  ABCI19	Low	Low	Very low	Low-moderate
  ABCI20	Moderate	Moderate	Low	High
  ABCI21	Moderate	Moderate	Low	High

• Phenotypic comparison:

  • Single abci21 knockouts may not show strong phenotypes due to redundancy

  • Double knockouts (abci20 abci21) show hypersensitivity to cytokinin

  • Triple knockouts (abci19 abci20 abci21) show similar but not enhanced phenotypes compared to double knockouts

• Protein complex analysis:

  • Use antibodies against all three proteins for reciprocal co-immunoprecipitation

  • Compare binding partners and complex composition

  • Analyze subcellular localization of individual proteins vs. the complex

Research has established that these three ABCI proteins form a complex at the ER membrane and function together to modulate cytokinin response. Their expression is regulated by light through the transcription factor HY5, with ABCI20 and ABCI21 showing light-induced expression in a HY5-dependent manner .

How can ABCI21 antibodies contribute to understanding plant hormone transport mechanisms?

ABCI21 antibodies can advance our understanding of hormone transport through:

• Transporter activity assays:

  • Immunopurify ABCI21 complexes from various developmental stages

  • Reconstitute in liposomes to measure transport activity

  • Compare affinities for different hormones (IAA vs. cytokinins)

  • Correlate with in vivo phenotypes

• Structural studies:

  • Use antibodies to purify native ABCI21 complexes

  • Perform cryo-EM analysis to determine structure

  • Map functional domains and interaction surfaces

  • Design specific inhibitors based on structural information

• Systems biology approaches:

  • Immunoprecipitate ABCI21 complexes from plants under various environmental conditions

  • Identify condition-specific interaction partners

  • Map hormone transport networks and regulatory mechanisms

Current research has established that ABCI21 is involved in IAA transport in an outward direction and also functions in modulating cytokinin response . Future research using ABCI21 antibodies can help reconcile these findings and establish a comprehensive model of how ABC transporters regulate hormone homeostasis in plants.

What methodological innovations might improve ABCI21 antibody applications in research?

Emerging methodologies that could enhance ABCI21 antibody applications include:

• Single-molecule imaging:

  • Develop high-affinity, minimally disruptive antibody fragments (Fabs, nanobodies)

  • Label with photostable fluorophores

  • Track single ABCI21 molecules in living cells

  • Analyze dynamics, clustering, and transport activity

• Proximity-dependent labeling:

  • Engineer ABCI21 fusions with proximity labeling enzymes

  • Use antibodies to purify labeled proteins

  • Map the dynamic ABCI21 interactome at different developmental stages

  • Identify transient interactions during hormone signaling

• Antibody-based biosensors:

  • Develop FRET-based sensors using ABCI21 antibodies

  • Monitor conformational changes during transport

  • Measure hormone binding in real-time

  • Correlate with physiological responses

These innovative approaches could overcome current limitations in studying membrane-bound transporters and provide unprecedented insights into ABCI21 function in hormone transport and signaling.

How might ABCI21 antibodies help resolve contradictions in current research findings?

ABCI21 antibodies can help address several unresolved questions:

• Subcellular localization discrepancy:

  • ABCI21 has been reported as both plasma membrane-localized and ER membrane-localized

  • Use super-resolution microscopy with ABCI21 antibodies

  • Perform immunogold electron microscopy for precise localization

  • Investigate potential trafficking between compartments

• Functional redundancy analysis:

  • Compare protein levels of ABCI19/20/21 in single, double, and triple mutants

  • Investigate compensatory upregulation

  • Correlate protein levels with phenotypic severity

  • Identify unique vs. redundant functions

• Hormone specificity question:

  • ABCI21 is implicated in both auxin transport and cytokinin response

  • Use antibodies to purify native complexes

  • Perform binding assays with different hormones

  • Compare complex composition in response to different hormones

Current research shows that abci21 knockout plants exhibit hypersensitivity to cytokinin but not to other major plant hormones including auxin , which seems contradictory to its reported role in IAA transport . Antibody-based approaches can help resolve this apparent contradiction by elucidating the precise molecular mechanisms linking ABCI21 to these hormone pathways.

What are the best practices for ABCI21 antibody selection and validation?

When selecting and validating ABCI21 antibodies for research:

• Match antibody to application:

  • For Western blotting: Select antibodies raised against denatured epitopes

  • For immunoprecipitation: Choose antibodies recognizing native conformations

  • For immunofluorescence: Test multiple antibodies for specificity in fixed tissues

• Comprehensive validation strategy:

  • Genetic validation: Test on abci21 null mutants (e.g., SALK_064144)

  • Biochemical validation: Peptide competition assays

  • Cross-reactivity assessment: Test on related proteins (ABCI19, ABCI20)

  • Application-specific validation: Optimize conditions for each technique

• Documentation requirements:

  • Record complete antibody information (source, catalog number, lot)

  • Document all validation experiments

  • Include knockout controls in publications

  • Report optimization parameters

Following these best practices ensures reproducible research and enables meaningful comparison of results across different studies investigating ABCI transporters in plant development.

What are the key methodological considerations for applying ABCI21 antibodies in developmental studies?

When using ABCI21 antibodies for developmental research:

• Developmental timing considerations:

  • ABCI21 expression varies throughout development

  • Expression is detected in both shoots and roots in early seedling stages (3-8 days)

  • Expression becomes primarily root-selective by 14 days after sowing

  • Design sampling strategies accordingly

• Tissue-specific analysis:

  • Use tissue-specific extraction methods to enrich for ABCI21

  • Compare with expression patterns from promoter:GUS studies

  • Consider cell-type specific variations in protein levels

• Environmental conditions:

  • ABCI21 expression is regulated by light in a HY5-dependent manner

  • Standardize light conditions when comparing samples

  • Document all growth conditions in detail

These methodological considerations ensure that ABCI21 antibody applications yield physiologically relevant results that can be interpreted in the context of plant development and environmental responses.