TCP21 Antibody

What is TCP21 and what are its known biological functions?

TCP21 (Accession No.: Q9FTA2) is a transcription factor in Arabidopsis thaliana (Mouse-ear cress) that belongs to the TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1 (TCP) family . Also known as Protein CCA1 HIKING EXPEDITION (CHE), it functions in the regulation of plant circadian rhythms and developmental processes . Recent research has uncovered that AtTCP21 also possesses remarkable antifungal properties, demonstrating the ability to inhibit growth of various pathogenic fungi . The protein can penetrate fungal cell walls and membranes where it generates intracellular reactive oxygen species (ROS) and mitochondrial superoxides, creating an environment that represses fungal cell growth and ultimately leads to apoptosis .

What types of TCP21 antibodies are currently available for research applications?

Based on current research, polyclonal antibodies against TCP21 are available for laboratory applications. Specifically:

This polyclonal antibody provides researchers with tools to detect and study TCP21 in plant systems, particularly in Arabidopsis thaliana models .

How can researchers validate the specificity of TCP21 antibodies?

Validating antibody specificity is critical for reliable experimental results. For TCP21 antibodies, researchers should implement multiple validation approaches:

• Positive and negative controls: Use wild-type Arabidopsis (positive) and TCP21 knockout/knockdown plants (negative) to confirm specificity.

• Western blot analysis: Verify that the antibody detects a protein of the expected molecular weight (~27 kDa for TCP21).

• Preabsorption tests: Pre-incubate the antibody with purified recombinant TCP21 protein before immunostaining to confirm that this blocks detection.

• Cross-reactivity assessment: Test against related TCP family members to ensure specificity within this transcription factor family.

These validation steps ensure that experimental observations genuinely reflect TCP21 biology rather than non-specific interactions .

How can TCP21 antibodies be used to study protein-DNA interactions in transcriptional regulation?

TCP21 antibodies can be instrumental in studying transcriptional regulation through several advanced techniques:

• Chromatin Immunoprecipitation (ChIP): TCP21 antibodies can precipitate chromatin fragments bound by TCP21, allowing identification of DNA sequences regulated by this transcription factor. The protocol requires:

  • Crosslinking proteins to DNA in vivo

  • Fragmenting chromatin

  • Immunoprecipitating with TCP21 antibody

  • Analyzing bound DNA sequences through sequencing or qPCR

• Electrophoretic Mobility Shift Assay (EMSA): Use TCP21 antibodies in supershift assays to confirm the identity of DNA-protein complexes containing TCP21.

• Protein-protein interaction studies: Combine TCP21 antibodies with co-immunoprecipitation to identify transcriptional complexes that include TCP21 and other regulatory proteins .

These approaches provide insights into how TCP21 participates in transcriptional networks regulating circadian rhythms and plant development.

What methodological considerations are important when using TCP21 antibodies to study its antifungal properties?

When investigating TCP21's recently discovered antifungal functions, researchers should consider:

• Immunolocalization studies: Use immunofluorescence with TCP21 antibodies to visualize:

  • TCP21 penetration into fungal cells

  • Co-localization with fungal cell structures

  • Temporal dynamics of TCP21 internalization

• Quantitative analysis: Measure TCP21 binding to fungi and correlate with:

  • ROS generation (measured by DCFH-DA or related probes)

  • Mitochondrial superoxide production (measured by MitoSOX)

  • Apoptotic markers

• Comparative studies: Compare wild-type TCP21 effects with:

  • Mutated versions of TCP21

  • Other TCP family members

  • Known antifungal proteins such as melittin

These approaches should account for the pH-dependent nature of TCP21's antifungal activity, as indicated by recent research findings .

How can researchers use TCP21 antibodies to investigate potential epitope variations?

Analysis of epitope variations requires specialized approaches:

• Epitope mapping: Use overlapping peptide arrays combined with TCP21 antibodies to identify specific binding regions.

• Chimera protein approaches: Design chimera proteins where segments of TCP21 are substituted with corresponding regions from related proteins to identify critical epitope regions. Tools like TCP (Tool for designing Chimera Proteins) can assist in designing appropriate chimeras based on tertiary structure information .

• High-throughput epitope scanning: Technologies like VirScan can be adapted to study TCP21 epitopes across different plant varieties or mutants .

These approaches are particularly valuable when studying TCP21 variants or when developing more specific monoclonal antibodies for future research .

What is the optimal protocol for using TCP21 antibodies in Western blot applications?

For optimal Western blot results with TCP21 antibodies:

Sample Preparation:

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

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% Triton X-100

  • 0.5% sodium deoxycholate

  • Protease inhibitor cocktail

Western Blot Protocol:

• Separate proteins on 12% SDS-PAGE

• Transfer to PVDF membrane (0.45 μm)

• Block with 5% non-fat milk in TBST for 1 hour

• Incubate with TCP21 primary antibody (1:1000 dilution) overnight at 4°C

• Wash 3× with TBST

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

• Wash 3× with TBST

• Develop using ECL substrate and appropriate imaging system

Expected Results:

• TCP21 should appear at approximately 27 kDa

• Consider running positive controls (recombinant TCP21) and negative controls (TCP21 knockout plant extracts) .

How should researchers optimize immunoprecipitation experiments using TCP21 antibodies?

For effective immunoprecipitation of TCP21:

Protocol:

• Prepare plant lysate in a gentle lysis buffer:

  • 20 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1 mM EDTA

  • 1% NP-40

  • Protease inhibitor cocktail

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

• Incubate pre-cleared lysate with TCP21 antibody (2-5 μg) overnight at 4°C

• Add fresh Protein G beads and incubate for 2-3 hours at 4°C

• Wash beads 4× with wash buffer (lysis buffer with reduced detergent)

• Elute proteins by boiling in SDS sample buffer

Optimization Tips:

• Adjust antibody concentration (1-10 μg) based on expression level

• Consider crosslinking antibody to beads to prevent antibody co-elution

• For weak interactions, use gentler detergents or chemical crosslinkers

• Validate results with reverse IP approaches or mass spectrometry .

What techniques can be employed to study TCP21 localization in plant tissues?

To investigate TCP21 localization:

Immunohistochemistry Protocol:

• Fix plant tissues in 4% paraformaldehyde

• Dehydrate and embed in paraffin or optimal cutting temperature compound

• Section tissues (5-10 μm)

• Rehydrate and perform antigen retrieval if necessary

• Block with 5% BSA in PBS with 0.1% Triton X-100

• Incubate with TCP21 antibody (1:100-1:500) overnight at 4°C

• Wash 3× with PBS-T

• Incubate with fluorophore-conjugated secondary antibody

• Counterstain for nuclei (DAPI) and cell walls (calcofluor)

• Mount and visualize using confocal microscopy

Alternative Approaches:

• Tissue clearing techniques for whole-mount immunostaining

• Co-localization studies with markers for specific cellular compartments

• Live-cell imaging using TCP21-fluorescent protein fusions to complement antibody-based approaches .

How can researchers address common issues when working with TCP21 antibodies?

Researchers should always include appropriate controls and consider that TCP21 expression may vary with circadian rhythms, developmental stages, and stress conditions .

How can TCP21 antibodies contribute to understanding plant-pathogen interactions?

The recently discovered antifungal properties of TCP21 open new research avenues:

• Mechanism studies: Use TCP21 antibodies to:

  • Track TCP21 movement from plant to fungal cells

  • Visualize subcellular localization in infected tissues

  • Measure TCP21 accumulation at infection sites

• Comparative analyses:

  • Quantify TCP21 levels across resistant vs. susceptible plant varieties

  • Compare TCP21 interactions with different fungal pathogens

  • Assess TCP21 activity against biotrophic vs. necrotrophic pathogens

• Functional studies:

  • Use TCP21 antibodies to block its function in infection assays

  • Identify mutations affecting TCP21's antifungal properties

  • Investigate whether TCP21's transcription factor and antifungal roles are linked

These approaches can reveal whether TCP21's dual functionality as both a transcription factor and antimicrobial protein represents a novel plant defense mechanism .

What future directions might enhance TCP21 antibody development and applications?

Several advanced approaches could enhance TCP21 research:

• Development of monoclonal antibodies: Creating highly specific monoclonal antibodies against different epitopes of TCP21 would enable more precise studies of protein domains and functions.

• Structure-based antibody design: Using computational approaches similar to those described for antibody loop structure prediction to design antibodies with enhanced specificity for TCP21 protein domains .

• Genetic approaches: Combining antibody-based studies with genetic analyses, similar to the twin and SNP-genotyped individual studies, to understand how genetic variants might influence TCP21 function in different plant varieties .

• Integration with omics technologies: Combining TCP21 antibody-based proteomics with transcriptomics and metabolomics to build comprehensive models of TCP21's roles in plant development and defense.

These approaches would facilitate deeper understanding of TCP21's dual roles as both a transcription factor regulating circadian rhythms and as an antimicrobial protein with potential biotechnological applications .