FH21B Antibody

What is the FH21B protein and why is it significant in plant research?

FH21B (Formin Homology 2 Domain-Containing Protein 21B) is a protein encoded in Arabidopsis thaliana (Mouse-ear cress) with the UniProt identifier P0C5K5. Formins play critical roles in regulating cytoskeletal dynamics, particularly in actin filament nucleation and elongation. These proteins are essential for various cellular processes including cytokinesis, cell polarity establishment, and morphogenesis in plants. Studying FH21B contributes to our understanding of plant cell architecture, development, and responses to environmental stimuli. Effective antibodies against this protein enable visualization and quantification of FH21B in various experimental contexts .

What are the key specifications of the commercially available FH21B Antibody?

The FH21B Antibody (Product Code: CSB-PA632782XA01DOA) is a polyclonal antibody raised in rabbits against recombinant Arabidopsis thaliana FH21B protein. It is supplied in liquid form in a buffer containing 0.03% Proclin 300 as a preservative, 50% glycerol, and 0.01M PBS at pH 7.4. The antibody has been antigen-affinity purified and is of IgG isotype. It has been validated for ELISA and Western Blot applications specifically for detecting FH21B in Arabidopsis thaliana samples .

How does the FH21B Antibody's specificity compare to other antibodies targeting plant cytoskeletal proteins?

Unlike antibodies targeting highly conserved cytoskeletal proteins such as actin or tubulin, the FH21B Antibody demonstrates high specificity for Arabidopsis thaliana FH21B protein with minimal cross-reactivity to other plant species. This specificity is advantageous for researchers focusing exclusively on Arabidopsis models but may limit cross-species studies. The polyclonal nature of this antibody means it recognizes multiple epitopes on the FH21B protein, potentially providing stronger signals than monoclonal alternatives, but with slightly higher batch-to-batch variability. When designing experiments requiring absolute specificity, researchers should consider performing additional validation using knockout/knockdown lines as negative controls .

What are the optimal conditions for using FH21B Antibody in Western Blot applications?

For optimal Western Blot results with the FH21B Antibody, follow this methodological approach:

• Sample preparation: Extract proteins from Arabidopsis tissues using a buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor cocktail.

• Protein separation: Load 20-40μg of total protein on 10% SDS-PAGE gels.

• Transfer: Use PVDF membranes (0.45μm pore size) with wet transfer at 100V for 1 hour.

• Blocking: Block with 5% non-fat dry milk in TBST (TBS + 0.1% Tween-20) for 1 hour at room temperature.

• Primary antibody incubation: Dilute FH21B Antibody 1:1000 to 1:2000 in blocking solution and incubate overnight at 4°C.

• Washing: Wash 3 times with TBST, 5 minutes each.

• Secondary antibody: Use anti-rabbit HRP-conjugated secondary antibody at 1:5000 dilution for 1 hour at room temperature.

• Detection: Develop using enhanced chemiluminescence reagents.

The FH21B protein appears at approximately 89 kDa. Include positive controls (wild-type Arabidopsis) and negative controls (FH21B knockout lines if available) to validate specificity .

How can FH21B Antibody be optimized for immunofluorescence microscopy in plant tissues?

For successful immunofluorescence with FH21B Antibody in plant tissues:

• Fixation: Fix fresh plant tissues in 4% paraformaldehyde in PBS for 2 hours at room temperature.

• Sectioning: Prepare 10-15μm thick sections of fixed tissue or use whole-mount preparations for root tips.

• Permeabilization: Treat sections with 0.2% Triton X-100 in PBS for 15 minutes.

• Cell wall digestion: For better antibody penetration, treat with a cocktail of 1% cellulase and 0.5% macerozyme in PBS for 10-15 minutes.

• Blocking: Block with 3% BSA and 0.1% Triton X-100 in PBS for 1 hour.

• Primary antibody: Dilute FH21B Antibody 1:100 to 1:200 in blocking solution and incubate overnight at 4°C.

• Washing: Wash 3 times with PBS containing 0.1% Triton X-100.

• Secondary antibody: Apply fluorophore-conjugated anti-rabbit secondary antibody (1:500) for 2 hours at room temperature.

• Counterstaining: Use DAPI (1μg/ml) for nuclear staining and phalloidin for F-actin visualization.

• Mounting: Mount in anti-fade mounting medium.

Image using confocal microscopy with appropriate excitation/emission settings. For co-localization studies, consider dual labeling with markers for actin filaments or other cytoskeletal components .

What methods can be used to validate the specificity of FH21B Antibody in experimental systems?

To rigorously validate FH21B Antibody specificity:

• Genetic validation:

  • Test on FH21B knockout or knockdown Arabidopsis lines

  • Compare with overexpression lines showing increased signal intensity

• Molecular validation:

  • Pre-absorption test: Pre-incubate antibody with purified recombinant FH21B protein before immunoassays; signal should be significantly reduced

  • Peptide competition assay: Co-incubate with the immunizing peptide/protein

• Technical validation:

  • Compare multiple detection methods (Western blot, immunofluorescence, ELISA)

  • Test reactivity against related formin family proteins to assess cross-reactivity

  • Perform immunoprecipitation followed by mass spectrometry

• Bioinformatic validation:

  • Confirm that molecular weight of detected protein matches predicted size (approximately 89 kDa)

  • Verify subcellular localization patterns match known/predicted distribution of FH21B

Comprehensive validation is especially important for polyclonal antibodies to ensure reproducibility across experiments and antibody batches .

What are common technical challenges when using FH21B Antibody in Western blots and how can they be resolved?

When troubleshooting Western blots with FH21B Antibody, consider these common issues and solutions:

For Arabidopsis samples, protein extraction can be particularly challenging due to cell wall components and proteases. Using a PVPP-containing extraction buffer can reduce interference from phenolic compounds that may affect antibody binding .

How can researchers optimize storage and handling of FH21B Antibody to maintain long-term reactivity?

For optimal maintenance of FH21B Antibody reactivity:

• Storage recommendations:

  • Store at -20°C or preferably -80°C for long-term stability

  • Avoid repeated freeze-thaw cycles by preparing small single-use aliquots (10-20μl)

  • Store in the original buffer containing 50% glycerol which prevents freeze damage

• Handling protocols:

  • Thaw aliquots on ice and return to -20°C immediately after use

  • Never vortex antibody solutions; mix by gentle inversion or flicking

  • Centrifuge briefly before opening tubes to collect solution at the bottom

  • Use sterile technique when handling to prevent microbial contamination

• Stability monitoring:

  • Include internal controls in each experiment to monitor antibody performance over time

  • Document lot numbers, dilutions, and signal intensity to track potential degradation

  • Consider preparing a standard curve with known quantities of recombinant FH21B for quantitative applications

• Buffer considerations:

  • The antibody is supplied in buffer containing 0.03% Proclin 300 as preservative

  • For applications requiring different buffers, minimize dialysis time to prevent activity loss

  • If diluting, use BSA (0.1-1%) as a stabilizing protein carrier

Following these guidelines can extend the functional lifetime of FH21B Antibody from several months to potentially years, ensuring experimental reproducibility and minimizing waste .

How can FH21B Antibody be utilized in co-immunoprecipitation studies to identify novel interacting proteins?

For successful co-immunoprecipitation (Co-IP) studies with FH21B Antibody:

• Sample preparation:

  • Harvest fresh Arabidopsis tissue (preferably 2-3g)

  • Homogenize in non-denaturing lysis buffer (50mM Tris-HCl pH 7.5, 150mM NaCl, 0.5% NP-40, 1mM EDTA) with protease/phosphatase inhibitors

  • Clarify lysate by centrifugation (14,000g, 10 minutes, 4°C)

• Antibody binding:

  • Pre-clear lysate with Protein A/G beads (1 hour, 4°C)

  • Conjugate FH21B Antibody to Protein A/G beads or magnetic beads (2-5μg antibody per 500μg total protein)

  • For covalent coupling, use cross-linking reagents like BS3 or DSS to prevent antibody co-elution

• Immunoprecipitation:

  • Incubate pre-cleared lysate with antibody-conjugated beads overnight at 4°C with gentle rotation

  • Wash 4-5 times with wash buffer (lysis buffer with reduced detergent)

  • Elute protein complexes with low pH buffer or SDS sample buffer

• Analysis methods:

  • Direct analysis by SDS-PAGE followed by silver staining and mass spectrometry

  • Western blot for suspected interacting partners

  • For transient interactions, consider chemical crosslinking before lysis

• Controls:

  • Input control (pre-IP lysate)

  • IgG control (non-specific rabbit IgG)

  • FH21B-knockout plant samples as negative control

This approach has potential to identify novel cytoskeletal regulators, signaling molecules, or other proteins that interact with FH21B in response to developmental cues or environmental stimuli. Quantitative proteomics can further elucidate the dynamic interactome of FH21B under different conditions .

What are the methodological considerations when using FH21B Antibody for chromatin immunoprecipitation (ChIP) studies?

While primarily used for cytoskeletal research, FH21B Antibody can potentially be adapted for ChIP studies if investigating nuclear functions or chromatin associations:

• Cross-linking optimization:

  • For plant tissues, use 1% formaldehyde for 10-15 minutes under vacuum

  • Quench with 125mM glycine for 5 minutes

  • Consider dual crosslinking with DSG or EGS followed by formaldehyde for protein-protein interactions

• Chromatin preparation:

  • Isolate nuclei using plant-specific buffers containing 0.25M sucrose, 10mM Tris-HCl pH 8.0, 10mM MgCl₂, 1% Triton X-100

  • Sonicate to achieve DNA fragments of 200-500bp (optimize cycles for plant tissues)

  • Confirm fragmentation by agarose gel electrophoresis

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Incubate 2-5μg FH21B Antibody with chromatin overnight at 4°C

  • Include appropriate controls: input DNA, non-specific IgG, and no-antibody controls

• Washing and elution:

  • Use stringent wash buffers with increasing salt concentrations

  • Elute DNA-protein complexes with elution buffer (1% SDS, 0.1M NaHCO₃)

  • Reverse crosslinks (65°C overnight) and purify DNA

• Analysis:

  • qPCR for candidate regions

  • ChIP-seq for genome-wide binding profile

  • Integrate with RNA-seq data to correlate binding with gene expression

• Validation strategies:

  • Confirm with secondary antibody against FH21B

  • Use tagged-FH21B transgenic lines for validation with anti-tag antibodies

This approach can reveal potential non-canonical functions of FH21B in transcriptional regulation or chromatin organization, though careful validation is essential given that formins are primarily cytoskeletal proteins .

How can FH21B Antibody be integrated into super-resolution microscopy studies of plant cytoskeleton dynamics?

To leverage FH21B Antibody in super-resolution microscopy studies:

• Sample preparation for different super-resolution techniques:

  • STED (Stimulated Emission Depletion):

    • Use fluorophores with good photostability (Alexa Fluor 647 or ATTO dyes)

    • Optimal primary antibody dilution: 1:50 to 1:100

    • Thin sections (<10μm) for best resolution

  • STORM/PALM:

    • Use photoswitchable fluorophores (Alexa Fluor 647, mEos)

    • Higher antibody concentration (1:50) to ensure dense labeling

    • Imaging buffer containing oxygen scavenging system and thiol

  • SIM (Structured Illumination Microscopy):

    • Standard immunofluorescence protocol with bright, photostable dyes

    • High signal-to-noise ratio is critical

• Co-visualization strategies:

  • Sequential labeling with primary antibodies from different species

  • For dual-color STORM, use activator-reporter dye pairs

  • DNA-PAINT approach for multi-color super-resolution imaging

• Live-cell adaptation considerations:

  • Consider creating nanobody derivatives of FH21B Antibody for live-cell applications

  • Develop FH21B-fluorescent protein fusions as alternative approach

  • Use microinjection of labeled Fab fragments for short-term dynamics

• Image analysis:

  • Specialized software (ThunderSTORM, Imaris, etc.) for reconstruction and analysis

  • Quantification of nanoscale distribution patterns

  • Correlation with other cytoskeletal elements using mathematical modeling

• Technical considerations:

  • Drift correction using fiducial markers

  • System calibration with DNA origami standards

  • Sample mounting with minimal autofluorescence

This approach can reveal previously unresolvable details of FH21B localization at actin nucleation sites, providing insights into the nanoscale organization of plant cytoskeletal networks and how FH21B contributes to their dynamics .

How should researchers quantitatively analyze Western blot data when studying FH21B expression levels across different plant tissues or conditions?

For rigorous quantitative analysis of FH21B expression via Western blot:

• Experimental design considerations:

  • Include biological replicates (minimum n=3)

  • Load equal amounts of total protein (verify with Ponceau S staining)

  • Include housekeeping protein controls (e.g., ACTIN, TUBULIN, GAPDH) on same membrane

  • Create a standard curve using recombinant FH21B if absolute quantification is needed

• Image acquisition:

  • Capture images within linear detection range (avoid saturation)

  • Use 16-bit TIFF format to preserve dynamic range

  • Include multiple exposure times to ensure optimum detection

• Quantification methodology:

  • Use densitometry software (ImageJ, Image Lab, etc.)

  • Measure integrated density within identical-sized regions for each band

  • Subtract local background from each measurement

  • Normalize to loading controls using ratio method:

    • FH21B relative expression = (FH21B density) / (Housekeeping protein density)

• Statistical analysis:

  • Apply appropriate statistical tests based on experimental design

  • For multiple tissue comparisons, use ANOVA with post-hoc tests

  • For treatment vs. control, t-tests or non-parametric alternatives

  • Present data as mean ± SD or SEM with significance levels

• Data visualization:

  • Create bar graphs showing relative expression across samples

  • Include representative blot images with molecular weight markers

  • Consider heatmaps for complex multi-tissue/condition experiments

This methodological approach ensures reproducible quantification of FH21B expression patterns, enabling meaningful comparisons across different experimental conditions or developmental stages .

What are the considerations when interpreting apparent contradictions between FH21B antibody results and gene expression data?

When encountering discrepancies between protein detection (using FH21B Antibody) and gene expression data (RNA-seq, qRT-PCR):

• Biological explanations:

  • Post-transcriptional regulation: mRNA levels may not correlate with protein abundance due to:

    • Differential translation efficiency

    • miRNA-mediated repression

    • RNA stability differences

  • Post-translational modifications affecting antibody recognition

  • Protein stability and turnover rates differing from mRNA dynamics

  • Tissue-specific or subcellular compartmentalization affecting extraction efficiency

• Technical considerations:

  • Antibody specificity: Verify the antibody recognizes the correct isoform(s)

  • Extraction methods: Different protocols may extract proteins with varying efficiency

  • Detection sensitivity: Western blotting may have different detection limits than qPCR

  • Temporal dynamics: mRNA changes often precede protein changes

• Resolution strategies:

  • Time-course experiments to capture temporal relationship between mRNA and protein

  • Polysome profiling to assess translation efficiency

  • Proteasome inhibitor treatments to assess protein turnover

  • Alternative detection methods (mass spectrometry, immunofluorescence)

  • Use of transgenic reporters (GFP-FH21B) as complementary approach

• Integrated analysis approach:

  • Correlate protein data with absolute transcript quantification

  • Apply mathematical models accounting for synthesis and degradation rates

  • Consider protein-protein interactions that might mask epitope recognition

These considerations help researchers interpret complex data patterns and develop hypotheses regarding the regulatory mechanisms controlling FH21B expression and function in different biological contexts .

How can researchers differentiate between FH21B and other formin family members when analyzing experimental results?

Distinguishing FH21B from other plant formin family members requires a multi-faceted approach:

• Antibody-based differentiation:

  • Epitope mapping: Determine the specific regions recognized by the FH21B Antibody

  • Cross-reactivity testing: Evaluate antibody against recombinant proteins of related formins

  • Competitive binding assays: Use specific peptides to block binding to related formins

  • Western blot analysis: Compare molecular weight patterns (FH21B ~89 kDa)

• Genetic approaches:

  • Use Arabidopsis knockout/knockdown lines for each formin as controls

  • Create transgenic lines with epitope-tagged versions of different formins

  • CRISPR-Cas9 generated mutations in specific domains

• Bioinformatic analysis:

  • Multiple sequence alignment to identify unique regions in FH21B

  • Domain structure analysis: FH1, FH2, and other domains may differ between family members

  • Phylogenetic analysis to determine evolutionary relationships

• Expression pattern analysis:

  • Tissue-specific expression profiles may differ between formin family members

  • Subcellular localization patterns (some formins may be membrane-associated while others cytoplasmic)

  • Differential responsiveness to environmental stimuli or developmental cues

• Functional differentiation:

  • In vitro actin polymerization assays with purified proteins

  • Microtubule binding/bundling assays

  • Protein-protein interaction profiles

By combining these approaches, researchers can confidently attribute experimental observations to FH21B specifically, rather than to related formin family members. This distinction is crucial for accurate functional characterization and avoiding misattribution of biological activities .