At2g33655 Antibody

What is the biological significance of At2g33655/Actin-7 in plant development?

Actin-7 plays a crucial role in plant growth and development, particularly in response to phytohormones like auxin. The ACT7 gene is rapidly and strongly induced by exogenous auxin and is essential for germination, root growth, and callus tissue formation. This particular actin isoform is predominantly expressed in rapidly developing tissues and responds dynamically to external stimuli such as hormonal exposure . The cytoskeletal protein encoded by At2g33655 is involved in directing specific changes in cell morphology and cytoarchitecture, which are fundamental to auxin-mediated developmental processes including cell division, expansion, differentiation, and organ initiation .

How are Anti-Actin-7 antibodies typically generated and characterized?

Anti-Actin-7 antibodies are commonly produced as mouse monoclonal antibodies by immunizing BALB/c mice with purified Arabidopsis thaliana Actin-7 protein . After hybridoma generation, antibodies are typically purified using Protein G affinity chromatography and supplied in PBS containing 0.05% sodium azide as a preservative . Characterization involves testing their reactivity in multiple applications including Western blotting, ELISA, and immunofluorescence. The most commonly used clones include 29G12.G5.G6, 33E8.C11.F5.D1, and 36H8.C12.H10.B6, each potentially offering different epitope recognition properties .

What applications can At2g33655/Actin-7 antibodies be reliably used for?

At2g33655/Actin-7 antibodies have been validated for multiple applications including:

• Western blotting (WB): For detecting Actin-7 in plant tissue homogenates

• Enzyme-linked immunosorbent assay (ELISA): For quantitative analysis

• Immunofluorescence (IF): For visualizing subcellular localization and distribution patterns

When initiating new research, it is advisable to test all three monoclonal antibody clones to determine which is most suitable for the specific experimental setup and application .

How can I rigorously validate the specificity of At2g33655/Actin-7 antibodies?

Thorough validation of antibody specificity is critical for reliable research outcomes. Based on established protocols for antibody validation, researchers should:

• Perform Western blot analyses comparing wild-type Arabidopsis tissues with tissues from act7 knockout mutants

• Compare band patterns produced by multiple anti-Actin-7 antibodies targeting different epitopes

• Verify expected molecular weight (approximately 41-42 kDa for Actin-7)

• Include samples with either overexpressed or silenced Actin-7 to confirm band intensity correlation with protein levels

• Extend exposure times during imaging to ensure no additional cross-reactive bands appear

Studies of other antibodies have demonstrated that commercial antibodies may cross-react with unintended proteins, producing identical banding patterns in both wild-type and knockout tissues . This emphasizes the importance of rigorous validation using genetic controls.

What controls are essential when using At2g33655/Actin-7 antibodies for immunohistochemistry?

Based on empirical evidence from antibody validation studies, the following controls are essential:

Studies have shown that some antibodies may produce identical immunostaining patterns in both wild-type and knockout tissues, suggesting non-specific binding . For instance, in AT1R antibody studies, smooth muscle cells, proximal tubule membranes, and collecting ducts showed positive staining even in tissues completely lacking the target protein .

How can I distinguish between Actin-7 and other actin isoforms in Arabidopsis?

Distinguishing between Actin-7 and other actin isoforms requires careful experimental design:

• Sequence alignment analysis to identify unique regions in Actin-7 compared to other isoforms

• Selection of antibodies raised against isoform-specific epitopes

• Two-dimensional gel electrophoresis followed by Western blotting to separate isoforms

• Validation using tissues from single and multiple actin isoform knockout plants

• Mass spectrometry analysis of immunoprecipitated proteins to confirm identity

The high homology between actin isoforms (often >90% sequence identity) makes specific detection challenging, similar to the difficulties encountered with AT1A and AT1B receptors which share approximately 94% amino acid identity .

What are the optimal parameters for Western blot detection of At2g33655/Actin-7?

Optimizing Western blot conditions for Actin-7 detection requires careful adjustment of several parameters:

For glycosylated forms of Actin-7, multiple bands at higher molecular weights than the predicted 41-42 kDa may be observed .

How should I optimize immunohistochemistry protocols for At2g33655/Actin-7 detection in plant tissues?

For optimal immunohistochemical detection of Actin-7 in plant tissues:

• Fixation: Use 4% paraformaldehyde for 12-24 hours to preserve cytoskeletal structures while maintaining antigen accessibility

• Embedding: Paraffin embedding works well for most plant tissues, though cryo-sectioning may preserve some epitopes better

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) can improve accessibility of some Actin-7 epitopes

• Blocking: 5-10% normal serum (from the species of secondary antibody) with 1% BSA to minimize background

• Primary antibody concentration: Start with 1:100-1:500 dilution and optimize

• Incubation: Overnight at 4°C to maximize specific binding

• Detection system: Amplification systems like tyramide signal amplification may be necessary for low-abundance regions

• Counterstaining: DAPI for nuclei visualization without interfering with cytoskeletal structures

Studies with other antibodies have shown that optimized immunohistochemistry can detect protein in various cellular compartments, including membrane structures and cytoskeletal elements .

What methods can accurately measure binding affinity of At2g33655/Actin-7 antibodies?

Accurate measurement of antibody binding affinity is essential for selecting optimal antibodies:

• Biolayer Interferometry (BLI): This technique can determine dissociation constants (KD) with high precision. Studies on other antibodies have achieved measurements in the picomolar range (e.g., 4.88 pM) .

• Surface Plasmon Resonance (SPR): Provides real-time binding kinetics, measuring both on-rate (kon) and off-rate (koff) constants.

• Enzyme-Linked Immunosorbent Assay (ELISA): Can be used for comparative binding studies.

Key parameters to measure and report:

• Dissociation constant (KD): Lower values indicate stronger binding

• Off-rate constant: Slower dissociation rates (e.g., 10−6/s) indicate stronger antigen-binding stability

• Optimal antigen loading concentration (e.g., 500 nM) to avoid avidity-based interactions

How can I address non-specific binding issues with At2g33655/Actin-7 antibodies?

Non-specific binding is a common challenge with antibodies. Studies have shown that even antibodies believed to be specific can bind to unintended proteins . To address this:

• Increase blocking time and concentration (try 5-10% BSA or milk)

• Add 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

• Include competitive blocking with non-immunized serum

• Perform pre-adsorption with tissue lysates from actin-7 knockout plants

• Test multiple antibody dilutions to find the optimal signal-to-noise ratio

• Increase washing duration and buffer volume between steps

• Consider epitope-specific antibodies that target unique regions of Actin-7

Research has demonstrated that three different antibodies to the same target protein can produce completely different banding patterns with no common bands at the expected molecular weight .

How can I use At2g33655/Actin-7 antibodies to study auxin-induced cytoskeletal remodeling?

To study auxin-induced cytoskeletal remodeling using Actin-7 antibodies:

• Time-course experiments:

  • Treat plant tissues with auxin (e.g., IAA or 2,4-D)

  • Fix tissues at multiple time points (0, 15, 30, 60, 120 minutes)

  • Process for immunofluorescence using validated Actin-7 antibodies

  • Quantify changes in filament organization, density, and orientation

• Co-localization studies:

  • Perform dual immunolabeling with Actin-7 antibodies and antibodies against auxin signaling components

  • Use confocal microscopy to analyze spatial relationships

  • Calculate Pearson's correlation coefficients to quantify co-localization

• Live-cell imaging approaches:

  • Use Actin-7 antibody fragments (Fab) conjugated to fluorophores for live imaging

  • Combine with fluorescently tagged auxin response factors

  • Perform FRET analysis to detect potential direct interactions

This approach builds on knowledge that the ACT7 promoter and protein are rapidly and strongly induced in response to exogenous auxin and that Actin-7 is essential for specific developmental processes .

What strategies can be employed for epitope mapping of At2g33655/Actin-7 antibodies?

Epitope mapping is crucial for understanding antibody specificity and functionality:

• Peptide array analysis:

  • Synthesize overlapping peptides (12-15 amino acids) spanning the Actin-7 sequence

  • Probe arrays with antibodies to identify reactive peptides

  • Confirm binding with competition assays

• Hydrogen-deuterium exchange mass spectrometry:

  • Compare deuterium uptake in Actin-7 alone versus Actin-7-antibody complex

  • Regions with reduced exchange rates indicate antibody binding sites

• Mutagenesis approaches:

  • Generate point mutations in recombinant Actin-7

  • Test antibody binding to identify critical residues

  • Construct deletion mutants to narrow down the epitope region

• X-ray crystallography or cryo-EM:

  • Determine the three-dimensional structure of the antibody-antigen complex

  • Provides atomic-level detail of interaction interfaces

The importance of epitope identification is highlighted by studies showing that antibodies targeting conserved regions (like the S2 region in SARS-CoV-2 studies) can provide broad recognition across variants .

How can I humanize mouse monoclonal At2g33655/Actin-7 antibodies for broader experimental applications?

For researchers seeking to reduce immunogenicity in certain experimental contexts:

• Identify variable regions:

  • Sequence the variable domains of mouse monoclonal antibody heavy and light chains

  • Use tools like IgBLAST to analyze somatic mutations from germline sequences

• Select human framework templates:

  • Identify closest human germline sequences (e.g., IGHV3-23*04 and IGKV4-1 as used in other studies)

  • Consider framework compatibility with CDR conformations

• CDR grafting and framework adaptation:

  • Transfer mouse CDRs to human framework

  • Retain key mouse framework residues that support CDR conformation

  • Generate multiple variants with different degrees of humanization

• Expression and screening:

  • Transiently express humanized variants

  • Test for antigen binding using ELISA and functional assays

  • Select candidates with preserved or improved affinity

• Advanced optimization:

  • Perform affinity maturation if needed

  • Characterize binding kinetics using BLI or SPR

  • Validate in intended applications

Studies with other antibodies have successfully maintained or even improved binding affinity after humanization, with some humanized antibodies achieving KD values as low as 13 pM while maintaining neutralizing capabilities .

How can At2g33655/Actin-7 antibodies be used to study stress responses in plants?

Actin-7 is involved in responses to external stimuli, making its antibodies valuable tools for studying stress responses:

• Design experiments to monitor Actin-7 distribution changes under various stresses:

  • Abiotic stresses (drought, salt, temperature, light)

  • Biotic stresses (pathogen infection, herbivory)

  • Hormone treatments (auxin, ABA, ethylene, jasmonic acid)

• Quantitative approaches:

  • Use image analysis software to quantify changes in Actin-7 filament architecture

  • Measure parameters like filament density, orientation, bundling, and fragmentation

  • Correlate cytoskeletal changes with physiological responses

• Integration with other methodologies:

  • Combine immunolocalization with live cell imaging of fluorescently tagged stress response proteins

  • Perform co-immunoprecipitation followed by mass spectrometry to identify stress-induced Actin-7 binding partners

  • Correlate with transcriptomic and proteomic data to develop comprehensive models

Research has established that Actin-7 is essential for root growth and responds to auxin , suggesting its involvement in adaptive responses to environmental challenges.

What are the considerations for developing multiplexed assays using At2g33655/Actin-7 antibodies?

Developing multiplexed assays requires careful antibody selection and validation:

• Antibody compatibility considerations:

  • Select antibodies raised in different host species to allow simultaneous detection

  • Ensure non-overlapping emission spectra for fluorophore-conjugated antibodies

  • Validate antibodies individually before combining in multiplexed formats

• Multiplexed immunofluorescence protocol optimization:

  • Sequential versus simultaneous antibody incubation

  • Concentration balancing to achieve comparable signal intensities

  • Spectral unmixing for closely overlapping fluorophores

• Advanced multiplexing technologies:

  • Cyclic immunofluorescence with antibody stripping and reprobing

  • Mass cytometry (CyTOF) using metal-conjugated antibodies

  • DNA-barcoded antibodies for highly multiplexed detection

When designing multiplexed assays, thorough validation is essential to ensure antibodies maintain specificity in the more complex detection environment, similar to the validation approaches described for single antibody applications .