ALF4 Antibody

What is ALF4 and why is it significant for plant research?

ALF4 (Aberrant Lateral Root Formation 4) is a protein in Arabidopsis thaliana that functions as an ortholog of human glomulin (GLMN) and plays a critical role in regulating cullin-ring ubiquitin ligases (CRLs). It is particularly significant because it binds to the RBX1 subunit of CRLs and inhibits SCF E3 ligase activity in vitro, thereby influencing auxin and gibberellin signaling pathways. The alf4 mutant exhibits a dramatic reduction in lateral root formation and other auxin-related developmental defects, making it an important target for understanding plant hormone signaling and developmental processes . Antibodies against ALF4 are essential tools for investigating these regulatory mechanisms and visualizing protein localization and interactions.

How does ALF4 function in plant hormone signaling pathways?

ALF4 functions as a regulator of SCF E3 ligases, which are crucial for plant hormone signaling. It interacts with RBX1 and influences the stability of hormone pathway components. Specifically:

• ALF4 stabilizes CUL1 in vivo, influencing the turnover of Aux/IAA proteins (auxin pathway repressors)

• In the absence of ALF4 (alf4 mutant), SCF substrates like IAA17 (auxin pathway) and RGA (gibberellin pathway) accumulate abnormally

• ALF4 inhibits CRL activity by competing with E2 enzymes for binding to RBX1

• This regulation affects the global levels of ubiquitinated proteins in the plant

The interaction between ALF4 and RBX1 has been confirmed through multiple experimental approaches, including yeast two-hybrid assays, co-immunoprecipitation, in vitro pulldown, and bimolecular fluorescence complementation (BiFC) .

What are the key experimental applications for ALF4 antibodies?

ALF4 antibodies serve several crucial functions in plant molecular biology research:

• Protein detection via Western blotting to quantify ALF4 protein levels

• Immunoprecipitation (IP) to study protein-protein interactions with components of the SCF complex

• Immunohistochemistry (IHC) to visualize tissue-specific expression patterns

• Chromatin immunoprecipitation (ChIP) if ALF4 is involved in transcriptional regulation

• Verification of protein knockout in mutant lines

When using antibodies for such applications, validation is essential to ensure specificity through techniques like testing in knockout lines and examining signal in different tissues with known expression patterns .

What are the optimal conditions for using ALF4 antibodies in Western blot experiments?

For optimal Western blot results with ALF4 antibodies, consider the following protocol:

• Sample preparation: Extract total protein from plant tissues using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, and protease inhibitor cocktail

• Protein separation: Use 10% SDS-PAGE gels to achieve optimal separation of ALF4 (~70 kDa)

• Transfer conditions: Transfer proteins to PVDF membranes at 100V for 90 minutes in cold transfer buffer

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

• Primary antibody: Dilute ALF4 antibody 1:1000 in blocking solution and incubate overnight at 4°C

• Detection system: Use HRP-conjugated secondary antibodies and ECL detection system

For particularly challenging samples, consider enriching the target protein via immunoprecipitation before Western blotting .

How can I validate the specificity of an ALF4 antibody?

Antibody validation is crucial for ensuring experimental reliability. For ALF4 antibodies, implement these validation strategies:

• Genetic validation: Test antibody reactivity in alf4 knockout/mutant lines, which should show no signal

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to samples

• Multiple antibody approach: Use antibodies raised against different epitopes of ALF4

• Overexpression validation: Test in samples overexpressing ALF4 (should show increased signal)

• Cross-species validation: Test reactivity against orthologous proteins if studying ALF4 in non-Arabidopsis species

These validation steps are particularly important for polyclonal antibodies, which may have batch-to-batch variation .

What are the best fixation methods for immunolocalization of ALF4 in plant tissues?

For effective immunolocalization of ALF4 in plant tissues:

• Fixation options:

  • For preserved protein structure: 4% paraformaldehyde in PBS for 2-4 hours

  • For better penetration: Add 0.1-0.2% glutaraldehyde to the fixative

  • For whole seedlings: Vacuum infiltration of fixative for 15-20 minutes

• Processing protocol:

  • Wash fixed tissues in PBS (3 × 10 minutes)

  • Dehydrate in ethanol series (30%, 50%, 70%, 90%, 100%)

  • Embed in suitable medium (paraffin or resin)

  • Section at 5-10 μm thickness

• Antigen retrieval:

  • Heat-mediated: Citrate buffer (pH 6.0) at 95°C for 10 minutes

  • Enzymatic: Proteinase K (1-5 μg/ml) for 5-10 minutes

• Antibody incubation:

  • Use 1:100 to 1:500 dilution for primary antibody

  • Incubate overnight at 4°C in humid chamber

  • Use fluorescent secondary antibodies for better quantification

How can ALF4 antibodies be used to investigate protein-protein interactions within the SCF complex?

ALF4 antibodies can be powerful tools for studying protein-protein interactions through these approaches:

• Co-immunoprecipitation (Co-IP):

  • Use ALF4 antibodies to pull down ALF4 protein complexes

  • Analyze co-precipitated proteins by Western blot or mass spectrometry

  • This approach confirmed the interaction between ALF4 and RBX1 in plant extracts

• Proximity ligation assay (PLA):

  • Allows visualization of protein interactions in situ

  • Combine ALF4 antibody with antibodies against suspected interaction partners

  • Quantify interaction signals in different subcellular compartments

• Bimolecular fluorescence complementation (BiFC) validation:

  • Use antibodies to confirm expression levels of fusion proteins

  • Validate BiFC results through immunofluorescence

  • Researchers used BiFC to demonstrate RBX1-ALF4 interaction in vivo

• Chromatin immunoprecipitation (ChIP):

  • If ALF4 associates with chromatin complexes

  • Identify DNA regions associated with ALF4-containing complexes

• Sequential Co-IP:

  • First IP with ALF4 antibody, then a second IP with antibody against another complex component

  • Useful for identifying specific subcomplexes within larger assemblies

What techniques can be used to measure ALF4-mediated inhibition of SCF activity in vitro?

To quantitatively assess ALF4's inhibition of SCF activity, researchers can employ these methodologies:

• In vitro ubiquitination assay:

  • Reconstitute the ubiquitination cascade with purified components:

    • E1 (UBE1/UBA1)

    • E2 (AtUBC8)

    • E3 (SCF complexes containing RBX1)

    • Substrate (e.g., GST-IAA7)

    • Ubiquitin

  • Add varying concentrations of purified ALF4 protein

  • Measure ubiquitination of substrate by Western blot

  • Quantify dose-dependent inhibition by ALF4

• E2 binding competition assay:

  • Use labeled E2 enzyme

  • Measure displacement by ALF4 through:

    • Fluorescence polarization

    • Surface plasmon resonance

    • Microscale thermophoresis (MST)

  • MST experiments showed ALF4 binding to RBX1-CUL1 with a Kd of 346.04 ± 77.05 nM

• RBX1 RING domain activity assay:

  • The RING domain of RBX1 is sufficient to promote ubiquitination

  • Wild-type ALF4 strongly inhibits this activity

  • Mutant versions (ALF4 A484A614 or ALF4 1-532stop) have little effect

  • This assay helps identify critical residues in ALF4 required for inhibition

How can I use ALF4 antibodies to investigate post-translational modifications of ALF4?

To study post-translational modifications (PTMs) of ALF4:

• IP-MS approach:

  • Immunoprecipitate ALF4 using validated antibodies

  • Analyze by mass spectrometry to identify PTMs

  • Compare PTM patterns under different conditions or treatments

• Modification-specific antibodies:

  • Use phospho-specific antibodies if phosphorylation sites are known

  • Combine with phosphatase treatments as controls

  • Western blot analysis comparing treated/untreated samples

• 2D gel electrophoresis:

  • Separate proteins by isoelectric point and molecular weight

  • Detect ALF4 isoforms using ALF4 antibody

  • Shifts in position indicate modifications

• Mobility shift assays:

  • Prepare samples with or without modification-removing enzymes

  • Changes in migration pattern indicate presence of modifications

  • Particularly useful for phosphorylation, ubiquitination, or SUMOylation

• Phos-tag SDS-PAGE:

  • Enhanced separation of phosphorylated proteins

  • Use ALF4 antibody for detection

  • Multiple bands indicate differentially phosphorylated forms

What are common issues when using ALF4 antibodies for immunoprecipitation and how can they be resolved?

Common immunoprecipitation issues and solutions for ALF4 antibodies:

For ALF4 specifically, use a lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA with protease inhibitors, as this has been successful in co-immunoprecipitation experiments with RBX1 .

How can I determine the optimal concentration of ALF4 antibody for different experimental applications?

Determining optimal antibody concentration requires systematic titration:

• Western blotting:

  • Start with 1:1000 dilution

  • Test range from 1:500 to 1:5000

  • Optimal concentration gives specific signal with minimal background

  • For plant samples, include positive control (wild-type) and negative control (alf4 mutant)

• Immunoprecipitation:

  • Begin with 2-5 μg antibody per 500 μg total protein

  • Test range from 1-10 μg

  • Assess efficiency by measuring percentage of target protein depleted from supernatant

• Immunohistochemistry/Immunofluorescence:

  • Start with 1:200 dilution

  • Test range from 1:50 to 1:500

  • Include controls for autofluorescence and secondary antibody specificity

• ChIP:

  • Begin with 5 μg antibody per reaction

  • Test range from 2-10 μg

  • Compare enrichment of target vs. control regions

For all applications, generate a standard curve plotting antibody concentration against signal intensity to identify the concentration that provides maximum specific signal before saturation .

How do I address cross-reactivity when using ALF4 antibodies in non-model plant species?

When using ALF4 antibodies in non-model plant species, follow these steps to address potential cross-reactivity:

• Sequence alignment analysis:

  • Compare ALF4 protein sequences between Arabidopsis and target species

  • Identify conservation level in the epitope region

  • Epitope conservation >70% suggests potential cross-reactivity

• Preliminary validation tests:

  • Western blot analysis using tissues from target species

  • Compare band pattern and molecular weight to predicted protein size

  • Test in tissues with expected high/low expression

• Specificity controls:

  • Pre-absorb antibody with recombinant ALF4 protein or peptide

  • Use genetic knockdowns/overexpression lines if available

  • Include heterologous expression systems as positive controls

• Epitope-specific antibody development:

  • Design peptides based on conserved regions across species

  • Generate new antibodies against these conserved epitopes

  • Validate across multiple species

• Alternative detection methods:

  • Consider using tagged versions of the protein where genetic transformation is possible

  • Use mass spectrometry-based approaches for protein identification

  • Employ RNA-based methods to complement protein studies

How can ALF4 antibodies be used to investigate the role of ALF4 in hormone cross-talk?

ALF4 antibodies can reveal mechanisms of hormone cross-talk through these approaches:

• Co-immunoprecipitation coupled with hormone treatments:

  • Treat plants with different hormones (auxin, gibberellin, etc.)

  • Immunoprecipitate ALF4 and identify interaction partners

  • Compare interactomes across hormone treatments

  • This approach can reveal how ALF4-protein interactions change during hormone responses

• Chromatin immunoprecipitation followed by sequencing (ChIP-seq):

  • If ALF4 associates with transcription factors or chromatin modifiers

  • Map genome-wide binding sites under different hormone treatments

  • Identify genes co-regulated by multiple hormone pathways

• Proximity-dependent labeling:

  • Generate ALF4 fusion with BioID or APEX2

  • Apply hormone treatments

  • Identify proteins in proximity to ALF4 during hormone responses

  • Validate interactions using co-IP with ALF4 antibodies

• Quantitative immunofluorescence:

  • Use ALF4 antibodies to track subcellular localization

  • Apply various hormone treatments

  • Quantify changes in nuclear/cytoplasmic distribution

  • Correlate with hormone-responsive phenotypes

The research has already established that loss of ALF4 affects both auxin (IAA7) and gibberellin (RGA) signaling pathways, suggesting it functions at the intersection of these hormone pathways .

What approaches can be used to study the temporal dynamics of ALF4 protein accumulation during plant development?

To study temporal dynamics of ALF4 protein levels during development:

• Developmental time course analysis:

  • Collect tissues at defined developmental stages

  • Perform quantitative Western blot using ALF4 antibodies

  • Normalize to appropriate loading controls

  • Create developmental expression profiles

• Tissue-specific immunohistochemistry:

  • Section plant tissues at various developmental stages

  • Perform immunostaining with ALF4 antibodies

  • Use confocal microscopy for high-resolution imaging

  • Quantify signal intensity across tissues and developmental time points

• Live imaging with fluorescent protein fusions:

  • Generate ALF4-GFP fusions under native promoter

  • Validate fusion protein function by complementation of alf4 mutant

  • Verify expression pattern matches endogenous protein using ALF4 antibodies

  • Perform live imaging during developmental processes

• Single-cell protein analysis:

  • Isolate protoplasts from different tissues/developmental stages

  • Perform flow cytometry with fluorescently labeled ALF4 antibodies

  • Sort cells based on ALF4 levels for further analysis

• Protein stability assays:

  • Treat samples with cycloheximide to block protein synthesis

  • Collect samples at time intervals

  • Quantify ALF4 protein degradation rate using antibodies

  • Compare stability across developmental stages

How can ALF4 antibodies be combined with proteomics approaches to understand ALF4's role in global protein ubiquitination?

Integrating ALF4 antibodies with proteomics can reveal global effects on the ubiquitin-proteasome system:

• Ubiquitinome analysis:

  • Compare wild-type and alf4 mutants using mass spectrometry

  • Identify differentially ubiquitinated proteins

  • Search results indicate that global ubiquitination levels are reduced in alf4 mutants

  • Verification of key targets with ALF4 antibodies

• Sequential immunoprecipitation approach:

  • First IP: Use anti-ubiquitin antibodies to capture ubiquitinated proteins

  • Second IP: Use ALF4 antibodies to identify ALF4-associated proteins

  • Mass spectrometry identification of proteins in the resulting fraction

  • This reveals proteins that are both ubiquitinated and associated with ALF4

• Proximity-dependent biotinylation:

  • Express ALF4-BioID fusion protein

  • Identify proteins in proximity to ALF4

  • Compare with ubiquitinome data

  • Validate using ALF4 antibodies for co-IP

• In vitro reconstitution systems:

  • Purify components of the ubiquitination machinery

  • Test effects of adding/removing ALF4

  • Use ALF4 antibodies to deplete or block ALF4 function

  • Research has shown that ALF4 inhibits SCF TIR1-mediated ubiquitination in a concentration-dependent manner

• Targeted quantitative proteomics:

  • Develop SRM/MRM assays for key ubiquitin pathway components

  • Monitor changes in abundance in presence/absence of ALF4

  • Correlate with immunoblot data using ALF4 antibodies