BIK1 Antibody

What is BIK1 and why is it important in plant research?

BIK1 (BOTRYTIS-INDUCED KINASE1) is a receptor-like cytoplasmic kinase that functions as a central regulator during plant immunity in Arabidopsis thaliana and other plant species. It plays a vital role in protecting plants from widespread pathogens in the environment as part of the plant immune system . The importance of BIK1 stems from its position as a critical signaling node that integrates multiple immune pathways, making BIK1 antibodies essential tools for studying plant immune responses, particularly pattern-triggered immunity (PTI) .

What types of BIK1 antibodies are available for research?

Researchers typically use polyclonal or monoclonal antibodies raised against either full-length BIK1 protein or specific peptide sequences. Anti-HA antibodies are commonly used to detect HA-tagged BIK1 in transgenic systems where BIK1-HA constructs are expressed . Phospho-specific antibodies that recognize activated (phosphorylated) forms of BIK1 are also available for studying BIK1 activation status in response to pathogen-associated molecular patterns (PAMPs) . These different antibody types serve distinct research purposes ranging from basic detection to studying specific post-translational modifications.

How can I confirm the specificity of my BIK1 antibody?

To confirm BIK1 antibody specificity, implement these methodological approaches:

• Include appropriate positive controls (purified recombinant BIK1 protein or extracts from plants overexpressing BIK1) and negative controls (bik1 mutant plants) in immunoblot analyses.

• Perform peptide competition assays where the antibody is pre-incubated with the immunizing peptide before immunoblotting.

• Compare band patterns between wild-type and bik1 mutant tissues to confirm absence of the specific band in mutant samples.

• Validate antibody specificity across multiple applications (immunoblotting, immunoprecipitation, and immunolocalization) to ensure consistent detection patterns .

What is the optimal protein extraction method for BIK1 detection in plant tissues?

For optimal BIK1 detection, use a protein extraction buffer containing:

• 50 mM Tris-HCl (pH 7.5)

• 150 mM NaCl

• 10% glycerol

• 1% Triton X-100

• 1 mM EDTA

• 1 mM DTT

• Protease inhibitor cocktail

• Phosphatase inhibitors (10 mM NaF, 1 mM Na₃VO₄)

Critical methodological considerations include:

• Rapid tissue harvesting and flash-freezing in liquid nitrogen to prevent protein degradation

• Maintaining samples at 4°C throughout extraction

• Including specific phosphatase inhibitors when studying BIK1 phosphorylation status

• Using appropriate detergent concentrations to solubilize membrane-associated BIK1

• Centrifuging extracts at high speed (>14,000 × g) to remove cell debris

This approach ensures preservation of both BIK1 protein integrity and its post-translational modifications for accurate immunodetection.

What are the recommended protocols for BIK1 immunoprecipitation?

For successful BIK1 immunoprecipitation:

• Extract proteins from 2-3 g of plant tissue using the buffer described in question 2.1

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

• Incubate pre-cleared lysate with 2-5 μg of BIK1 antibody overnight at 4°C with gentle rotation

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

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

• Elute bound proteins by boiling in SDS-PAGE sample buffer

For co-immunoprecipitation studies investigating BIK1 interactions with partners like WRKY transcription factors or PRRs, use milder wash conditions to preserve protein-protein interactions . This protocol enables the isolation of BIK1 complexes while maintaining their native interactions.

How should I optimize immunoblotting conditions for detecting BIK1?

For optimal BIK1 detection via immunoblotting:

• Transfer proteins to PVDF membranes (preferred over nitrocellulose for phosphorylated BIK1)

• Block membranes with 5% non-fat dry milk in TBST (for general BIK1 detection) or 5% BSA in TBST (for phospho-BIK1 detection)

• Use primary antibody at 1:1000-1:2000 dilution and incubate overnight at 4°C

• Apply secondary antibody at 1:5000-1:10000 dilution for 1-2 hours at room temperature

• Include appropriate loading controls (Coomassie brilliant blue staining or non-specific bands)

• For detecting phosphorylated BIK1, include phosphatase inhibitors throughout the procedure

The molecular weight of BIK1 is approximately 42-45 kDa, but post-translational modifications like ubiquitination can increase its apparent molecular weight by approximately 8 kDa or more .

How can I detect ubiquitinated forms of BIK1 using antibodies?

To detect ubiquitinated BIK1:

• Perform protein extraction with buffer containing 50 μM MG132 and 10 mM N-ethylmaleimide to inhibit proteasome activity and deubiquitinating enzymes, respectively

• Immunoprecipitate BIK1 using anti-BIK1 antibodies

• Perform immunoblotting with anti-ubiquitin antibodies to detect ubiquitinated forms

• For specific ubiquitination types, use antibodies that recognize K48-linked (degradative) or K63-linked (non-degradative) polyubiquitin chains

Expected results include detection of mono- and polyubiquitinated BIK1 forms appearing as higher molecular weight bands or smears above the main BIK1 band . Different E3 ligases generate distinct ubiquitination patterns: PUB25/26 and PUB4 primarily mediate polyubiquitination for degradation, while RHA3A/B and RGLG1/2 catalyze monoubiquitination for non-degradative signaling .

What methods can I use to detect phosphorylated BIK1?

For detecting phosphorylated BIK1:

• Use phospho-specific antibodies targeting known BIK1 phosphorylation sites

• Alternatively, perform immunoprecipitation with anti-BIK1 antibodies followed by immunoblotting with anti-phosphothreonine or anti-phosphoserine antibodies

• Include lambda phosphatase treatment as a negative control to confirm phosphorylation specificity

• Use the phosphorylation-dependent mobility shift of BIK1 in SDS-PAGE (phosphorylated BIK1 migrates more slowly)

Activation of BIK1 upon PAMP perception (e.g., flg22, elf18, chitin) results in phosphorylation that can be detected through these methods . Phosphorylation status is crucial for BIK1 function and stability, as phosphorylated BIK1 is protected from degradation by certain E3 ubiquitin ligases like PUB25/26 .

How can I detect S-nitrosylated BIK1 in plant samples?

To detect S-nitrosylated BIK1:

• Perform the biotin switch technique:
  a. Block free thiols with methylmethanethiosulfonate (MMTS)
  b. Reduce S-nitrosylated cysteines with ascorbate
  c. Label newly exposed thiols with biotin-HPDP
  d. Purify biotinylated proteins using streptavidin beads
  e. Detect BIK1 by immunoblotting with anti-BIK1 antibodies

• Alternatively, treat samples with GSNO (S-nitrosoglutathione) as a positive control for S-nitrosylation

Research has shown that BIK1 is S-nitrosylated in response to GSNO treatment in vivo, and this modification is important for BIK1 phosphorylation during pattern-triggered immunity . The specific cysteine residues involved in S-nitrosylation can be identified through site-directed mutagenesis followed by the biotin switch assay.

How can I study BIK1 subcellular localization using antibodies?

For immunolocalization of BIK1:

• Fix plant tissues in 4% paraformaldehyde

• Embed in paraffin or prepare for cryosectioning

• Perform antigen retrieval if necessary

• Block with 5% BSA or normal serum

• Incubate with primary anti-BIK1 antibody (1:100-1:500 dilution)

• Apply fluorophore-conjugated secondary antibody

• Counterstain nuclei with DAPI

• Image using confocal microscopy

This approach has revealed that BIK1 localizes to both the plasma membrane and the nucleus . The nuclear localization of BIK1 is particularly important for its role in regulating defense hormone expression through interaction with WRKY transcription factors, demonstrating a direct link between pattern recognition receptor signaling and transcriptional reprogramming .

What approaches can I use to study the BIK1 interactome?

To study the BIK1 interactome:

• Perform co-immunoprecipitation with anti-BIK1 antibodies followed by mass spectrometry analysis

• Use proximity-dependent biotin identification (BioID) with BIK1-BioID fusion proteins

• Implement split-luciferase complementation assays to validate specific interactions in Nicotiana benthamiana

• Conduct yeast two-hybrid screens using BIK1 as bait

These methods have identified various BIK1-interacting proteins including:

• Pattern recognition receptors (FLS2, EFR, PEPR1/2)

• Co-receptors (BAK1)

• E3 ubiquitin ligases (PUB25/26, RHA3A/B, RGLG1/2, PUB4)

• MAP kinases (MAP4K3/4)

• Transcription factors (WRKY)

The specific choice of method depends on whether you're investigating stable or transient interactions, and whether the interaction occurs at the membrane, cytoplasm, or nucleus.

How can I monitor BIK1 dynamics during immune responses?

To monitor BIK1 dynamics during immune responses:

• Perform time-course experiments after PAMP treatment (flg22, elf18, chitin)

• Collect samples at multiple time points (0, 5, 15, 30, 60 minutes, etc.)

• Extract proteins and analyze by immunoblotting with anti-BIK1 and anti-phospho-BIK1 antibodies

• Quantify band intensities using densitometry software

• For live-cell imaging, use BIK1-GFP fusion proteins and monitor localization changes

This approach reveals the rapid phosphorylation of BIK1 upon PAMP perception, followed by its dissociation from the receptor complex and subsequent signaling events . The dynamics of BIK1 activation, relocalization, and eventual attenuation provide crucial insights into the temporal regulation of plant immune responses.

Why might I be unable to detect BIK1 in my plant samples?

Several methodological issues could prevent BIK1 detection:

• Inappropriate extraction buffer: BIK1 associates with membranes, requiring detergent for solubilization

• Protein degradation: Insufficient protease inhibitors or sample heating

• Low BIK1 abundance: Consider concentration through immunoprecipitation

• Antibody specificity: Verify antibody effectiveness with positive controls

• Post-translational modifications: Modified BIK1 may migrate differently or epitopes might be masked

• Tissue specificity: BIK1 expression varies across tissues and developmental stages

To troubleshoot, first validate the antibody using recombinant BIK1 or BIK1-overexpressing plants, then optimize extraction conditions to preserve BIK1 integrity .

How can I differentiate between different post-translationally modified forms of BIK1?

To differentiate between modified BIK1 forms:

• Use Phos-tag™ SDS-PAGE for enhanced separation of phosphorylated forms

• Employ 2D gel electrophoresis (isoelectric focusing followed by SDS-PAGE)

• Treat samples with specific enzymes:

  • Lambda phosphatase to remove phosphorylation

  • Deubiquitinating enzymes to remove ubiquitination

  • Reducing agents to disrupt S-nitrosylation

• Compare migration patterns before and after treatments

• Use modification-specific antibodies when available

This approach helps distinguish between:

• Unmodified BIK1 (~42-45 kDa)

• Phosphorylated BIK1 (slightly higher molecular weight with mobility shift)

• Monoubiquitinated BIK1 (~8 kDa larger than unmodified BIK1)

• Polyubiquitinated BIK1 (high molecular weight smear)

What controls should I include when studying BIK1 phosphorylation status?

Essential controls for BIK1 phosphorylation studies include:

• Positive controls:

  • Wild-type plants treated with flg22/elf18 to induce BIK1 phosphorylation

  • Constitutively active BIK1 phosphomimetic mutants

• Negative controls:

  • Untreated samples

  • bik1 mutant plants

  • BIK1 kinase-dead mutants

  • Lambda phosphatase-treated samples

• Additional controls:

  • BAK1 phosphorylation status (upstream of BIK1)

  • MAPK activation (downstream of BIK1)

  • ROS burst assays to correlate phosphorylation with functional outputs

These controls help distinguish specific BIK1 phosphorylation events from background signals and confirm the biological relevance of observed phosphorylation patterns .

How can I study the differential roles of nuclear versus cytoplasmic BIK1?

To investigate compartment-specific BIK1 functions:

• Perform subcellular fractionation to separate nuclear and cytoplasmic/membrane fractions

• Immunoblot fractions with anti-BIK1 antibodies

• Include fraction-specific markers (histone H3 for nuclear, plasma membrane ATPase for membrane)

• Use nuclear export/import inhibitors to block BIK1 translocation

• Generate BIK1 variants with mutated nuclear localization/export signals

Research has shown that nuclear BIK1 interacts with WRKY transcription factors to regulate defense hormone expression, particularly jasmonic acid (JA) and salicylic acid (SA), while cytoplasmic/membrane-associated BIK1 mediates immediate early immune responses like the ROS burst . This compartmentalization represents an important regulatory mechanism in plant immunity.

What methodological approaches can I use to study the relationship between BIK1 phosphorylation and ubiquitination?

To investigate the interplay between BIK1 phosphorylation and ubiquitination:

• Generate phosphomimetic (S/T→D/E) and phospho-dead (S/T→A) BIK1 mutants

• Express these variants in plant systems

• Perform in vivo ubiquitination assays

• Conduct cycloheximide chase experiments to measure protein stability

• Implement in vitro reconstitution assays with purified components

This methodological approach has revealed that PUB25/26 preferentially target hypophosphorylated BIK1 for degradation, while phosphorylated BIK1 is protected. In contrast, other E3 ligases like RHA3A/B mediate monoubiquitination of activated BIK1 . The integration of phosphorylation and ubiquitination creates a sophisticated regulatory network controlling BIK1 homeostasis during immune responses.

How can I investigate the role of BIK1 in cross-talk between different immune signaling pathways?

To study BIK1's role in immune pathway cross-talk:

• Perform co-immunoprecipitation with BIK1 antibodies after treatment with various elicitors:

  • Bacterial: flg22, elf18

  • Fungal: chitin

  • Endogenous: AtPep1

  • Hormonal: methyl jasmonate, salicylic acid

• Compare phosphorylation, ubiquitination, and S-nitrosylation patterns across treatments

• Employ genetic approaches by crossing bik1 mutants with mutants in other pathways:

  • map4k3 map4k4

  • Hormone signaling mutants

  • Other PRR pathway mutants

Studies have shown that double mutants like map4k3 map4k4 and map4k4-1 bik1 exhibit more severe phenotypes than single mutants, indicating functional relationships between these components . The map4k4-1 bik1 double mutant shows stronger autoimmune responses with higher upregulation of PR1 expression, demonstrating BIK1's integrative role in immune regulation .

How can I use BIK1 antibodies to study protein complexes at the plasma membrane?

For studying membrane-localized BIK1 complexes:

• Perform blue-native PAGE after mild detergent solubilization

• Implement single-molecule pull-down (SiMPull) assays

• Use in situ proximity ligation assays on plant tissues

• Employ super-resolution microscopy techniques like STORM or PALM

• Conduct quantitative co-localization analysis using confocal microscopy

These approaches help visualize and quantify BIK1-containing complexes at the plasma membrane, including associations with pattern recognition receptors (PRRs) and their co-receptors before and after PAMP perception . The dynamic assembly and disassembly of these complexes is critical for proper immune signal transduction.

What techniques can I use to identify specific BIK1 ubiquitination sites?

To identify BIK1 ubiquitination sites:

• Perform immunoprecipitation of BIK1 followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

• Look for peptides with a characteristic GG remnant (from ubiquitin) on lysine residues

• Generate lysine-to-arginine point mutants at candidate sites

• Conduct in vivo and in vitro ubiquitination assays with wild-type and mutant BIK1

• Use ubiquitin remnant motif antibodies for enrichment of ubiquitinated peptides before MS analysis

Mass spectrometry analysis has identified specific ubiquitination sites on BIK1 that are targeted by different E3 ligases, but there may be additional sites that remain undiscovered, particularly those on activated versus non-activated BIK1 . The identification of these sites is crucial for understanding how different ubiquitination events specifically regulate BIK1 function.

How can I apply quantitative approaches to study BIK1 turnover rates during immunity?

For quantitative analysis of BIK1 dynamics:

• Implement SILAC (Stable Isotope Labeling with Amino acids in Cell culture) or TMT (Tandem Mass Tag) labeling for quantitative proteomics

• Perform pulse-chase experiments with inducible BIK1 expression systems

• Use cycloheximide chase assays with immunoblotting at multiple time points

• Apply mathematical modeling to determine degradation rate constants

• Employ fluorescence recovery after photobleaching (FRAP) with BIK1-FP fusions

These approaches provide quantitative measurements of BIK1 turnover rates under different conditions, revealing how post-translational modifications and E3 ligase activities dynamically regulate BIK1 homeostasis . The balance between BIK1 synthesis, activation, and degradation is critical for proper immune response amplitude and duration.

Table 1: BIK1 Post-Translational Modifications and Regulatory Functions

Table 2: Comparative Analysis of BIK1 Antibody Applications in Research