HERK1 Antibody

What is HERK1 and why is it important for research?

HERK1 is a receptor-like kinase belonging to the CrRLK1L family with approximately 75% identity and 86% similarity at the amino acid level to ANJEA (ANJ), another member of this family . HERK1 is significant in research because it plays critical roles in cellular signaling, particularly in plant reproductive processes. Studies involving T-DNA insertion lines (such as herk1-1) have demonstrated that HERK1, especially when studied in conjunction with ANJ, affects seed development . For researchers, HERK1 represents an important model for understanding receptor kinase function in signaling cascades and developmental biology.

What types of HERK1 antibodies are available for laboratory use?

While the search results don't specifically enumerate commercial HERK1 antibody types, based on research protocols for similar proteins like ERK1, researchers typically have access to several antibody formats. These include primary antibodies targeting specific HERK1 epitopes, phospho-specific antibodies that recognize activated HERK1, and tagged antibody conjugates for visualization techniques. Both polyclonal and monoclonal antibodies may be available, with the former offering broader epitope recognition and the latter providing higher specificity for particular domains or post-translational modifications.

What are the recommended applications for HERK1 antibodies?

Based on similar research with ERK1 antibodies, HERK1 antibodies are suitable for multiple applications including western blotting, immunofluorescence, immunoprecipitation, and flow cytometry . For western blotting, these antibodies can detect HERK1 in cell or tissue lysates to confirm expression levels or knockout efficiency in mutant lines like herk1-1 . In immunofluorescence studies, HERK1 antibodies can visualize protein localization and potential interactions with other proteins. HERK1 antibodies are also valuable for co-immunoprecipitation experiments to identify protein-protein interactions within signaling complexes.

What is the optimal protocol for detecting HERK1 in plant tissues using immunofluorescence?

For optimal immunofluorescence detection of HERK1 in plant tissues, researchers should follow these methodological steps:

• Fix tissue samples in 4% paraformaldehyde to preserve protein structure and cellular architecture

• Permeabilize samples with an appropriate detergent (0.1% Triton X-100 is often effective)

• Block with 3-5% BSA to reduce non-specific binding

• Incubate with primary anti-HERK1 antibody (typically 1:100 to 1:500 dilution)

• Wash thoroughly to remove unbound primary antibody

• Incubate with fluorophore-conjugated secondary antibody (e.g., Cy3 or Cy5 conjugates)

• Counterstain subcellular compartments as needed (similar to MitoTracker for mitochondria )

• Mount samples and image using confocal microscopy

This protocol is based on successful immunofluorescence techniques used for related proteins like ERK1, where researchers effectively visualized protein localization in subcellular compartments .

How can I validate HERK1 antibody specificity in my experimental system?

To validate HERK1 antibody specificity:

• Genetic controls: Compare antibody staining between wild-type and herk1 knockout/knockdown lines. The T-DNA insertion line herk1-1 could serve as a negative control, though note that RT-qPCR analysis has shown some residual transcripts may be present (approximately 20% of wild-type levels 3' of the T-DNA insertion) .

• Western blot validation: Perform western blots on samples from wild-type and mutant tissues to confirm the antibody detects bands of the expected molecular weight that are reduced or absent in mutants.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to samples; this should neutralize specific binding.

• Immunoprecipitation-mass spectrometry: Perform IP followed by mass spectrometry to confirm the antibody pulls down HERK1 and identify any cross-reactive proteins.

• Heterologous expression systems: Test antibody against recombinant HERK1 expressed in systems like E. coli or insect cells.

What are the recommended fixation and permeabilization conditions for HERK1 immunostaining?

For optimal HERK1 immunostaining, fixation and permeabilization conditions should be carefully selected:

Fixation options:

• 4% paraformaldehyde for 15-20 minutes at room temperature preserves protein epitopes while maintaining cellular structure

• Methanol fixation (-20°C for 10 minutes) may be beneficial for certain antibodies but can destroy some epitopes

Permeabilization options:

• 0.1% Triton X-100 for 5-10 minutes is suitable for most plant tissues

• 0.05-0.1% saponin may provide gentler permeabilization for preserving membrane structures

• Digitonin at low concentrations (0.01-0.05%) for selective permeabilization of plasma membrane while preserving internal membranes

These recommendations are based on successful protocols used for detecting related kinases in cellular contexts, as demonstrated in studies of ERK1 localization .

How can I study HERK1 interactions with other proteins like ANJEA using antibody-based methods?

To study HERK1-ANJEA interactions or other protein partnerships, several antibody-based methods are effective:

• Co-immunoprecipitation (Co-IP): Precipitate HERK1 using specific antibodies and analyze co-precipitated proteins by western blot using anti-ANJ antibodies. This approach can identify stable interactions between HERK1 and ANJ or other partners.

• Proximity Ligation Assay (PLA): This technique can detect protein interactions within 40nm distance in situ. It requires specific antibodies for both HERK1 and ANJ from different host species.

• FRET analysis with antibodies: Similar to the FRET techniques used for ERK1 and VDAC1 , researchers can employ antibodies conjugated to appropriate fluorophores (e.g., Cy3 and Cy5) to detect HERK1-ANJ interactions. When proteins interact, energy transfer occurs between fluorophores, which can be quantified:

  FRET efficiency = 1 - (F_DA/F_D)

  Where F_DA is the donor fluorescence in the presence of acceptor and F_D is the donor fluorescence after acceptor photobleaching .

• Bimolecular Fluorescence Complementation (BiFC): Though this requires genetic constructs rather than antibodies directly, it can complement antibody-based approaches to visualize interactions in living cells.

What approaches can be used to detect phosphorylated forms of HERK1?

For detecting phosphorylated HERK1, researchers should consider these methodological approaches:

• Phospho-specific antibodies: Use antibodies specifically raised against phosphorylated residues of HERK1. Similar to phospho-ERK1/2 detection , these antibodies can identify activated HERK1 in various experimental contexts.

• Phos-tag SDS-PAGE: This technique involves supplementing polyacrylamide gels with Phos-tag reagent, which retards the migration of phosphorylated proteins, creating a mobility shift detectable with standard HERK1 antibodies.

• Lambda phosphatase treatment: Treat samples with lambda phosphatase before western blotting to confirm that mobility shifts or phospho-antibody signals are genuinely due to phosphorylation.

• Mass spectrometry: Following immunoprecipitation with HERK1 antibodies, use mass spectrometry to identify and quantify specific phosphorylation sites.

• In vitro kinase assays: Use recombinant HERK1 and potential substrates to assess kinase activity, detecting phosphorylation with phospho-specific antibodies or radioactive ATP incorporation.

How can I examine HERK1 subcellular localization changes during signaling events?

To examine dynamic changes in HERK1 subcellular localization:

• Time-course immunofluorescence: Process samples at different time points after stimulus application, using HERK1 antibodies for immunofluorescence. This approach allows visualization of protein redistribution, similar to the ERK1 translocation studies where movement between cytosol, nucleus, and mitochondria was quantified following serum stimulation .

• Live-cell imaging with fluorescent protein fusions: While not directly using antibodies, generating HERK1-GFP constructs (similar to hERK1-GFP ) allows real-time visualization of protein movement. This can be complemented with immunostaining of fixed samples using HERK1 antibodies to validate observations.

• Subcellular fractionation and western blotting: Isolate different cellular compartments (plasma membrane, cytosol, nucleus) at various time points post-stimulation and analyze HERK1 distribution by western blotting with specific antibodies.

• Quantitative image analysis: Employ advanced image analysis methods to quantify HERK1 redistribution among subcellular compartments, as demonstrated with ERK1:

  • Create masks for different cellular compartments

  • Quantify fluorescence intensity within each compartment

  • Normalize by total cellular fluorescence

  • Plot redistribution over time

Why might I observe non-specific binding with my HERK1 antibody, and how can I reduce it?

Non-specific binding with HERK1 antibodies can result from several factors:

• Antibody concentration: Excessive antibody leads to increased background. Perform titration experiments to determine optimal concentrations (typically 1:100 to 1:1000 for immunofluorescence and 1:500 to 1:5000 for western blots).

• Inadequate blocking: Increase blocking agent concentration (5-10% BSA or normal serum) and duration (1-2 hours at room temperature or overnight at 4°C).

• Cross-reactivity: HERK1 shares high similarity with other CrRLK1L family members like ANJ (86% similarity at amino acid level) . Select antibodies raised against unique epitopes or validate specificity against recombinant proteins.

• Sample preparation issues: Incomplete fixation or excessive permeabilization can increase non-specific binding. Optimize fixation time and reduce detergent concentration.

• Secondary antibody problems: Test secondary antibodies alone (without primary) to check for non-specific binding. Consider using highly cross-adsorbed secondary antibodies.

What are the potential pitfalls when using HERK1 antibodies for co-immunoprecipitation experiments?

When performing co-immunoprecipitation with HERK1 antibodies, researchers should be aware of these methodological challenges:

• Epitope masking: Protein-protein interactions may block antibody access to HERK1 epitopes. Try different antibodies recognizing distinct epitopes or employ different precipitation strategies.

• Weak or transient interactions: HERK1 may form weak or transient complexes that dissociate during standard Co-IP procedures. Consider using chemical crosslinking (e.g., DSP or formaldehyde) before lysis to stabilize interactions.

• Buffer compatibility: Inappropriate buffers may disrupt protein interactions. Test different lysis buffers with varying salt concentrations, detergents, and pH levels to preserve complexes while ensuring effective extraction.

• Antibody orientation: If the antibody binds regions involved in protein-protein interactions, it might prevent co-precipitation. Use constructs with epitope tags located away from functional domains as alternative precipitation targets.

• Antibody cross-reactivity: Given the similarity between HERK1 and ANJ (86% similarity) , antibodies might not distinguish between these related proteins. Confirm results using complementary approaches like mass spectrometry.

How should I optimize antibody concentrations for different applications?

For optimal antibody concentrations across applications:

Western Blotting optimization:

• Start with manufacturer's recommended dilution (typically 1:1000)

• Perform a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000)

• Select concentration that provides specific bands with minimal background

• For phospho-specific antibodies, include positive control samples (e.g., stimulated tissues) and negative controls (phosphatase-treated samples)

Immunofluorescence optimization:

• Begin with 1:200 dilution for most antibodies

• Create a dilution matrix of primary and secondary antibodies (e.g., primary: 1:100, 1:200, 1:500; secondary: 1:200, 1:500, 1:1000)

• Evaluate signal-to-noise ratio at each combination

• Include appropriate negative controls, such as samples from herk1 knockout plants

Immunoprecipitation optimization:

• Start with 2-5 μg antibody per 500 μg protein lysate

• Test both direct antibody coupling to beads and protein A/G-mediated binding

• Compare different antibody amounts while keeping lysate concentration constant

• Verify precipitation efficiency by western blotting input, unbound, and precipitated fractions

What controls should I include when performing knockdown/knockout validation with HERK1 antibodies?

When validating HERK1 knockdown or knockout lines with antibodies, include these essential controls:

• Genetic controls:

  • Wild-type sample (positive control)

  • Known HERK1 knockout line (negative control)

  • For T-DNA insertion lines like herk1-1, verify disruption using both RT-qPCR and protein detection, as residual transcripts may be present

  • Heterozygous plants to demonstrate dose-dependency

• Technical controls:

  • Secondary antibody only (background control)

  • Competing peptide control (pre-incubate antibody with immunizing peptide)

  • Loading controls for western blots (housekeeping proteins)

  • Multiple antibodies targeting different HERK1 epitopes to confirm results

• Complementation controls:

  • Rescue experiments expressing HERK1 in knockout background to confirm phenotype causality, as described for herk1 anj double mutants

  • Visualization of both native and complemented protein expression using antibodies

How can I distinguish between closely related protein family members when using HERK1 antibodies?

Distinguishing HERK1 from related proteins like ANJ (which shares 86% similarity) requires careful methodological consideration:

• Epitope selection: Use antibodies raised against unique regions of HERK1 that differ from ANJ and other family members. N- or C-terminal domains often show greater divergence than catalytic domains.

• Western blot migration patterns: Slight differences in molecular weight may allow distinction on high-resolution gels. Consider using Phos-tag gels to separate based on phosphorylation differences.

• Genetic verification: Always include samples from verified herk1 and anj single mutants as controls when analyzing protein expression or localization.

• Immunodepletion approach: Sequential immunoprecipitation with specific antibodies can deplete one family member before analyzing the remaining proteins.

• Mass spectrometry validation: Following immunoprecipitation, use mass spectrometry to identify unique peptides specific to HERK1 versus ANJ or other family members.

How can HERK1 antibodies be applied in proximity labeling techniques?

HERK1 antibodies can be integrated into emerging proximity labeling techniques through these methodological approaches:

• Antibody-enzyme fusion constructs: Conjugate HERK1 antibodies to enzymes like APEX2 (ascorbate peroxidase) or BioID (biotin ligase) to label proteins in close proximity to HERK1 in fixed or living cells.

• Proximity-dependent biotinylation: After fixation and permeabilization, use HERK1 antibodies followed by secondary antibodies conjugated to HRP or APEX2, then perform biotin-phenol labeling to identify proximal proteins.

• Immuno-APEX: A two-step approach where HERK1 is first bound by primary antibodies, followed by secondary antibodies conjugated to APEX2, allowing spatially-restricted biotinylation of proximal proteins.

• Split-enzyme complementation: Combine antibody-based targeting with split enzyme reporters (like split-BioID) to achieve proximity labeling only when two proteins of interest (e.g., HERK1 and ANJ) interact.

These techniques would allow researchers to map the HERK1 interactome in various cellular contexts, potentially revealing new interaction partners beyond those currently known, such as ANJ .

What are the considerations for using HERK1 antibodies in super-resolution microscopy?

When applying HERK1 antibodies in super-resolution microscopy, researchers should consider these methodological factors:

• Antibody quality and specificity: Super-resolution techniques magnify not only signals but also background and artifacts. Use highly specific antibodies validated in knockout controls like herk1-1 .

• Fluorophore selection:

  • For STORM/PALM: Select bright, photoswitchable fluorophores (e.g., Alexa Fluor 647)

  • For STED: Choose fluorophores resistant to high-intensity depletion lasers (e.g., STAR RED, ATTO 647N)

  • For SIM: Conventional fluorophores are suitable but should have high photostability

• Sample preparation optimization:

  • Fixation must preserve nanoscale structures without introducing artifacts

  • Consider using smaller probes (Fab fragments, nanobodies) for improved resolution

  • Optimize antibody concentration to ensure appropriate labeling density

• Multicolor considerations: When studying HERK1 interactions with proteins like ANJ , carefully select fluorophore pairs with minimal crosstalk and compatible for the chosen super-resolution technique.

• Quantitative analysis: Develop rigorous analysis methods to quantify nanoscale distribution patterns, clustering, or colocalization with interaction partners.

How can mass cytometry (CyTOF) be used with HERK1 antibodies for multi-parameter analysis?

To implement HERK1 antibodies in mass cytometry (CyTOF) studies:

• Metal conjugation: Conjugate purified HERK1 antibodies with rare earth metals (e.g., lanthanides) using commercial conjugation kits. Different isotopes can be used for antibodies targeting total HERK1 versus phosphorylated forms.

• Panel design: Design comprehensive panels including:

  • HERK1 and related family members like ANJ

  • Potential interaction partners

  • Phosphorylation-specific markers

  • Cell type and cell state markers

  • Subcellular markers to track localization

• Sample preparation considerations:

  • Optimize fixation and permeabilization for simultaneous detection of surface and intracellular proteins

  • Include cell barcoding to minimize batch effects

  • Prepare single-stained controls for each metal-conjugated antibody

• Data analysis approaches:

  • Apply dimension reduction techniques (tSNE, UMAP) to visualize complex relationships

  • Use clustering algorithms to identify cell populations with distinct HERK1 expression or activation patterns

  • Implement trajectory analysis to map HERK1 activity changes during developmental or signaling processes

This approach allows simultaneous examination of HERK1 expression, activation state, and dozens of other parameters at single-cell resolution.