ASK3 Antibody

What is ASK3 and why are antibodies against it important for research?

ASK3 (Apoptosis Signal-regulating Kinase 3) is a reported synonym of the MAP3K15 gene, which encodes mitogen-activated protein kinase kinase kinase 15. This protein primarily functions in protein phosphorylation pathways and various cellular signaling cascades . The human version of ASK3 has a canonical amino acid length of 1313 residues and a protein mass of approximately 147.4 kilodaltons, with three distinct isoforms identified to date . As a member of the STE Ser/Thr protein kinase family, ASK3 plays crucial roles in multiple biological processes, making antibodies against it essential tools for investigating these pathways, protein-protein interactions, and expression patterns in various experimental models.

What are the common applications for ASK3 antibodies in research settings?

ASK3 antibodies are versatile research tools applicable across multiple experimental platforms. The most common applications include:

• Western Blotting (WB): For detecting and quantifying ASK3 protein in cell or tissue lysates

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative measurement of ASK3 in solution

• Flow Cytometry (FCM): For analyzing ASK3 expression in individual cells

• Immunohistochemistry (IHC): For visualizing ASK3 distribution in tissue sections

These applications enable researchers to investigate ASK3 expression, localization, and function in various biological contexts, from cell culture systems to animal models and human specimens.

How do I select the appropriate ASK3 antibody for my specific research application?

Selecting the optimal ASK3 antibody requires consideration of several key factors:

• Species reactivity: Ensure the antibody recognizes ASK3 in your experimental model. Available options include antibodies reactive with human, mouse, or Arabidopsis ASK3 .

• Application compatibility: Verify that the antibody has been validated for your specific application (WB, ELISA, FCM, or IHC). Some antibodies may perform well in certain applications but not others .

• Antibody type: Consider whether a monoclonal or polyclonal antibody better suits your needs. Monoclonals offer high specificity for a single epitope, while polyclonals recognize multiple epitopes and may provide stronger signals.

• Conjugation requirements: Determine if you need an unconjugated antibody or one conjugated to a specific tag (biotin, fluorophore, enzyme) based on your detection method .

• Isotype and host species: Consider the antibody isotype and host species to avoid potential cross-reactivity issues in your experimental system.

What validation methods should I use to confirm the specificity of my ASK3 antibody?

Rigorous validation is essential for ensuring antibody specificity. Recommended validation approaches include:

• Positive and negative controls: Test the antibody on samples known to express or lack ASK3.

• Knockdown/knockout verification: Compare antibody staining between wild-type samples and those with ASK3 genetically silenced or deleted.

• Cross-reactivity testing: Evaluate potential cross-reactivity with related proteins, particularly other MAP kinase family members.

• Western blot analysis: Confirm that the antibody detects a protein of the expected molecular weight (approximately 147.4 kDa for canonical human ASK3) .

• Epitope blocking: Pre-incubate the antibody with its purified antigen to demonstrate signal specificity.

Similar to antibody validation approaches used for other targets, these methods can significantly enhance confidence in experimental results when working with ASK3 antibodies .

What are the optimal conditions for Western blotting with ASK3 antibodies?

For successful Western blot detection of ASK3, consider the following protocol recommendations:

• Sample preparation:

  • Use RIPA or NP-40 buffer supplemented with protease and phosphatase inhibitors

  • Include 1-2 mM DTT or β-mercaptoethanol to maintain protein integrity

  • Heat samples at 95°C for 5 minutes in Laemmli buffer

• Gel electrophoresis:

  • Use 6-8% acrylamide gels due to ASK3's large molecular weight (147.4 kDa)

  • Run at 100-120V to ensure proper protein separation

• Transfer conditions:

  • Transfer to PVDF membrane at 25V overnight at 4°C for large proteins

  • Use 10-20% methanol in transfer buffer to facilitate transfer

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk or BSA in TBS-T for 1 hour at room temperature

  • Incubate with primary ASK3 antibody at manufacturer-recommended dilution (typically 1:500-1:2000) overnight at 4°C

  • Wash thoroughly with TBS-T (3-5 times, 5 minutes each)

  • Incubate with appropriate secondary antibody (e.g., alkaline phosphatase-labeled anti-mouse Fc at approximately 160 ng/ml)

• Signal detection:

  • Use enhanced chemiluminescence for optimal sensitivity

  • Expose membrane for 1-10 minutes depending on expression level

How can I optimize immunoprecipitation protocols using ASK3 antibodies?

For effective immunoprecipitation of ASK3, follow these optimization strategies:

• Lysis buffer selection:

  • Use gentle non-ionic detergent buffers (e.g., 1% NP-40 or 0.5% Triton X-100)

  • Include protease and phosphatase inhibitors to preserve protein integrity

  • Maintain physiological pH (7.2-7.5) to preserve protein-protein interactions

• Antibody binding conditions:

  • Pre-clear lysates with Protein A/G beads to reduce non-specific binding

  • Use 2-5 μg of ASK3 antibody per 500 μg of total protein

  • Incubate antibody with lysate overnight at 4°C with gentle rotation

• Bead selection and handling:

  • Choose magnetic beads for cleaner preparations and higher yields

  • Wash beads 3-5 times with decreasing salt concentrations to maintain specificity while removing contaminants

  • Elute proteins using gentle conditions (low pH buffer or SDS sample buffer) depending on downstream applications

• Verification techniques:

  • Confirm successful immunoprecipitation by Western blot

  • Include negative controls (isotype-matched irrelevant antibody) and input samples

How can I use ASK3 antibodies to study protein-protein interactions within signaling pathways?

ASK3 antibodies can be powerful tools for investigating protein-protein interactions using these advanced approaches:

• Co-immunoprecipitation (Co-IP):

  • Immunoprecipitate ASK3 using specific antibodies and analyze co-precipitating proteins

  • Alternatively, immunoprecipitate potential interacting partners and probe for ASK3

  • Use mild lysis and washing conditions to preserve protein complexes

• Proximity ligation assay (PLA):

  • Employ ASK3 antibodies alongside antibodies against potential interacting partners

  • This technique allows visualization of protein interactions within 40 nm distance in situ

  • Quantify interaction signals in different cellular compartments or conditions

• Bimolecular Fluorescence Complementation (BiFC):

  • Use ASK3-specific antibodies to confirm expression of fusion constructs

  • Validate interactions observed through complementary techniques

• Chromatin Immunoprecipitation (ChIP):

  • If investigating transcriptional regulation roles, use ASK3 antibodies to identify DNA binding sites

  • Follow with sequencing (ChIP-seq) to map genome-wide binding patterns

What approaches can be used to study ASK3 phosphorylation status using antibodies?

To investigate ASK3 phosphorylation, researchers can implement these methodological approaches:

• Phospho-specific antibodies:

  • Utilize antibodies that specifically recognize phosphorylated residues of ASK3

  • Compare results with total ASK3 antibodies to determine phosphorylation ratios

• Phosphatase treatment controls:

  • Treat sample aliquots with lambda phosphatase prior to immunoblotting

  • Compare signal between treated and untreated samples to verify phosphorylation

• Phos-tag™ SDS-PAGE:

  • Use Phos-tag™ acrylamide gels with ASK3 antibodies to separate phosphorylated and non-phosphorylated forms

  • This technique retards phosphorylated protein migration, creating distinct bands

• Mass spectrometry validation:

  • Immunoprecipitate ASK3 using specific antibodies

  • Perform mass spectrometry to identify phosphorylation sites

  • Confirm findings using phospho-specific antibodies if available

What are the key differences between human and mouse ASK3 antibodies?

When working with different species, researchers should consider these important distinctions:

Researchers should carefully evaluate the suitability of each antibody for their specific experimental system and consider epitope mapping information when available.

How do plant ASK3 antibodies differ from mammalian ASK3 antibodies?

Plant ASK3 antibodies, particularly those targeting Arabidopsis ASK3, differ significantly from mammalian counterparts:

• Target protein:

  • Plant ASK3 refers to a different protein than mammalian ASK3

  • Plant ASK3 is part of the SKP1-like protein family involved in protein ubiquitination

  • No structural homology with mammalian ASK3/MAP3K15

• Applications:

  • Plant ASK3 antibodies are primarily validated for Western blot and ELISA applications

  • Less frequently used in immunofluorescence or immunohistochemistry

• Specificity considerations:

  • Plant ASK3 antibodies show no cross-reactivity with mammalian tissues

  • These antibodies specifically recognize Arabidopsis ASK3

• Commercial availability:

  • Multiple suppliers offer Arabidopsis-specific ASK3 antibodies

  • These include Biorbyt, CUSABIO, and MyBioSource products

When working with plant systems, researchers must ensure they select antibodies specifically designed for plant ASK3 rather than mammalian homologs.

What strategies can resolve weak or absent signals when using ASK3 antibodies?

When facing detection challenges with ASK3 antibodies, consider these troubleshooting approaches:

• Sample preparation optimization:

  • Ensure complete protein extraction using appropriate lysis buffers

  • Add protease inhibitors to prevent degradation

  • For membrane-associated fractions, use stronger detergents to solubilize proteins

• Antibody concentration adjustments:

  • Titrate antibody concentrations to identify optimal working dilution

  • Consider longer incubation times (overnight at 4°C) for primary antibody

• Detection system enhancement:

  • Switch to more sensitive detection systems (e.g., from colorimetric to chemiluminescent)

  • Use signal amplification methods (e.g., biotinylated secondary antibodies with streptavidin-HRP)

  • Consider tyramide signal amplification for immunohistochemistry

• Cross-validation:

  • Test alternative ASK3 antibodies targeting different epitopes

  • Verify protein expression using complementary methods (qPCR, overexpression)

How can I minimize background and non-specific binding when using ASK3 antibodies?

To improve signal-to-noise ratio and reduce non-specific binding:

• Blocking optimization:

  • Test different blocking agents (BSA, non-fat milk, normal serum)

  • Increase blocking time or concentration

  • Add 0.1-0.3% Triton X-100 or Tween-20 to reduce hydrophobic interactions

• Washing protocol enhancement:

  • Increase washing frequency and duration

  • Use higher salt concentration in wash buffers (150-500 mM NaCl)

  • Add 0.05-0.1% Tween-20 to wash buffers

• Antibody specificity measures:

  • Pre-absorb antibodies with related proteins

  • Use antibody diluent containing 1-5% of the same protein as the blocking buffer

  • Consider using monoclonal antibodies for higher specificity

• Tissue/cell preparation:

  • Block endogenous peroxidase activity for IHC applications

  • Perform antigen retrieval optimization for fixed tissues

  • Block endogenous biotin if using biotin-streptavidin detection systems

How can ASK3 antibodies be used in single-cell analysis techniques?

ASK3 antibodies can be incorporated into cutting-edge single-cell analysis methods:

• Single-cell Western blotting:

  • Use ASK3 antibodies to detect protein expression in individual cells

  • Correlate with other signaling molecules at single-cell resolution

• Mass cytometry (CyTOF):

  • Conjugate ASK3 antibodies with rare earth metals

  • Simultaneously analyze ASK3 expression alongside dozens of other markers

• Imaging mass cytometry:

  • Apply metal-labeled ASK3 antibodies to tissue sections

  • Achieve subcellular localization data with multiplexed marker analysis

• Microfluidic antibody capture:

  • Immobilize ASK3 antibodies in microfluidic channels

  • Capture and analyze secreted or released ASK3 from single cells

These techniques provide unprecedented resolution for studying ASK3 expression heterogeneity and functional roles at the individual cell level.

What considerations are important when using ASK3 antibodies in multiplexed immunoassays?

When incorporating ASK3 antibodies into multiplexed platforms, consider these methodological factors:

• Antibody compatibility:

  • Ensure ASK3 antibodies are compatible with other antibodies in the panel

  • Select antibodies raised in different host species to avoid cross-reactivity

  • Use isotype-specific secondary antibodies for clean separation of signals

• Signal separation strategies:

  • For fluorescence-based multiplexing, choose fluorophores with minimal spectral overlap

  • In chromogenic multiplexing, select enzymes producing distinct colors

  • Consider sequential detection for co-localization studies

• Validation requirements:

  • Validate each antibody individually before combining

  • Compare multiplexed results with single-antibody controls

  • Test for potential antigen masking effects when multiple antibodies bind nearby epitopes

• Data analysis considerations:

  • Implement appropriate compensation controls for fluorescence spectra overlap

  • Use image analysis software capable of unmixing complex signals

  • Consider artificial intelligence-assisted analysis for highly multiplexed data