DSK2A Antibody

What is DSK2 and what cellular functions does it participate in?

DSK2 is a reported synonym of the UBQLN1 gene, which encodes ubiquilin 1 protein. This protein functions in several critical cellular pathways including the cellular response to hypoxia and autophagy. The human version of DSK2 has a canonical amino acid length of 589 residues and a protein mass of 62.5 kilodaltons, with four distinct isoforms identified to date .

In plants such as Arabidopsis, two DSK2 paralogs exist as a result of tandem duplication (DSK2A and DSK2B), with 87% amino acid identity . Both DSK2 proteins contain an N-terminal ubiquitin-like (UBL) domain that mediates their interaction with the proteasome and a C-terminal ubiquitin-associated (UBA) domain that can bind both K48 and K63 polyubiquitin chains . DSK2 plays a critical role in selective autophagy and stress response mechanisms by targeting specific proteins for degradation.

What is the subcellular localization of DSK2?

DSK2 displays a broad subcellular distribution pattern. It is reported to be localized in multiple cellular compartments including the cell membrane, nucleus, cytoplasmic vesicles, endoplasmic reticulum (ER), and cytoplasm . This widespread distribution reflects its diverse functions in various cellular processes and signaling pathways. When visualized using immunofluorescence techniques with anti-DSK2 antibodies, the protein can be observed as both diffuse signals and distinct puncta, with the latter often representing autophagosomal structures particularly under stress conditions .

How should I select the appropriate anti-DSK2 antibody for my experiments?

When selecting an anti-DSK2 antibody, consider the following methodology-focused approach:

• Target species specificity: Determine whether you need antibodies that recognize human, plant (Arabidopsis), yeast (Saccharomyces), or other species-specific DSK2 orthologs. Available antibodies show varying reactivity patterns across species .

• Domain specificity: Choose between antibodies targeting different regions of DSK2:

  • N-terminal antibodies (recognizing the UBL domain)

  • C-terminal antibodies (recognizing the UBA domain)

  • Middle region antibodies

• Application compatibility: Verify the antibody has been validated for your specific application:

  • Western blot (WB)

  • Immunohistochemistry (IHC)

  • Immunoprecipitation (IP)

  • ELISA

  • Immunofluorescence (IF)

• Validation data: Request validation data showing specificity using knockout/knockdown controls and expected molecular weight detection .

What are the technical considerations for using DSK2A antibodies in Western blot analyses?

When conducting Western blot analyses with DSK2A antibodies, follow these methodological guidelines:

• Sample preparation:

  • For plant samples: Grind tissue in liquid nitrogen and extract in buffer containing protease inhibitors and phosphatase inhibitors if phosphorylation is being studied

  • For cellular samples: Use lysis buffers containing 1% NP-40 or similar detergents

• Protocol optimization:

  • Use 10-12% SDS-PAGE gels for optimal resolution

  • Transfer to PVDF membranes (preferred over nitrocellulose for this protein)

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

  • Incubate with primary antibody at 1:1000 to 1:2000 dilution overnight at 4°C

• Detection considerations:

  • Human DSK2/UBQLN1 migrates at approximately 62.5 kDa

  • Plant DSK2A appears at approximately 55-60 kDa

  • Look for potential post-translational modifications that may cause shifts in migration

  • When studying ubiquitinated forms, high molecular weight smears may be observed

• Controls:

  • Include positive controls (tissues/cells known to express DSK2)

  • Include negative controls (DSK2 knockout/knockdown samples where available)

How can I effectively study the interaction between DSK2A and ATG8 in autophagy pathways?

To investigate the DSK2A-ATG8 interaction in autophagy pathways, researchers can employ several complementary approaches:

• Co-immunoprecipitation (Co-IP):

  • Use anti-DSK2A antibodies to pull down the protein complex followed by Western blot with anti-ATG8 antibodies

  • Include appropriate controls including IgG controls and reverse Co-IP

  • Treatment with autophagy inducers (e.g., starvation or rapamycin) can enhance the detection of this interaction

• Bimolecular Fluorescence Complementation (BiFC):

  • Generate constructs with DSK2A-YFPN and ATG8-YFPC (or vice versa)

  • Co-express in protoplasts or plant cells

  • Visualize interaction using confocal microscopy

  • Colocalization experiments can be performed using Cerulean-ATG8e as a marker for autophagosomes

• GST pull-down assays:

  • Express recombinant DSK2A-MBP and GST-ATG8

  • Conduct in vitro binding assays to determine direct interaction

  • Analyze using anti-MBP antibodies to detect DSK2A-MBP

• Mutational analysis:

  • Generate DSK2A variants with mutations in potential ATG8-interaction motifs

  • Test the effect on binding capacity and autophagy function

  • Compare wild-type and mutant protein interactions using the methods described above

Research findings indicate that DSK2 can interact with ATG8, similar to human DSK2 homologs (Ubiquilins) that function in autophagy as LC3-interacting partners .

What are the key considerations when studying DSK2A phosphorylation and how does it affect function?

DSK2A phosphorylation represents a critical regulatory mechanism affecting its function in selective autophagy. When investigating this process:

• Detection methods:

  • Use phospho-specific antibodies if available

  • Apply Phos-tag SDS-PAGE to separate phosphorylated from non-phosphorylated forms

  • Employ mass spectrometry to identify precise phosphorylation sites

  • Utilize lambda phosphatase treatment to confirm phosphorylation status

• Kinase interaction studies:

  • Research indicates that BIN2 kinase phosphorylates DSK2

  • Perform in vitro kinase assays using recombinant BIN2 and DSK2A

  • Analyze how phosphorylation affects protein-protein interactions (e.g., with BES1)

• Functional impact assessment:

  • Generate phosphomimetic (S/T to D/E) and phospho-null (S/T to A) mutants

  • Compare their ability to interact with autophagy machinery and target proteins

  • Assess subcellular localization changes using immunofluorescence

  • Evaluate effects on selective autophagy flux

• Stress response correlation:

  • Analyze phosphorylation status under different stress conditions (e.g., nutrient starvation, osmotic stress)

  • Compare results from wild-type and kinase-deficient backgrounds

Research has demonstrated that BIN2 phosphorylation of DSK2 regulates its interaction with ATG8, which is crucial for targeting growth regulators like BES1 to the autophagy pathway during stress responses .

How can I establish an effective experimental system to study DSK2A-mediated selective autophagy?

Establishing an experimental system for DSK2A-mediated selective autophagy requires a multi-faceted approach:

• Genetic tools:

  • Generate DSK2A/B knockout or knockdown lines (T-DNA insertion mutants or RNAi lines)

  • Create inducible DSK2A overexpression lines

  • Design reporter constructs (e.g., DSK2A-GFP, BES1-RFP) for live imaging

• Autophagy induction protocols:

  • Establish reliable methods for inducing selective autophagy (e.g., sucrose starvation, mannitol-induced osmotic stress)

  • Compare effects in wild-type vs. DSK2-deficient backgrounds

  • Include appropriate controls such as autophagy inhibitors (e.g., ConA) and general autophagy mutants (e.g., atg7-2)

• Cargo recognition analysis:

  • Study interaction between DSK2A and specific cargo proteins (e.g., BES1)

  • Utilize immunoprecipitation with DSK2A antibodies to identify novel cargo

  • Apply domain deletion experiments (e.g., DSK2ΔUBA) to determine domains required for cargo interaction

• Visualization techniques:

  • Use confocal microscopy to track DSK2A-cargo colocalization with autophagy markers

  • Apply electron microscopy to visualize autophagosomes containing DSK2A and cargo

  • Quantify autophagy through standardized methods (e.g., counting puncta formation)

Research findings demonstrate that DSK2 is required to target specific proteins like BES1 to autophagy but is not required for proper function of the core autophagy machinery, as evidenced by normal ATG8e puncta formation in DSK2-deficient backgrounds .

What strategies can address non-specific binding of DSK2A antibodies in immunoprecipitation experiments?

Non-specific binding represents a significant challenge when using DSK2A antibodies for immunoprecipitation. Researchers can implement these advanced troubleshooting strategies:

• Antibody validation and optimization:

  • Test multiple antibodies targeting different epitopes of DSK2A

  • Titrate antibody concentrations to determine optimal amounts

  • Validate specificity using DSK2A knockout/knockdown controls

  • Pre-clear lysates with Protein A/G beads before immunoprecipitation

• Buffer optimization:

  • Adjust salt concentration in wash buffers (150-500 mM NaCl) to reduce non-specific interactions

  • Include mild detergents (0.1-0.5% NP-40 or Triton X-100)

  • Add competing proteins (0.1-1% BSA) to block non-specific binding sites

  • Test different buffer compositions based on subcellular localization of target interactions

• Advanced controls:

  • Include isotype-matched control antibodies

  • Perform reciprocal immunoprecipitations when studying protein-protein interactions

  • Utilize blocking peptides specific to the antibody's epitope

  • Include samples from DSK2A-deficient cells/tissues

• Cross-linking strategies:

  • Apply DSP (dithiobis[succinimidyl propionate]) or formaldehyde cross-linking to stabilize transient interactions

  • Optimize cross-linking conditions (time, concentration) for specific experimental contexts

  • Include appropriate controls to ensure cross-linking specificity

Research involving DSK2A-GFP immunoprecipitation has demonstrated successful pulldown of high molecular weight forms of interacting proteins like BES1 when using optimized conditions with autophagy inhibitors like ConA .

How can I differentiate between the roles of DSK2A and DSK2B in selective autophagy pathways?

Differentiating between DSK2A and DSK2B functions requires sophisticated experimental approaches:

• Paralog-specific tools:

  • Develop highly specific antibodies that can distinguish between the 87% identical DSK2A and DSK2B proteins

  • Generate single and double knockout/knockdown lines (DSK2A-KO, DSK2B-KO, and DSK2A/B-KO)

  • Create paralog-specific tagged versions for localization studies

• Complementation studies:

  • Express DSK2A in DSK2B-KO backgrounds and vice versa

  • Assess rescue of phenotypes to determine functional redundancy

  • Design chimeric proteins swapping domains between paralogs to identify paralog-specific functional regions

• Cargo specificity analysis:

  • Perform paralog-specific immunoprecipitation followed by mass spectrometry

  • Compare interacting partners between DSK2A and DSK2B

  • Quantify binding affinities for shared cargo proteins

• Stress-specific responses:

  • Compare DSK2A vs. DSK2B expression and activity under different stress conditions

  • Analyze phenotypes of single mutants under various stresses

  • Assess post-translational modifications specific to each paralog

• Evolutionary analysis:

  • Compare DSK2 paralogs across plant species to identify conserved and divergent features

  • Infer functional specialization through phylogenetic approaches

Research findings indicate that while both DSK2A and DSK2B can interact with targets like BES1 and participate in selective autophagy, there may be context-specific functions that distinguish their roles in stress responses and plant growth regulation .

What are the common causes of false positive/negative results when using DSK2A antibodies?

When using DSK2A antibodies, researchers frequently encounter false positive/negative results that can be addressed through systematic troubleshooting:

• False positives:

  • Cross-reactivity: DSK2A antibodies may detect related proteins containing UBL or UBA domains

    • Solution: Validate using knockout controls and peptide competition assays

  • Non-specific binding: High antibody concentrations may lead to background signals

    • Solution: Optimize antibody dilutions and blocking conditions

  • Secondary antibody issues: Cross-reactions with endogenous immunoglobulins

    • Solution: Use isotype-specific secondary antibodies and pre-absorb against host species proteins

• False negatives:

  • Epitope masking: Post-translational modifications or protein interactions may block antibody binding sites

    • Solution: Use multiple antibodies targeting different epitopes

  • Low expression levels: Endogenous DSK2A may be below detection threshold

    • Solution: Employ signal amplification methods or concentrate samples

  • Protein degradation: DSK2A may be rapidly degraded during sample preparation

    • Solution: Include protease inhibitors and handle samples at 4°C

• Protocol-specific issues:

  • Western blot: Inadequate transfer of high molecular weight complexes

    • Solution: Extend transfer time or use semi-dry transfer systems

  • Immunoprecipitation: Harsh washing conditions disrupting interactions

    • Solution: Optimize salt and detergent concentrations in wash buffers

  • Immunofluorescence: Fixation affecting epitope accessibility

    • Solution: Compare different fixation methods (PFA vs. methanol)

Researchers have successfully detected DSK2-BES1 interactions by using approaches that preserve protein complexes, such as in vivo BiFC assays and careful coIP protocols with appropriate inhibitors .

How can I optimize DSK2A antibody performance in different experimental conditions?

Optimizing DSK2A antibody performance requires systematic adjustment of experimental parameters:

• Western blot optimization:

  • Blocking agents: Compare 5% milk vs. 3-5% BSA for reduced background

  • Antibody dilution series: Test primary antibody at 1:500, 1:1000, 1:2000, and 1:5000

  • Incubation conditions: Compare 1 hour at room temperature vs. overnight at 4°C

  • Detection systems: Compare chemiluminescence, fluorescent, and colorimetric detection methods

• Immunofluorescence optimization:

  • Fixation methods: Test paraformaldehyde (4%), methanol, and acetone fixation

  • Permeabilization: Optimize detergent type (Triton X-100, Tween-20, saponin) and concentration

  • Antigen retrieval: Apply citrate buffer or EDTA-based methods for improved epitope accessibility

  • Signal amplification: Utilize tyramide signal amplification for low abundance targets

• Immunoprecipitation optimization:

  • Antibody coupling: Compare direct coupling to beads vs. indirect capture methods

  • Lysis conditions: Test different detergents (NP-40, CHAPS, digitonin) for membrane protein extraction

  • Pre-clearing strategies: Optimize pre-clearing steps to reduce non-specific binding

  • Elution methods: Compare harsh (boiling in SDS) vs. mild (peptide competition) elution

• Environmental factors:

  • Sample treatment: Analyze how stress conditions affect antibody performance

  • Cell/tissue type: Optimize protocols for different experimental materials

  • Storage conditions: Evaluate antibody stability under different storage conditions

Research on DSK2-mediated selective autophagy has demonstrated successful antibody applications when protocols are optimized for specific experimental contexts, particularly when studying stress-induced changes in protein localization and interactions .

How can DSK2A antibodies be used to study selective autophagy in stress response mechanisms?

DSK2A antibodies offer powerful tools for investigating selective autophagy in stress response pathways:

• Stress-induced translocation studies:

  • Monitor DSK2A subcellular redistribution during different stresses using immunofluorescence

  • Quantify autophagosome association under conditions like nutrient starvation or osmotic stress

  • Perform time-course experiments to track the kinetics of DSK2A recruitment to autophagic structures

• Cargo selection mechanisms:

  • Use DSK2A antibodies in proximity labeling approaches (BioID or APEX) to identify stress-specific cargoes

  • Perform quantitative co-immunoprecipitation to measure changes in DSK2A-cargo interactions under stress

  • Apply FRET/FLIM techniques to analyze DSK2A-cargo binding dynamics in living cells

• Regulatory pathway analysis:

  • Study how phosphorylation by stress-activated kinases (e.g., BIN2) modulates DSK2A function

  • Investigate ubiquitination patterns of DSK2A using chain-specific antibodies

  • Analyze DSK2A interactome changes during stress using antibody-based pulldown followed by proteomics

• Therapeutic implications:

  • Explore potential roles of DSK2-mediated selective autophagy in stress-related disorders

  • Investigate pharmacological modulators of DSK2A activity in disease models

  • Develop screening assays using DSK2A antibodies to identify compounds affecting selective autophagy

Research has demonstrated that DSK2 plays crucial roles in targeting growth regulators like BES1 for autophagy-mediated degradation during stress, providing a mechanism by which plants coordinate growth and stress responses .

What emerging technologies can enhance the utility of DSK2A antibodies in research?

Several cutting-edge technologies are expanding the research applications of DSK2A antibodies:

• Advanced imaging approaches:

  • Super-resolution microscopy: Apply techniques like STORM, PALM, or STED for nanoscale visualization of DSK2A-positive structures

  • Lattice light-sheet microscopy: Enable long-term live imaging of DSK2A dynamics with minimal phototoxicity

  • Correlative light-electron microscopy (CLEM): Combine immunofluorescence with ultrastructural analysis of DSK2A-labeled compartments

• Single-cell analysis methods:

  • Mass cytometry (CyTOF): Utilize metal-conjugated DSK2A antibodies for high-dimensional single-cell profiling

  • Imaging mass cytometry: Analyze DSK2A expression and localization in tissue contexts with subcellular resolution

  • Single-cell Western blot: Quantify DSK2A levels in individual cells to assess population heterogeneity

• Proximity-based interaction mapping:

  • APEX/BioID approaches: Identify proteins in close proximity to DSK2A through biotinylation and affinity purification

  • Split-protein complementation: Develop new reporters for DSK2A interactions with autophagy machinery

  • Proximity ligation assay (PLA): Detect and quantify endogenous DSK2A-protein interactions in situ

• Engineered antibody derivatives:

  • Nanobodies/single-domain antibodies: Develop smaller DSK2A-binding reagents for improved tissue penetration

  • Bispecific antibodies: Create reagents simultaneously targeting DSK2A and interaction partners

  • Intrabodies: Express functional antibody fragments in living cells to monitor or perturb DSK2A function

These technologies can significantly enhance our understanding of DSK2A's role in processes like selective autophagy of growth regulators during stress responses .

What are the key considerations for validating and publishing research using DSK2A antibodies?

When validating and publishing research using DSK2A antibodies, researchers should address these critical considerations:

• Antibody validation:

  • Document complete antibody information (source, catalog number, lot, dilutions)

  • Provide evidence of specificity through knockout/knockdown controls

  • Include positive controls showing expected patterns in tissues/cells known to express DSK2A

  • Present raw, unprocessed images alongside processed data

• Experimental controls:

  • Include appropriate negative controls (secondary antibody only, isotype controls)

  • Show technical replicates demonstrating reproducibility

  • Validate key findings with multiple independent antibodies when possible

  • Confirm results using complementary, antibody-independent approaches

• Method transparency:

  • Provide detailed protocols including blocking conditions, incubation times, and washing steps

  • Specify image acquisition parameters and processing methods

  • Describe quantification methods with statistical analyses

  • Disclose any limitations of the antibodies or approaches used

• Data interpretation:

  • Discuss potential cross-reactivity with DSK2B or other related proteins

  • Address how post-translational modifications might affect antibody recognition

  • Consider how experimental conditions (stress, fixation) might influence results

  • Interpret findings in context of current knowledge about DSK2 function

Rigorous validation and transparent reporting ensure that research findings on DSK2A-mediated processes, such as selective autophagy in stress responses, can be effectively reproduced and extended by the broader scientific community .

How might our understanding of DSK2A function evolve with improved antibody technologies?

As antibody technologies advance, our understanding of DSK2A function is likely to evolve in several important directions:

• Spatiotemporal dynamics:

  • Advanced live-cell imaging with next-generation antibody-based biosensors will reveal real-time DSK2A activity during stress responses

  • Intravital microscopy using tissue-penetrating antibody derivatives will enable visualization of DSK2A function in intact organisms

  • These approaches will provide insights into the precise timing and subcellular locations of DSK2A-mediated selective autophagy events

• Interaction networks:

  • Proximity labeling techniques using engineered antibodies will map the complete DSK2A interactome under different conditions

  • Single-molecule tracking with antibody fragments will characterize the dynamics of DSK2A-cargo recognition

  • These methods will uncover previously unknown DSK2A functions beyond its established role in targeting proteins like BES1 for degradation

• Structural insights:

  • Conformation-specific antibodies will detect different functional states of DSK2A

  • Antibody-assisted cryo-EM will reveal the structure of DSK2A-containing complexes

  • These structural insights will explain how phosphorylation and other modifications regulate DSK2A function in selective autophagy

• Translational applications:

  • Highly specific antibodies distinguishing between DSK2 paralogs and modified forms will enable precise manipulation of selective autophagy pathways

  • Therapeutic antibodies or antibody-mimetics targeting specific DSK2 functions may be developed for stress-related disorders

  • Diagnostic applications may emerge from understanding DSK2A's role in coordinating growth and stress responses