dnaK-A Antibody

What is DnaK and why is it significant in research?

DnaK is a highly conserved bacterial chaperone protein belonging to the heat-shock protein 70 (Hsp70) family. It plays essential roles in protein folding, quality control, and stress response mechanisms. Research significance includes:

• Functions as an ATP-dependent molecular chaperone that assists in protein folding

• Plays critical roles in phage lambda DNA replication by releasing lambda O and P proteins from the preprimosomal complex

• Participates in chromosomal DNA replication through interaction with DnaA protein

• Acts in bacterial stress response to conditions like hyperosmotic shock

• Serves as a moonlighting protein on bacterial cell surfaces, mediating adhesion to host cells and extracellular matrix components

DnaK's involvement in bacterial survival under stress conditions (including antibiotic exposure) makes it a valuable research target for understanding bacterial pathogenesis and developing therapeutic strategies.

DnaK is the bacterial homolog of human Hsp70, but important structural and functional differences exist:

• While highly conserved, DnaK has specific domains that differ from human Hsp70

• DnaK forms a transient dimer upon ATP binding that is essential for efficient interaction with Hsp40 co-chaperones

• Specific DnaK-targeting peptides like ARV-1502 can bind bacterial DnaK without interacting with human Hsp70

This distinction is crucial for antibody development and application in research, as it allows for:

• Selective targeting of bacterial proteins without cross-reactivity with human proteins

• Development of antibacterial strategies that don't interfere with human Hsp70 function

• Use of DnaK antibodies as diagnostic tools for bacterial infections

What are the optimal protocols for Western blot detection of DnaK?

For optimal Western blot detection of DnaK, researchers should follow these methodological steps:

• Sample preparation:

  • For bacterial samples, use fresh cultures and standardize cell number

  • For recombinant protein, use purified protein at 10-50 ng per lane

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

• Transfer and detection:

  • Transfer to PVDF membrane using semi-dry transfer (250 mA, 1 h)

  • Block with PBS containing 0.2% Tween-20 and 5% BSA for 1 hour at room temperature

  • Incubate with primary antibody (e.g., mouse monoclonal 8E2/2 at 1:5000 dilution) overnight at 4°C

  • Wash with TBS or PBS-T buffer 3-4 times for 10-15 minutes each

  • Incubate with appropriate HRP-conjugated secondary antibody (typically 1:2500 to 1:10,000 dilution)

  • Develop using ECL substrate and image with a digital imaging system

• Optimization considerations:

  • For polyclonal antibodies, dilutions of 1:500 to 1:5000 are recommended for Western blot

  • Include positive and negative controls to validate specificity

  • Expected molecular weight of E. coli DnaK is approximately 70 kDa

How can I validate the specificity of a DnaK antibody?

Validating antibody specificity is critical for reliable research. Follow these methodological approaches:

• Positive and negative controls:

  • Use purified recombinant DnaK protein as a positive control

  • Use samples from DnaK knockout strains or cells transformed with empty vectors as negative controls

  • Compare with another validated DnaK antibody if available

• Cross-reactivity testing:

  • Test against purified human Hsp70 to ensure bacterial specificity

  • Test against closely related bacterial species to determine cross-species reactivity

  • Perform peptide competition assays using the immunizing peptide

• Multiple detection methods:

  • Compare results across different techniques (Western blot, ELISA, immunofluorescence)

  • Perform immunoprecipitation followed by mass spectrometry to confirm target identity

  • Colony blot analysis can confirm surface expression in bacterial colonies

These validation steps ensure that experimental results accurately reflect DnaK biology rather than non-specific interactions.

What methods are effective for detecting surface-expressed DnaK in bacteria?

Surface-expressed DnaK can be detected using several complementary approaches:

• Colony blot analysis:

  • Place PVDF membranes on bacterial colonies for 5 minutes

  • Block membranes with TBS containing 5% skim milk

  • Incubate with anti-DnaK serum (1:1,000 dilution)

  • Detect using appropriate HRP-conjugated secondary antibody

  • Include positive controls (such as GAPDH antibody) whose surface expression has been previously confirmed

• Immunofluorescence assay:

  • Fix bacterial cells with cold ethanol for 30 minutes at 4°C

  • Block with PBS containing 5% BSA for 1 hour at 37°C

  • Incubate with rabbit anti-DnaK serum (1:500 dilution)

  • Detect using FITC-labeled anti-rabbit IgG (1:500 dilution)

  • Counterstain nuclei with DAPI and visualize using fluorescence microscopy

• Flow cytometry:

  • Incubate non-permeabilized bacterial cells with anti-DnaK antibody

  • Detect using fluorophore-conjugated secondary antibody

  • Compare surface staining with total (permeabilized) staining to confirm surface localization

These methods can reveal DnaK's moonlighting functions on bacterial surfaces, including adhesion to host cells and extracellular matrix components.

How can DnaK antibodies be used to study the role of bacterial DnaK in cancer therapy resistance?

Recent research has revealed that bacterial DnaK can reduce the efficacy of anti-cancer drugs. DnaK antibodies are valuable tools for investigating this phenomenon:

• Detection of bacterial DnaK in tumor microenvironment:

  • Use immunohistochemistry with anti-DnaK antibodies on tumor sections to detect cancer-associated bacteria expressing DnaK

  • Correlate DnaK levels with response to chemotherapy in patient samples

  • Distinguish bacterial DnaK from human Hsp70 using specific antibodies

• Mechanistic studies:

  • Use Western blotting to detect DnaK-p53 interactions in cancer cells exposed to bacterial DnaK

  • Apply immunofluorescence to track intracellular localization of DnaK in cancer cells

  • Monitor changes in DnaK levels following chemotherapy treatment

• Therapeutic targeting:

  • Test DnaK-binding peptides like ARV-1502 to restore drug sensitivity

  • Research has shown that both Mycoplasma fermentans and Fusobacterium nucleatum DnaKs reduce effectiveness of cisplatin and 5FU, while ARV-1502 peptide restored drug efficacy

  • In mouse models expressing bacterial DnaK, treatment with DnaK inhibitor ARV-1502 improved anti-cancer effect of cisplatin and 5FU by approximately 25%

These applications highlight the potential for DnaK antibodies in developing personalized microbiome-based cancer therapies.

What approaches can be used to study DnaK dimerization and its functional significance?

Research has demonstrated that DnaK forms functional dimers essential for its interaction with Hsp40. Methods to study this include:

• Mutation analysis combined with antibody detection:

  • Introduce mutations (R56A, T301A, N537A, D540A) that affect dimer formation

  • Use Western blot with anti-DnaK antibody (mAb 8E2/2) to confirm protein expression

  • Correlate dimer formation with chaperone activity

• Biochemical characterization:

  • Analyze ATP-induced allosteric coupling in dimer mutants

  • Examine co-chaperone interactions, particularly with Hsp40 and GrpE

  • Research has shown that mutations compromising dimer formation specifically affect interaction with Hsp40 co-chaperone

• Functional assays:

  • Test chaperone activity using aggregation prevention assays

  • Assess in vivo function through growth tests under stress conditions

  • Compare ATP hydrolysis rates between wild-type and dimer-deficient mutants

These approaches provide insight into how DnaK dimerization affects its cellular functions and interactions with other chaperone system components.

How can peptide binding to DnaK be accurately detected and characterized?

Several methodological approaches can be used to study DnaK-peptide interactions:

• Cellulose-immobilized peptide binding assays:

  • Immobilize synthetic peptides on cellulose membranes (PepSpot membranes)

  • Incubate membranes with purified DnaK protein (typically 100 nM)

  • Detect bound DnaK using specific antibodies (mouse monoclonal anti-DnaK antibody clone 8E2/2)

  • Visualize using HRP-conjugated secondary antibody and chemiluminescent detection

• Computational prediction and validation:

  • Use algorithms like TANGO to predict potential DnaK binding sites

  • Design peptide arrays based on predictions

  • Validate binding experimentally using antibody detection approaches

• Therapeutic peptide interaction studies:

  • Test binding of peptides like ARV-1502 to DnaK

  • Evaluate effects on DnaK ATPase activity

  • ARV-1502 (derived from pyrrhocoricin and drosocin) binds to DnaK substrate-binding domain and reduces its ATPase activity

These methods allow for accurate characterization of DnaK-peptide interactions, which is crucial for understanding DnaK function and developing therapeutic strategies targeting this protein.

What are common issues when working with DnaK antibodies and how can they be resolved?

Researchers may encounter several challenges when working with DnaK antibodies:

• Cross-reactivity issues:

  • Problem: Antibody cross-reacts with human Hsp70 or other bacterial heat shock proteins

  • Solution: Use carefully validated antibodies with demonstrated specificity; include appropriate controls; perform peptide competition assays

• Weak or inconsistent signal in Western blots:

  • Problem: Poor antibody binding or low protein abundance

  • Solution: Optimize antibody concentration (try 1:500-1:5000 range for polyclonal antibodies) ; increase protein loading; extend primary antibody incubation time; use enhanced detection systems

• False positives in immunofluorescence:

  • Problem: Non-specific binding to cell surface structures

  • Solution: Include additional blocking steps; use more stringent washing; include appropriate controls (preimmune serum, isotype controls)

• Variable expression levels:

  • Problem: DnaK expression varies with growth conditions

  • Solution: Standardize growth conditions; induce stress response if needed; quantify using recombinant protein standards

How do I design experiments to investigate DnaK as a potential antibiotic target?

DnaK's role in bacterial stress response and survival makes it a promising antibiotic target. Research approaches include:

• Hot spot residue analysis:

  • Identify critical residues in DnaK structure using bioinformatics

  • Focus on residues unique to bacterial DnaK versus human Hsp70

  • DnaK mutations increase bacterial sensitivity to fluoroquinolones, oxacillin, and methicillin in normally resistant strains

• Inhibitor screening and validation:

  • Screen for small molecules or peptides that bind DnaK

  • Validate binding using antibody-based displacement assays

  • Test effects on DnaK chaperone function and ATPase activity

• Phenotypic assays:

  • Assess bacterial survival under antibiotic stress with/without DnaK inhibitors

  • Monitor changes in DnaK expression using Western blot with anti-DnaK antibodies

  • Evaluate synergistic effects between DnaK inhibitors and conventional antibiotics

• In vivo studies:

  • Test efficacy and toxicity in animal infection models

  • Use DnaK antibodies to confirm target engagement

Research suggests DnaK inhibition could be particularly effective against antibiotic-resistant pathogens like Acinetobacter baumannii, where the heat-shock response is involved in resistance mechanisms .

What considerations are important when developing immunotherapeutic applications using DnaK?

DnaK has potential immunotherapeutic applications, requiring several methodological considerations:

• Recombinant DnaK production and quality control:

  • Express in suitable bacterial systems with appropriate tags for purification

  • Confirm identity and activity using Western blot with anti-DnaK antibodies (8E2/2)

  • Verify purity using SDS-PAGE and assess endotoxin levels

• Delivery systems development:

  • Encapsulate DnaK in appropriate carriers (e.g., calcium alginate microparticles)

  • Characterize particle size and encapsulation efficiency

  • Research has shown that 200 mg (wet weight) of microparticles can contain 300 μg of antigen (1.5 μg antigen per mg microparticles)

• Immunogenicity assessment:

  • Monitor antibody development using ELISA with anti-DnaK serum

  • Research shows DnaK elicits antibody responses in immunized animals, indicating good immunogenicity

• Application-specific considerations:

  • For fish immunostimulation, oral administration protocols have been established

  • For cancer therapy approaches, combination with chemotherapeutic agents requires careful timing protocols

These considerations ensure effective development of DnaK-based immunotherapeutic approaches with appropriate quality control and validation.

What emerging technologies could enhance DnaK antibody applications in research?

Several emerging technologies hold promise for advancing DnaK antibody research:

• Single-cell protein analysis:

  • Apply mass cytometry to analyze DnaK levels in heterogeneous bacterial populations

  • Use single-cell Western blotting to detect DnaK in individual cells

  • Correlate DnaK levels with antibiotic resistance phenotypes at single-cell resolution

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize DnaK distribution and interactions

  • Live-cell imaging using fluorescently-tagged antibody fragments to track DnaK dynamics

  • Correlative light and electron microscopy to link DnaK localization with ultrastructural features

• Antibody engineering approaches:

  • Develop nanobodies against DnaK for improved tissue penetration

  • Create bispecific antibodies targeting DnaK and complementary bacterial targets

  • Engineer antibody-drug conjugates for targeted delivery to bacteria expressing surface DnaK

These technologies could significantly expand our understanding of DnaK biology and enhance therapeutic targeting approaches.

How might DnaK antibodies contribute to microbiome-based personalized medicine?

Recent research suggests important applications of DnaK antibodies in personalized medicine:

• Cancer therapy optimization:

  • Screen patient tumors for cancer-associated bacteria expressing DnaK

  • Identify patients likely to experience reduced chemotherapy efficacy due to bacterial DnaK

  • TCGA dataset analysis has revealed correlation between bacteria carrying DnaK (like F. nucleatum) and response to chemotherapy

• Diagnostic applications:

  • Develop antibody-based assays to detect specific bacterial DnaKs in clinical samples

  • Distinguish between different bacterial species based on DnaK epitope patterns

  • Create rapid diagnostic tools for bacterial infections based on DnaK detection

• Therapeutic monitoring:

  • Use anti-DnaK antibodies to monitor microbiome changes during treatment

  • Track efficacy of DnaK-targeting therapeutic approaches

  • Monitor development of anti-DnaK immune responses

These applications highlight the potential for DnaK antibodies to contribute to more personalized and effective therapeutic approaches in the context of the human microbiome.