DNAJB6 Antibody

What are the main applications of DNAJB6 antibodies in research?

DNAJB6 antibodies are versatile research tools with multiple applications in molecular and cellular biology. The primary applications include:

• Western Blotting (WB): Detects DNAJB6 protein in cell or tissue lysates with high specificity. Most DNAJB6 antibodies show reactivity in WB applications with recommended dilutions typically ranging from 1:2000 to 1:8000 .

• Immunoprecipitation (IP): Isolates DNAJB6 protein complexes from cell lysates to study protein-protein interactions, particularly useful when examining DNAJB6's interactions with other chaperone proteins .

• Immunofluorescence (IF): Visualizes subcellular localization of DNAJB6 in fixed cells, enabling distinction between nuclear DNAJB6a and cytoplasmic DNAJB6b isoforms .

• Immunohistochemistry (IHC): Detects DNAJB6 expression in tissue sections, with most antibodies requiring antigen retrieval with TE buffer (pH 9.0) or citrate buffer (pH 6.0) .

• Enzyme-linked Immunosorbent Assay (ELISA): Quantifies DNAJB6 levels in various biological samples .

• Flow Cytometry (FACS): Analyzes DNAJB6 expression in individual cells within heterogeneous cell populations .

Multiple validated DNAJB6 antibodies are available with reactivity to human, mouse, and rat proteins, making them suitable for diverse experimental models .

How do I select the appropriate DNAJB6 antibody for my research?

Selecting the optimal DNAJB6 antibody requires careful consideration of several key factors:

1. Isoform specificity:

• DNAJB6 exists in two main spliced isoforms (DNAJB6a and DNAJB6b) with distinct subcellular localizations and functions

• For isoform-specific detection, select antibodies targeting unique regions, such as anti-DNAJB6b antibodies that recognize the CKEQLLRLDNK sequence at amino acids 232-240

• General DNAJB6 antibodies typically recognize common epitopes present in both isoforms

2. Application compatibility:

• Verify the antibody is validated for your specific application (WB, IP, IF, IHC, ELISA, or FACS)

• Some antibodies perform better in certain applications than others; for example, monoclonal DNAJB6 antibodies like B-5 (sc-365574) are validated for WB, IP, IF, and ELISA

3. Species reactivity:

• Confirm reactivity with your experimental model (human, mouse, rat, etc.)

• Many DNAJB6 antibodies detect conserved epitopes across species

4. Clonality considerations:

• Monoclonal antibodies offer high specificity but recognize single epitopes

• Polyclonal antibodies provide broader recognition but may have batch-to-batch variation

• Both types are available for DNAJB6 detection

5. Format and conjugation:

• Choose between unconjugated or conjugated antibodies (HRP, PE, FITC, Alexa Fluor®) based on your detection system

• For multiplex staining, consider fluorescently conjugated antibodies

Review product validation data, published literature, and supplementary resources to guide your selection decision .

What is the recommended protocol for using DNAJB6 antibodies in Western blotting?

The following protocol outlines the optimal steps for detecting DNAJB6 using Western blotting:

Sample preparation:

• Lyse cells or tissues in appropriate lysis buffer (RIPA buffer containing protease inhibitors is commonly used)

• Determine protein concentration using standard methods (BCA or Bradford assay)

• Prepare samples by mixing with Laemmli buffer and heating at 95°C for 5 minutes

Gel electrophoresis and transfer:

• Load 20-40 μg of protein per lane on 10-12% SDS-PAGE gels

• Separate proteins at 100-120V until adequate resolution is achieved

• Transfer proteins to PVDF or nitrocellulose membrane (wet transfer recommended)

Antibody incubation:

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

• Incubate with primary DNAJB6 antibody at recommended dilution (typically 1:2000-1:8000) overnight at 4°C

• Wash membrane 3-5 times with TBST, 5 minutes each

• Incubate with appropriate HRP-conjugated secondary antibody for 1 hour at room temperature

• Wash membrane 3-5 times with TBST, 5 minutes each

Detection:

• Apply ECL substrate and detect signal using film or digital imaging systems

• Expected molecular weights:

  • DNAJB6a: approximately 38 kDa

  • DNAJB6b: approximately 27 kDa

Notes:

• For optimal isoform separation, use 10% gels and extend running time

• When analyzing DNAJB6 protein half-life, cycloheximide chase assays can be employed with Western blotting as the detection method

• For validation, include positive controls (brain tissue extracts show high expression of DNAJB6)

What are the troubleshooting steps for DNAJB6 antibody immunostaining?

When experiencing issues with DNAJB6 immunostaining, consider the following troubleshooting approaches:

1. High background or non-specific staining:

• Increase blocking time or concentration (try 5% normal serum matching the species of secondary antibody)

• Reduce primary antibody concentration (test serial dilutions)

• Include additional washing steps with 0.025-0.5% Triton X-100 in PBS

• Use appropriate controls (secondary antibody only, isotype controls)

2. Weak or no signal:

• Optimize antigen retrieval method:

  • For DNAJB6 in tissue sections, use Tris/EDTA buffer (10 mM Tris-HCl, 1 mM EDTA, pH 9.0) at 80°C for 30 minutes

  • Alternatively, try citrate buffer (pH 6.0) for certain tissue types

• Increase primary antibody concentration or incubation time

• Test different fixation methods (4% PFA for 20 minutes is standard for cultured cells)

• For cells, include membrane permeabilization step with 0.5% Triton-X-100 for 5 minutes

3. Unexpected staining pattern:

• Verify antibody specificity (validate with KO/KD controls)

• Consider isoform-specific staining - DNAJB6a is nuclear while DNAJB6b is cytoplasmic

• F-actin counterstain (phalloidin-atto550) can help visualize cell boundaries

4. Tissue-specific considerations:

• For brain tissue, co-staining with tyrosine hydroxylase (TH) can help identify specific neuronal populations

• When staining disease models (e.g., synucleinopathies), consider that DNAJB6b may be downregulated

5. Technical optimization:

• For immunofluorescence, minimize exposure to light during secondary antibody incubation

• Consider using automated staining platforms for consistent results

• Document all optimization steps systematically

For difficult samples, pre-absorption of antibody with antigenic peptide can be used as a specificity control .

How can I distinguish between DNAJB6a and DNAJB6b isoforms in my experiments?

Distinguishing between DNAJB6 isoforms requires specific approaches given their high sequence similarity:

Isoform-specific antibodies:

• Use isoform-specific antibodies like anti-DNAJB6b antibodies that target unique C-terminal sequences (amino acids 232-240: CKEQLLRLDNK)

• Commercial antibodies may recognize both isoforms; verify specificity with recombinant proteins expressing either isoform

• For custom antibody generation, target the unique C-terminal regions of each isoform

Immunofluorescence patterns:

• DNAJB6a localizes predominantly to the nucleus

• DNAJB6b shows cytoplasmic distribution

• Counterstain with DAPI for nuclear visualization and F-actin stain (phalloidin-atto550) for cytoplasmic boundaries

• Co-localization studies with nuclear and cytoplasmic markers confirm isoform identities

Western blotting differentiation:

• DNAJB6a appears at approximately 38 kDa

• DNAJB6b appears at approximately 27 kDa

• Use 10-12% SDS-PAGE gels with extended running time for optimal separation

• Include appropriate positive controls (brain tissue expresses both isoforms)

Functional validation:

• DNAJB6a effectively suppresses nuclear polyQ protein aggregation

• DNAJB6b is a potent suppressor of cytoplasmic polyQ aggregation

• Design experiments testing these differential activities

RT-PCR approach:

• Design primers flanking the alternatively spliced regions

• Amplification produces distinct band sizes for each isoform

• Combine with Western blotting for comprehensive analysis

Implementing these strategies allows precise discrimination between the functionally distinct DNAJB6 isoforms, critical for accurately interpreting research findings .

What are the best methods to study DNAJB6 interactions with other proteins?

Multiple complementary techniques can effectively characterize DNAJB6 protein-protein interactions:

Co-immunoprecipitation (Co-IP):

• Use DNAJB6 antibodies coupled to agarose beads for pull-down experiments

• For higher specificity, use DNAJB6 Antibody AC (sc-365574 AC) containing 25% agarose

• After immunoprecipitation, analyze complexes by Western blotting with antibodies against potential interaction partners

• Verify interactions bidirectionally by immunoprecipitating with antibodies against the partner protein

• Controls should include non-specific IgG and DNAJB6 or partner antibodies alone

Proximity ligation assay (PLA):

• Enables visualization of protein interactions in situ with single-molecule sensitivity

• Requires specific antibodies raised in different species against DNAJB6 and its potential partners

• Particularly useful for detecting interactions between DNAJB6 and other chaperones like Hsp70, BAG3, and HSPB8

Bimolecular fluorescence complementation (BiFC):

• Express DNAJB6 and potential interactor fused to complementary fragments of fluorescent proteins

• Interaction brings fragments together, restoring fluorescence

• Allows visualization of interaction sites within cells

Mass spectrometry-based approaches:

• Tandem affinity purification coupled with mass spectrometry identifies novel interaction partners

• SILAC or TMT labeling enables quantitative comparison of interactomes between wild-type and mutant DNAJB6

• Particularly valuable for studying disease-causing mutations that may alter protein interactions

Yeast two-hybrid screening:

• Identifies direct protein-protein interactions

• Can be used to screen libraries for novel DNAJB6 interactors

• Validate hits with other methods due to potential false positives

In vitro binding assays:

• Recombinant DNAJB6 and potential partners can be used in pull-down assays

• Surface plasmon resonance (SPR) quantifies binding kinetics and affinities

• Especially important for characterizing DNAJB6's interaction with Hsp70 and its role in chaperoning activity

These methodologies have revealed DNAJB6's interactions with key partners in protein quality control pathways, including components of the chaperone-assisted selective autophagy (CASA) complex .

How can I assess the functional impact of DNAJB6 mutations using antibody-based methods?

Investigating DNAJB6 mutations requires a comprehensive approach combining multiple antibody-based techniques:

Protein stability and turnover:

• Cycloheximide chase assay: Transfect cells with wild-type or mutant DNAJB6, block protein synthesis with cycloheximide, and quantify remaining protein over time using Western blotting

• Research shows mutations p.Phe93Leu and p.Phe89Ile significantly decrease DNAJB6 turnover rates

• Complement with proteasome inhibitors (lactacystin) or lysosome inhibitors (bafilomycin A1) to determine degradation pathways

Subcellular localization:

• Immunofluorescence microscopy to detect altered localization patterns of mutant DNAJB6

• Co-staining with organelle markers (nuclear, ER, mitochondrial) to identify mislocalization

• Live-cell imaging with fluorescently-tagged DNAJB6 variants to track dynamic localization changes

Protein aggregation:

• Filter trap assays with anti-DNAJB6 antibodies to detect SDS-insoluble aggregates

• Immunofluorescence to visualize aggregation patterns and co-localization with other proteins

• Sequential extraction protocols to assess solubility changes in mutant DNAJB6

Chaperone activity:

• Luciferase refolding assays with immunoprecipitation of DNAJB6 complexes

• Polyglutamine aggregation suppression assays (nuclear vs. cytoplasmic) to evaluate isoform-specific functions

• Co-immunoprecipitation to assess Hsp70 binding efficiency of mutant DNAJB6

Autophagy dysregulation:

• Immunoblotting for autophagy markers (LC3-II, p62/SQSTM1) in cells expressing mutant DNAJB6

• Immunofluorescence co-localization studies with autophagy markers

• Analysis of CASA complex formation using proximity ligation assays

Disease model analysis:

• Immunohistochemistry in patient samples or animal models with DNAJB6 mutations

• DNAJB6 expression levels by quantitative ELISA in clinical specimens

• Mitochondrial dysfunction assessment via co-localization with mitochondrial markers

These methodologies have revealed that DNAJB6 mutations associated with limb-girdle muscular dystrophy (LGMD D1) cause protein accumulation and disrupt protein homeostasis, potentially contributing to mitochondrial abnormalities and muscle weakness .

What are the considerations for studying DNAJB6 in neurodegenerative disease models?

Investigating DNAJB6 in neurodegenerative disease contexts requires specialized approaches:

Tissue-specific considerations:

• Brain tissue requires optimized fixation and antigen retrieval protocols:

  • Tris/EDTA buffer (10 mM Tris-HCl, 1 mM EDTA, pH 9.0) at 80°C for 30 minutes

  • Perform additional permeabilization with 0.025% Triton X-100 in PBS

• Use co-staining with neuronal markers (e.g., tyrosine hydroxylase) to identify specific populations

Pathological protein aggregates:

• DNAJB6 is present in Lewy bodies in Parkinson's disease patients

• Use dual immunofluorescence to co-localize DNAJB6 with α-synuclein, tau, or polyQ proteins

• Apply sequential extraction protocols to evaluate DNAJB6 in soluble versus insoluble fractions

Isoform-specific alterations:

• DNAJB6b is downregulated in synucleinopathies

• Use isoform-specific antibodies for precise quantification

• Apply quantitative ELISA methods developed specifically for DNAJB6 detection

Protein-protein interactions in disease:

• Investigate DNAJB6 interaction with:

  • α-synuclein in Parkinson's disease models

  • Polyglutamine proteins in Huntington's disease

  • RNA-binding proteins (TDP-43, hnRNPA1, hnRNPA2)

• Use proximity ligation assays in tissue sections for in situ interaction detection

Functional rescue experiments:

• Overexpress wild-type DNAJB6 in disease models and assess:

  • Restoration of mitophagy in Parkinson's models (DNAJB6 promotes relocation of Parkin and LC3 to depolarized mitochondria)

  • Suppression of polyQ aggregation (DNAJB6 requires C-terminal domain, amino acids 152-232)

  • Impact on autophagy markers and protein clearance

Autophagy pathway involvement:

• Monitor DNAJB6's interaction with chaperone-assisted selective autophagy (CASA) components

• Assess co-localization with LC3-positive autophagosomes

• Evaluate impact of autophagy modulators (e.g., LiCl, which may work through GSK3β inhibition and autophagy activation)

These approaches have revealed that DNAJB6 plays protective roles in neurodegenerative diseases by preventing protein aggregation and promoting clearance of misfolded proteins .

What controls should be included when using DNAJB6 antibodies?

Comprehensive control strategies ensure reliable and interpretable results when using DNAJB6 antibodies:

Positive controls:

• Tissues/cells known to express DNAJB6:

  • Brain tissue (highest expression levels)

  • Jurkat cells, HEK-293 cells, HeLa cells

  • Neuro-2a cells for mouse studies

• Recombinant DNAJB6 protein for Western blotting standardization

• DNAJB6-overexpressing cells as technical positive controls

Negative controls:

• Primary antibody omission (secondary antibody only)

• Isotype control antibodies matching the DNAJB6 antibody host species and class

• DNAJB6 knockdown/knockout cells or tissues (validated by genotyping)

• Non-expressing tissues (use tissue panels to identify suitable negative controls)

Specificity controls:

• Pre-absorption with immunizing peptide should abolish specific signal

• Comparison of staining patterns between different DNAJB6 antibodies targeting distinct epitopes

• Western blotting to confirm single bands at expected molecular weights (27 kDa for DNAJB6b, 38 kDa for DNAJB6a)

Isoform validation:

• Compare antibodies targeting common regions versus isoform-specific regions

• Subcellular localization patterns (nuclear for DNAJB6a, cytoplasmic for DNAJB6b)

• Simultaneous detection with isoform-specific primers in RT-PCR

Application-specific controls:

• For immunoprecipitation: IgG control and DNAJB6 antibody alone

• For immunofluorescence: Counterstains to verify subcellular localization (DAPI for nucleus, phalloidin for cytoskeleton)

• For FACS: Single-stained and unstained populations

Disease model controls:

• Age-matched and gender-matched controls for patient samples

• Wild-type littermates for transgenic animal models

• Vehicle-treated samples for drug intervention studies

Rigorous implementation of these controls ensures that observed signals represent genuine DNAJB6 detection rather than technical artifacts or non-specific binding.

How can I quantify DNAJB6 expression levels in tissue samples?

Multiple complementary approaches enable accurate quantification of DNAJB6 expression:

Western blotting quantification:

• Use standard curves with recombinant DNAJB6 protein

• Normalize DNAJB6 signal to housekeeping proteins (β-actin, GAPDH, or α-tubulin)

• Employ image analysis software for densitometry (ImageJ, Image Lab)

• For isoform-specific quantification, distinguish 27 kDa (DNAJB6b) from 38 kDa (DNAJB6a) bands

• Include multiple technical and biological replicates (n≥3)

DNAJB6-specific ELISA:

• Novel quantitative ELISA methods developed specifically for DNAJB6 detection provide greater sensitivity

• Establish standard curves using purified recombinant DNAJB6

• For isoform-specific quantification, use capture or detection antibodies targeting unique epitopes

• Validate results against Western blotting measurements

Immunohistochemistry/Immunofluorescence quantification:

• Digital image analysis of stained tissue sections

• Measure:

  • Staining intensity (mean fluorescence intensity)

  • Percentage of positive cells

  • Subcellular distribution patterns

• Use automated tissue analysis platforms for consistent results

• Consider regional variations in expression, particularly in brain tissue

Flow cytometry:

• Quantify DNAJB6 expression in single cells within heterogeneous populations

• Establish gating strategies based on negative controls

• Calculate mean fluorescence intensity (MFI) for population-level comparisons

• Particularly useful for blood cells or disaggregated tissue samples

qRT-PCR for mRNA quantification:

• Design primers specific to DNAJB6 (for total expression) or spanning unique regions (for isoform-specific detection)

• Normalize to stable reference genes

• Correlate mRNA levels with protein expression to assess post-transcriptional regulation

• Note that protein levels may not directly correlate with mRNA levels due to post-transcriptional regulation

Mass spectrometry-based proteomics:

• Selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) for absolute quantification

• Use stable isotope-labeled peptide standards for precise measurement

• Can distinguish isoforms based on unique peptide sequences

These methods have revealed differential expression patterns of DNAJB6 in disease states, such as downregulation of DNAJB6b in synucleinopathies and altered expression in cancer tissues .

What are the considerations for generating and validating custom DNAJB6 antibodies?

Creating custom DNAJB6 antibodies requires careful planning and comprehensive validation:

Antigen design strategies:

• For pan-DNAJB6 antibodies:

  • Target conserved regions between isoforms, typically within amino acids 1-217

  • The J-domain (amino acids ~1-70) is highly conserved among DnaJ proteins and may cause cross-reactivity

• For isoform-specific antibodies:

  • DNAJB6a-specific: Target unique C-terminal region

  • DNAJB6b-specific: Target the unique C-terminal sequence (amino acids 232-240: CKEQLLRLDNK)

• Avoid regions with post-translational modifications that might mask epitopes

Immunization considerations:

• Conjugate peptides to carrier proteins (KLH, BSA) to enhance immunogenicity

• For DNAJB6b-specific antibodies, include an N-terminal cysteine for conjugation

• Use multiple immunization protocols with varied schedules (5 doses over 12 weeks shows good results)

• Consider host species that differ from experimental models to minimize background

Purification strategies:

• Two-step purification process:

  • Protein G/A purification for IgG isolation

  • Affinity purification using peptide-linked agarose matrix

• Elute bound antibodies with glycine pH 2.7 into Tris-containing vials

• Buffer exchange into PBS by gel filtration

Validation requirements:

• Western blotting: Confirm single bands at expected molecular weights (27 kDa for DNAJB6b, 38 kDa for DNAJB6a)

• Immunoprecipitation: Verify ability to pull down endogenous DNAJB6

• Immunofluorescence: Confirm expected subcellular localization patterns

• Knockout/knockdown controls: Test antibody specificity in DNAJB6-depleted samples

• Cross-reactivity testing: Evaluate against related DNAJ family proteins

• Peptide competition assays: Pre-incubation with immunizing peptide should abolish signal

Documentation requirements:

• Detailed information on immunogen sequence and position

• Host species and clonality

• Validation data across multiple applications

• Recommended storage conditions and working dilutions

• Species reactivity information

Quality control measures:

• Batch-to-batch consistency testing

• Stability testing under various storage conditions

• Application-specific optimization

• Detailed documentation of validation experiments

Successful custom antibody generation has enabled critical discoveries, such as the development of DNAJB6b-specific antibodies that demonstrated downregulation of this isoform in synucleinopathies .

How does fixation and sample preparation affect DNAJB6 antibody performance?

Optimal fixation and sample preparation protocols significantly impact DNAJB6 detection quality:

Fixation considerations:

• For cell cultures:

  • 4% paraformaldehyde (PFA) for 20 minutes at 4°C preserves DNAJB6 antigenicity while maintaining cellular architecture

  • Methanol fixation may be superior for preserving certain epitopes but can disrupt membrane structures

• For tissue sections:

  • 4% PFA perfusion followed by post-fixation provides optimal results

  • Fixation time should be optimized based on tissue thickness (typically 24-48 hours)

  • Cryopreservation after fixation maintains many epitopes better than paraffin embedding

Antigen retrieval requirements:

• For paraffin-embedded tissues:

  • Tris/EDTA buffer (10 mM Tris-HCl, 1 mM EDTA, pH 9.0) at 80°C for 30 minutes is optimal for DNAJB6 detection

  • Alternative: citrate buffer (pH 6.0) for certain tissue types

  • Heat-induced epitope retrieval (pressure cooker or microwave) is generally superior to enzymatic methods

• For frozen sections:

  • Brief fixation in cold acetone may be sufficient

  • Mild antigen retrieval may still improve signal intensity

Permeabilization protocols:

• For cell cultures:

  • 0.5% Triton X-100 in PBS for 5 minutes effectively permeabilizes cell membranes

  • For more sensitive epitopes, use 0.025% Triton X-100 with extended incubation

• For tissue sections:

  • Permeabilization time may need extension for dense tissues

  • Detergent concentration should be optimized based on tissue type

Blocking conditions:

• 5% normal serum matching the species of secondary antibody reduces background

• 5% BSA in PBS is effective for most applications

• For highly autofluorescent tissues (brain, liver), include additional blocking steps:

  • Sudan Black B treatment

  • Autofluorescence quenching reagents

  • Photobleaching prior to antibody incubation

Special considerations for subcellular compartments:

• Nuclear DNAJB6a detection may require stronger permeabilization

• Cytoplasmic DNAJB6b detection benefits from counterstaining with F-actin (phalloidin-atto550)

• Mitochondrial co-localization studies require careful membrane preservation

Tissue-specific adaptations:

• Brain tissue: More aggressive antigen retrieval may be needed

• Muscle tissue: Extended fixation time can cause overfixation and epitope masking

• Clinical specimens: Standardize processing time to minimize pre-analytical variables

These optimized protocols have been critical for discoveries such as the identification of DNAJB6 in Lewy bodies in Parkinson's disease patients and the characterization of DNAJB6 expression patterns in various cell types.

How can DNAJB6 antibodies be used to study neurodegenerative diseases?

DNAJB6 antibodies serve as powerful tools for investigating neurodegenerative disorders through multiple approaches:

Parkinson's disease research:

• Immunohistochemistry reveals DNAJB6 localization in Lewy bodies

• Co-immunostaining with α-synuclein antibodies demonstrates direct association

• Proximity ligation assays detect interactions between DNAJB6 and parkinsonian proteins

• Western blotting shows DNAJB6b downregulation in synucleinopathies

• Use isoform-specific antibodies to track differential regulation of DNAJB6a versus DNAJB6b

Huntington's disease applications:

• Filter trap assays with anti-DNAJB6 antibodies assess polyQ protein aggregation suppression

• DNAJB6's C-terminal domain (amino acids 152-232) is crucial for suppressing polyQ aggregation

• Immunofluorescence distinguishes between DNAJB6a's role in nuclear versus DNAJB6b's role in cytoplasmic polyQ protein aggregation

• Co-immunoprecipitation reveals polyQ protein interactions with DNAJB6

Protein clearance mechanisms:

• Immunofluorescence tracks DNAJB6's role in mitophagy by promoting relocation of Parkin and LC3 to depolarized mitochondria

• Western blotting for autophagy markers (LC3-II, p62/SQSTM1) in relation to DNAJB6 levels

• Co-immunoprecipitation identifies interactions with chaperone-assisted selective autophagy (CASA) components

Stress response visualization:

• Stress granule co-localization studies using immunofluorescence

• Heat shock response experiments tracking DNAJB6 relocalization

• RNA-binding protein (TDP-43, hnRNPA1, hnRNPA2) co-localization with DNAJB6 under stress conditions

Therapeutic intervention assessment:

• Western blotting quantifies DNAJB6 upregulation in response to potential therapeutics

• Immunohistochemistry evaluates changes in aggregation patterns following treatment

• ELISA measures DNAJB6 levels in patient fluids as potential biomarkers

• LiCl treatment effects can be monitored through DNAJB6 antibody-based techniques

These methods have revealed critical roles for DNAJB6 in neurodegenerative diseases, including its presence in the core of Lewy bodies, upregulation in astrocytes of Parkinson's disease patients, and differential isoform activities in suppressing protein aggregation .

What role does DNAJB6 play in muscular dystrophies and how can antibodies help investigate this?

DNAJB6 antibodies are essential tools for investigating its critical role in limb-girdle muscular dystrophy (LGMD D1):

Pathological assessment:

• Immunohistochemistry identifies characteristic pathological features in muscle biopsies:

  • Rimmed vacuoles

  • Protein aggregates

  • Abnormal fiber morphology

• Co-immunostaining with sarcomeric proteins (desmin, α-actinin, FHL1) reveals their accumulation in aggregates

• Z-disc protein co-localization studies help characterize structural abnormalities

Mutation impact analysis:

• Western blotting quantifies mutant DNAJB6 protein stability:

  • Cycloheximide chase assays show mutations p.Phe93Leu and p.Phe89Ile significantly decrease DNAJB6 turnover rates

  • Proteasome inhibitor (lactacystin) and lysosome inhibitor (bafilomycin A1) treatments help determine degradation pathways

• Immunoprecipitation assesses how mutations affect interactions with other proteins

• Filter trap assays measure aggregation-prone protein accumulation

Mitochondrial dysfunction:

• Co-immunostaining with mitochondrial markers evaluates structural and functional changes

• Loss of DNAJB6 causes mitochondrial defects that contribute to muscle weakness in LGMD D1

• Proximity ligation assays detect altered interactions between DNAJB6 and mitochondrial proteins

Therapeutic monitoring:

• Western blotting quantifies changes in DNAJB6 levels following treatment

• LiCl treatment effects can be tracked through antibody-based methods:

  • Reduced aggregation

  • Improved muscle fiber morphology

  • Changes in downstream GSK3β signaling pathways

• Immunofluorescence assesses changes in protein aggregation patterns

Autophagy pathway investigation:

• Co-immunostaining with autophagy markers (LC3, p62/SQSTM1) reveals accumulation in patient muscle

• Proximity ligation assays detect DNAJB6 interactions with chaperone-assisted selective autophagy (CASA) components BAG3 and HSPB8

• Western blotting quantifies changes in autophagy markers following treatment

Animal model validation:

• Immunohistochemistry confirms DNAJB6 mutant mouse models recapitulate human pathology

• Western blotting compares protein levels between models and human samples

• Developmental studies track DNAJB6 expression during muscle formation and maintenance

These approaches have revealed that dominant mutations in DNAJB6 disrupt protein homeostasis through effects on Hsp70 function, leading to accumulation of misfolded proteins and myopathic changes .

How can researchers use DNAJB6 antibodies to investigate its role in cancer biology?

DNAJB6 antibodies enable multifaceted investigation of its emerging roles in cancer:

Differential expression analysis:

• Immunohistochemistry on tissue microarrays compares DNAJB6 levels across cancer types and stages

• Western blotting quantifies isoform-specific expression changes:

  • DNAJB6a levels are significantly reduced in aggressive breast cancer cells and advanced grade infiltrating ductal carcinoma

  • Isoform ratio alterations may serve as prognostic indicators

• ELISA methods provide quantitative measurement in patient samples

Subcellular localization studies:

• Immunofluorescence reveals compartment-specific changes in cancer cells

• Nuclear-cytoplasmic distribution of DNAJB6 isoforms may shift during cancer progression

• Co-localization with cancer-associated proteins identifies potential functional interactions

Functional mechanism investigation:

• Co-immunoprecipitation identifies cancer-specific protein interaction partners

• Chromatin immunoprecipitation (ChIP) assesses DNAJB6a's role in transcriptional regulation

• Proximity ligation assays visualize protein-protein interactions in situ

• Analysis of the secreted proteome from DNAJB6a-expressing cells reveals:

  • Reduced levels of tumor progression and metastasis-promoting secreted proteins

  • Increased levels of secreted metastasis suppressor proteins

Metastasis and invasion studies:

• Immunohistochemistry on primary tumors versus metastatic lesions tracks DNAJB6 changes

• DNAJB6a overexpression in aggressive breast cancer cell lines decreases migration and invasion

• Xenograft model analysis shows DNAJB6a restricts orthotopic tumor growth in nude mice

• Co-immunostaining with epithelial-mesenchymal transition markers correlates with invasion capacity

Clinical correlation analysis:

• Tissue microarray staining with DNAJB6 antibodies enables correlation with:

  • Patient survival outcomes

  • Response to specific therapies

  • Cancer molecular subtypes

• Multi-parameter immunofluorescence combines DNAJB6 with other prognostic markers

Therapeutic response monitoring:

• Western blotting tracks DNAJB6 expression changes following treatment

• Immunofluorescence assesses subcellular redistribution after drug exposure

• Co-immunoprecipitation identifies altered protein interactions in response to therapy

These approaches have revealed that DNAJB6a functions as a metastasis suppressor in breast cancer, with its expression significantly reduced in aggressive breast cancer cells, while therapeutic restoration of DNAJB6a expression may represent a potential treatment strategy .

What techniques can be used to study DNAJB6's role in viral infections?

DNAJB6 antibodies enable detailed investigation of its emerging roles in viral pathogenesis:

Viral infection models:

• Immunofluorescence tracks DNAJB6 redistribution during viral infection

• Co-localization studies with viral proteins identify direct interactions

• Western blotting quantifies expression changes in response to infection

• The chaperone-co-chaperone couple, Hsp70 and DNAJB6, play a determinative role in dengue virus (DENV) virion production

Virion assembly analysis:

• Immunoprecipitation of DNAJB6 complexes from infected cells identifies associated viral components

• Proximity ligation assays visualize DNAJB6-viral protein interactions in situ

• Subcellular fractionation followed by Western blotting locates DNAJB6 in relation to viral assembly sites

• Analysis of protein assembly processes responsible for maintaining viral proteostasis

Mechanistic investigation:

• Cycloheximide chase assays determine if DNAJB6 affects viral protein stability

• Co-immunoprecipitation identifies interactions with viral proteins and other host factors

• Time-course immunofluorescence tracks DNAJB6 dynamics throughout viral life cycle

• Assessment of how viruses highjack the chaperoning activity of DNAJB6

Therapeutic target exploration:

• Antibody-based screening identifies compounds that disrupt DNAJB6-viral protein interactions

• Western blotting quantifies changes in viral protein levels following modulation of DNAJB6 activity

• Immunofluorescence assesses viral replication efficiency in DNAJB6-modulated cells

• Co-immunoprecipitation evaluates therapeutic disruption of chaperone complexes

Host response characterization:

• Immunohistochemistry on infected tissues maps DNAJB6 distribution

• Flow cytometry quantifies DNAJB6 levels in infected versus uninfected cells

• Western blotting tracks post-translational modifications of DNAJB6 during infection

• Time-course analysis reveals dynamic changes in DNAJB6 expression and localization

Cross-viral comparisons:

• Comparative analysis across multiple virus families:

  • DENV (dengue virus) has been shown to utilize DNAJB6

  • Assessment of other viruses that may employ similar mechanisms

  • Identification of virus-specific versus general mechanisms

These approaches have revealed that DNAJB6 plays a crucial role in dengue virus propagation, with potential implications for other viral infections, suggesting that DNAJB6-virus interactions may represent promising drug targets for antiviral therapy .

How should DNAJB6 antibodies be validated for reproducible research?

A comprehensive validation strategy ensures reliable DNAJB6 detection across applications:

Specificity validation:

• Genetic approach:

  • Test antibody in DNAJB6 knockout/knockdown models

  • Compare signal in wild-type versus DNAJB6-deficient samples

  • Rescue experiments with DNAJB6 re-expression

• Peptide competition:

  • Pre-incubate antibody with immunizing peptide before application

  • Specific signal should be blocked or significantly reduced

• Orthogonal detection:

  • Verify consistent results using multiple antibodies targeting different DNAJB6 epitopes

  • Compare monoclonal versus polyclonal antibody performance

Application-specific validation:

• Western blotting:

  • Confirm single bands at expected molecular weights (27 kDa for DNAJB6b, 38 kDa for DNAJB6a)

  • Validate in multiple cell/tissue types with varying expression levels

  • Include positive controls (brain tissue) and negative controls (knockdown samples)

• Immunoprecipitation:

  • Verify pull-down of DNAJB6 by mass spectrometry

  • Confirm co-immunoprecipitation of known interaction partners

  • Include IgG control and DNAJB6 antibody alone controls

• Immunohistochemistry/Immunofluorescence:

  • Compare staining patterns with literature-reported localization

  • Verify subcellular distribution (nuclear for DNAJB6a, cytoplasmic for DNAJB6b)

  • Test multiple fixation and antigen retrieval protocols

Quantitative performance metrics:

• Sensitivity assessment:

  • Determine limit of detection using serial dilutions

  • Compare detection threshold across applications

• Dynamic range evaluation:

  • Test antibody performance across wide concentration ranges

  • Establish linear detection ranges for quantification

• Reproducibility testing:

  • Inter-lot consistency evaluation

  • Intra-laboratory and inter-laboratory reproducibility assessment

  • Stability over time under recommended storage conditions

Multi-parameter validation:

• Multiplexing compatibility:

  • Test performance in multi-color immunofluorescence

  • Validate with multiple detection systems (fluorescent, chromogenic)

• Cross-platform consistency:

  • Compare results between Western blotting, immunohistochemistry, and ELISA

  • Reconcile any discrepancies with biological explanations

Documentation standards:

• Comprehensive reporting of validation experiments

• Inclusion of positive and negative controls in published images

• Disclosure of antibody source, catalog number, lot number, and dilution

• Detailed methodological description enabling reproduction by other researchers

Implementation of these validation steps ensures reliable, reproducible DNAJB6 antibody performance across diverse experimental conditions.

What are the key considerations for designing experiments to study DNAJB6 function?

Effective experimental design for DNAJB6 functional studies requires careful planning:

Isoform-specific considerations:

• Design studies to distinguish between DNAJB6a and DNAJB6b functions:

  • DNAJB6a: Nuclear localization, transcriptional regulation, nuclear protein quality control

  • DNAJB6b: Cytoplasmic localization, protein aggregation suppression, stress response

• Use isoform-specific antibodies or tagged constructs for precise tracking

• Include both isoforms in functional analyses when possible

Loss-of-function approaches:

• siRNA/shRNA knockdown:

  • Design targeting sequences common to both isoforms or isoform-specific regions

  • Validate knockdown by Western blotting and qRT-PCR

  • Include scrambled siRNA controls

• CRISPR/Cas9 gene editing:

  • Design guide RNAs targeting critical functional domains

  • Create knockout and knock-in cell lines

  • Validate using DNAJB6 antibodies

• Dominant-negative mutants:

  • H31Q mutation disrupts J-domain function

  • Validate expression by Western blotting and functional impact by chaperone assays

Gain-of-function strategies:

• Overexpression systems:

  • Use inducible promoters for temporal control

  • Create stable cell lines for consistent expression

  • Include both wild-type and disease-associated mutant forms

  • Validate expression levels by Western blotting with DNAJB6 antibodies

• Domain-specific constructs:

  • J-domain (amino acids ~1-70)

  • Glycine/phenylalanine-rich region

  • C-terminal domain (amino acids 152-232) critical for polyQ aggregation suppression

Disease-relevant models:

• Neurodegenerative disease models:

  • PolyQ protein aggregation assays

  • α-synuclein aggregation models

  • Patient-derived iPSCs differentiated into neurons

• Muscular dystrophy models:

  • LGMD D1 patient-derived myoblasts

  • DNAJB6 mutant mice

  • C2C12 myoblast differentiation system

Protein quality control assays:

• Luciferase refolding assays to measure chaperone activity

• Filter trap assays for aggregation suppression

• Cycloheximide chase experiments for protein stability analysis

• Proteasome and autophagy inhibitor studies to determine degradation pathways

Stress response analyses:

• Heat shock experiments with temperature and duration optimization

• Oxidative stress exposure (H₂O₂, paraquat)

• ER stress induction (tunicamycin, thapsigargin)

• Time-course immunofluorescence to track DNAJB6 relocalization

Interaction studies:

• Co-immunoprecipitation with key partners (Hsp70, BAG3, HSPB8)

• Proximity ligation assays for in situ interaction visualization

• FRET/BRET approaches for real-time interaction dynamics

• Mass spectrometry-based interactome analysis

These design considerations enable comprehensive functional characterization of DNAJB6 across diverse cellular contexts and disease states.

How can DNAJB6 antibodies be used for biomarker development in neurodegenerative diseases?

DNAJB6 represents a promising biomarker candidate for neurodegenerative diseases, with antibody-based detection methods enabling clinical translation:

Tissue-based biomarker approaches:

• Immunohistochemistry on post-mortem brain tissue:

  • Quantify DNAJB6 expression patterns across disease states

  • Assess DNAJB6 localization in protein aggregates (Lewy bodies, polyQ inclusions)

  • Correlate with disease severity and progression markers

• Multi-parameter immunofluorescence:

  • Combine DNAJB6 with disease-specific markers (α-synuclein, tau, TDP-43)

  • Create diagnostic algorithms based on co-localization patterns

  • Digital pathology quantification for objective assessment

Fluid biomarker development:

• Quantitative ELISA methods for DNAJB6 detection in:

  • Cerebrospinal fluid (CSF)

  • Plasma/serum

  • Extracellular vesicles

• Isoform-specific quantification using antibodies targeting unique epitopes:

  • DNAJB6b downregulation in synucleinopathies could serve as a specific marker

  • DNAJB6a/DNAJB6b ratio may provide diagnostic information

• Multiplex immunoassays combining DNAJB6 with established neurodegeneration markers

Single-cell analysis approaches:

• Flow cytometry on peripheral blood mononuclear cells:

  • Measure DNAJB6 levels in specific immune cell populations

  • Correlate with disease state and progression

• Mass cytometry (CyTOF) for high-dimensional analysis:

  • Combine DNAJB6 with multiple cellular markers

  • Identify disease-specific cellular signatures

Digital biomarker integration:

• Correlate DNAJB6 levels with:

  • Neuroimaging data (MRI, PET)

  • Clinical scales and progression rates

  • Genetic risk factors

  • Other protein biomarkers

• Machine learning approaches to identify DNAJB6-based prediction models

Longitudinal monitoring applications:

• Serial sampling to track DNAJB6 changes during disease progression

• Assessment of treatment response using DNAJB6 as a pharmacodynamic marker

• Correlation with clinical outcomes and disease milestones

Validation requirements:

• Multi-center cohort studies with standardized protocols

• Analytical validation of DNAJB6 detection methods:

  • Precision, accuracy, reproducibility

  • Pre-analytical variable assessment

• Clinical validation against gold-standard diagnosis

These approaches build on research showing DNAJB6b downregulation in synucleinopathies and DNAJB6's presence in pathological protein aggregates, suggesting its potential as both a diagnostic and prognostic biomarker for neurodegenerative diseases.

What are the emerging technologies for studying DNAJB6 protein interactions and dynamics?

Cutting-edge technologies are transforming our ability to investigate DNAJB6's functions and interactions:

Advanced imaging approaches:

• Super-resolution microscopy:

  • STORM/PALM imaging achieves 10-20 nm resolution of DNAJB6 nanoscale organization

  • SIM reveals DNAJB6 distribution within subcellular structures

  • Expansion microscopy physically enlarges specimens for enhanced visualization

• Live-cell imaging techniques:

  • FRAP (Fluorescence Recovery After Photobleaching) measures DNAJB6 mobility

  • Single-molecule tracking follows individual DNAJB6 molecules in real-time

  • Optogenetic control of DNAJB6 localization and interactions

Proximity-based interaction detection:

• BioID/TurboID proximity labeling:

  • Fuse DNAJB6 to biotin ligase to identify proximal proteins

  • Map interaction neighborhoods in different cellular compartments

  • Compare interactomes between wild-type and mutant DNAJB6

• APEX2 proximity labeling:

  • Higher spatial and temporal resolution than BioID

  • Compatible with electron microscopy visualization

• Split-protein complementation assays:

  • NanoBiT for sensitive detection of protein-protein interactions

  • SPARK (Specific Protein Association tool giving transcriptional Readout with rapid Kinetics) for detecting transient interactions

Proteomics innovations:

• Crosslinking mass spectrometry (XL-MS):

  • Maps interaction interfaces between DNAJB6 and client proteins

  • Identifies conformational changes in disease-associated mutations

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Measures structural dynamics and conformational changes

  • Identifies regions involved in client binding

• Thermal proteome profiling:

  • Assesses DNAJB6 thermal stability changes upon client binding

  • Identifies small molecules that stabilize or destabilize DNAJB6

Single-cell technologies:

• Single-cell proteomics:

  • Measures DNAJB6 levels and modifications in individual cells

  • Correlates with cellular phenotypes and states

• Spatial transcriptomics combined with immunofluorescence:

  • Maps DNAJB6 protein expression alongside transcriptome data

  • Provides spatial context for DNAJB6 function

Structural biology approaches:

• Cryo-electron microscopy:

  • Determines structures of DNAJB6 complexes with client proteins and Hsp70

  • Visualizes conformational changes during chaperone cycle

• Integrative structural biology:

  • Combines multiple data types (SAXS, NMR, XL-MS, cryo-EM)

  • Creates comprehensive structural models of DNAJB6 function

Functional genomics integration:

• CRISPR screens with DNAJB6 antibody-based readouts

• Parallel reporter assays to study DNAJB6 transcriptional effects

• Synthetic genetic interaction mapping to identify functional pathways

These emerging technologies provide unprecedented insights into DNAJB6's dynamic functions across cellular contexts and disease states, enabling mechanistic understanding that may guide therapeutic development.