DNAJC4 Antibody

What is DNAJC4 and what cellular functions does it perform?

DNAJC4 is a member of the DNAJ/HSP40 family of proteins primarily involved in chaperone activity and protein folding. This 28-30 kDa protein (observed molecular weight) interacts with HSP70 and plays a crucial role in protein quality control of misfolded proteins . DNAJC4 is implicated in various cellular processes, including DNA damage repair, apoptosis, and cell proliferation . The protein has potential significance in research related to neurodegenerative diseases, cancer, and other conditions associated with protein misfolding .

What are the recommended dilutions for DNAJC4 antibodies across different experimental applications?

Based on validated protocols, the following dilutions are recommended for DNAJC4 antibodies:

These recommendations should be optimized for your specific experimental conditions .

In what cell lines or tissues has DNAJC4 antibody been validated?

DNAJC4 antibodies have been validated in multiple cell lines and tissues:

This demonstrated reactivity across multiple systems indicates the versatility of DNAJC4 antibodies for various experimental models .

What antigen retrieval methods are optimal for DNAJC4 immunohistochemistry?

For optimal antigen retrieval in IHC applications with DNAJC4 antibodies, the following methods are recommended:

• Primary method: TE buffer pH 9.0

• Alternative method: Citrate buffer pH 6.0

These retrieval conditions have been validated specifically for DNAJC4 detection in human breast cancer tissue . Optimization may be necessary depending on your specific tissue type and fixation method.

How should DNAJC4 antibodies be stored to maintain optimal activity?

DNAJC4 antibodies should be stored at -20°C for long-term preservation . The storage buffer typically contains:

• PBS with 0.02% sodium azide

• 50% glycerol at pH 7.3

Most antibodies are stable for one year after shipment when stored properly . Repeated freeze/thaw cycles should be avoided to maintain antibody performance . For short-term storage (less than one week), antibodies can be kept at 4°C after reconstitution or initial use .

What strategies can mitigate non-specific binding when using DNAJC4 antibodies?

To reduce non-specific binding in DNAJC4 antibody applications:

• Optimize blocking conditions (5% BSA or normal serum from the secondary antibody host species)

• Titrate primary antibody concentration (test multiple dilutions within the recommended range)

• Reduce incubation time or temperature if background is excessive

• Include additional washing steps with PBS-T (0.05-0.1% Tween-20)

• Pre-absorb the antibody with non-relevant tissue extracts if cross-reactivity is observed

For Western blot applications specifically, maintaining the antibody dilution within 1:500-1:2000 range has been shown to provide optimal signal-to-noise ratio .

How can DNAJC4 antibodies be validated using genetic knockout/knockdown systems?

A comprehensive validation strategy for DNAJC4 antibodies using genetic manipulation should include:

• CRISPR/Cas9 knockout validation:

  • Generate DNAJC4 knockout cell lines

  • Compare antibody signal between wild-type and knockout samples

  • Confirm complete absence of signal in knockout samples by Western blot

• siRNA/shRNA knockdown validation:

  • Use 2-3 different siRNA sequences targeting DNAJC4

  • Confirm knockdown efficiency by qRT-PCR (>80% reduction)

  • Demonstrate corresponding reduction in antibody signal intensity

  • Include scrambled/non-targeting controls

• Overexpression validation:

  • Transfect cells with tagged DNAJC4 construct

  • Confirm co-localization of anti-DNAJC4 antibody with tag-specific antibody

  • Verify increased signal in overexpressing cells

Several publications have successfully employed knockdown/knockout strategies to validate DNAJB4 antibodies, which use similar validation methodologies applicable to DNAJC4 .

What are the key differences between monoclonal and polyclonal DNAJC4 antibodies for specific research applications?

Currently, most commercially available DNAJC4 antibodies are polyclonal (rabbit) antibodies . These polyclonal antibodies have been successfully validated for multiple applications including WB, IHC, IF/ICC, and ELISA .

How can DNAJC4 antibodies be utilized in co-immunoprecipitation studies to identify protein interaction partners?

For effective co-immunoprecipitation of DNAJC4 and its interaction partners:

• Lysis buffer optimization:

  • Use gentle, non-denaturing buffers (e.g., RIPA without SDS or modified NP-40 buffer)

  • Include protease inhibitors and phosphatase inhibitors

  • Consider crosslinking if interactions are transient

• Antibody selection:

  • Choose antibodies validated for immunoprecipitation

  • Consider using 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

• Experimental design:

  • Include appropriate negative controls (non-specific IgG, lysate from DNAJC4 knockout cells)

  • Validate successful IP of DNAJC4 before proceeding to co-IP studies

  • Consider reverse co-IP to confirm interactions

• Analysis of interaction partners:

  • Western blot for suspected interaction partners (e.g., HSP70)

  • Mass spectrometry for unbiased identification of novel partners

  • Validate key interactions using alternative methods (proximity ligation assay, FRET)

The protocol can be adapted from successful co-IP studies using related DNAJB4 antibodies, which have been demonstrated to effectively immunoprecipitate their target proteins .

What experimental approaches can investigate DNAJC4's role in protein quality control pathways?

To study DNAJC4's function in protein quality control:

• Proteostasis stress models:

  • Heat shock experiments (41-43°C for 1-2 hours)

  • Proteasome inhibition (MG132 treatment)

  • ER stress induction (tunicamycin, thapsigargin)

  • Oxidative stress models (H₂O₂, paraquat)

• Protein aggregation studies:

  • Co-transfection with aggregation-prone proteins (polyQ, α-synuclein)

  • Monitoring aggregate formation with/without DNAJC4 manipulation

  • Filter trap assays to quantify insoluble protein fractions

• Client protein identification:

  • DNAJC4 immunoprecipitation followed by mass spectrometry

  • Proximity labeling approaches (BioID, APEX)

  • Analysis of the proteome in DNAJC4 knockout/knockdown cells

• Co-chaperone activity assessment:

  • In vitro ATPase assays with purified HSP70 and DNAJC4

  • FRET-based assays to monitor client protein interactions

  • Luciferase refolding assays with/without DNAJC4

These approaches can leverage DNAJC4 antibodies for detection, localization, and isolation of the protein and its complexes .

How can subcellular localization of DNAJC4 be accurately determined using immunofluorescence techniques?

For precise subcellular localization of DNAJC4:

• Sample preparation optimization:

  • Test multiple fixation methods (4% PFA, methanol, or combination)

  • Optimize permeabilization (0.1-0.5% Triton X-100 or 0.05-0.2% saponin)

  • Use antibody dilutions of 1:50-1:200 for optimal signal-to-noise ratio

• Co-localization with organelle markers:

  • Include established markers for relevant compartments (ER, Golgi, mitochondria)

  • Calculate Pearson's correlation coefficient or Manders' overlap coefficient

  • Perform Z-stack imaging for three-dimensional co-localization analysis

• Controls and validation:

  • Include peptide competition controls

  • Compare staining patterns in multiple cell types (A549, MCF-7, HeLa)

  • Validate with orthogonal approaches (subcellular fractionation)

• Advanced imaging techniques:

  • Super-resolution microscopy (STORM, STED) for precise localization

  • Live-cell imaging with fluorescently tagged DNAJC4 to confirm antibody staining patterns

DNAJC4 antibodies have been successfully used in immunofluorescence applications with A549 and MCF-7 cells, demonstrating specific subcellular staining patterns .

What considerations are important when designing experiments to study DNAJC4 in disease models?

When investigating DNAJC4 in disease contexts:

• Neurodegenerative disease models:

  • Evaluate DNAJC4 expression in patient-derived samples vs. controls

  • Assess co-localization with disease-specific protein aggregates

  • Test DNAJC4 modulation as a therapeutic approach in cellular models

• Cancer research applications:

  • Compare DNAJC4 expression between normal and cancer tissues

  • Correlate expression with clinical parameters and patient outcomes

  • Investigate how DNAJC4 affects cancer cell response to proteotoxic stress

• Experimental design considerations:

  • Include appropriate physiological controls

  • Consider cell type-specific effects (test multiple relevant cell lines)

  • Design time-course experiments to capture dynamic changes

  • Use concentration gradients for drug treatment studies

• Technical considerations:

  • Optimize antibody dilutions for each experimental system (1:20-1:200 for IHC)

  • Include positive control tissues with known DNAJC4 expression (breast cancer tissue, colon cancer tissue)

  • Consider using multiple antibodies targeting different epitopes for validation

DNAJC4 antibodies have been validated in cancer tissue contexts (breast and colon), making them suitable tools for oncology research applications .

What emerging techniques could enhance DNAJC4 research beyond traditional antibody applications?

Cutting-edge approaches for DNAJC4 research include:

• Proximity labeling proteomics:

  • BioID or TurboID fusion with DNAJC4 to identify proximal interactors

  • APEX2-based approaches for temporal resolution of interactions

  • Quantitative analysis of the DNAJC4 proximity interactome under stress conditions

• CRISPR-based approaches:

  • CRISPRi/CRISPRa for tunable DNAJC4 expression modulation

  • CRISPR screens to identify genetic interactions with DNAJC4

  • Knock-in of fluorescent tags at endogenous loci for physiological imaging

• Single-cell techniques:

  • scRNA-seq to identify cell populations with differential DNAJC4 expression

  • Antibody-based mass cytometry for protein-level analysis

  • Single-cell proteomics to correlate DNAJC4 with client proteins

• Structural biology integration:

  • Combine antibody epitope mapping with structural predictions

  • Develop conformation-specific antibodies to distinguish functional states

  • Structure-guided design of DNAJC4 modulators

These approaches can complement traditional antibody-based methods to provide deeper insights into DNAJC4 biology and function.

How should researchers approach the challenge of distinguishing between DNAJC4 and other closely related DnaJ family proteins?

To ensure specificity for DNAJC4 over other DnaJ family members:

• Antibody selection:

  • Choose antibodies raised against unique regions of DNAJC4

  • Avoid antibodies targeting highly conserved J-domains

  • Check cross-reactivity data with related proteins (e.g., DNAJB4)

• Validation strategies:

  • Test antibody specificity in overexpression systems with multiple DnaJ proteins

  • Perform peptide competition assays with DNAJC4-specific peptides

  • Include DNAJC4 knockout controls to confirm signal specificity

• Experimental design:

  • Include related DnaJ proteins (e.g., DNAJB4) as controls in expression studies

  • Use siRNA targeting DNAJC4-specific sequences to confirm specificity

  • Consider phylogenetic analysis when interpreting evolutionary conservation

• Bioinformatic approaches:

  • Perform sequence alignments to identify unique regions of DNAJC4

  • Design experiments targeting unique domains or post-translational modifications

  • Use available structural data to understand epitope accessibility

Researchers should be particularly careful to distinguish between DNAJC4 and DNAJB4, as both have been studied in similar contexts and may share functional redundancy .