DNAJC2 Antibody

What is DNAJC2 and why is it important in cellular research?

DNAJC2 (DnaJ Heat Shock Protein Family (Hsp40) Member C2) serves dual critical functions in cells, acting both as a molecular chaperone in the cytosol and as a chromatin regulator in the nucleus. As a cytosolic protein, it functions as a component of the ribosome-associated complex (RAC) involved in folding nascent polypeptides. In the nucleus, it mediates the switching from polycomb-repressed genes to an active state by being recruited to histone H2A ubiquitinated at Lys-119 .

DNAJC2 is also known by several alternative names, including:

• ZRF1 (Zuotin-related factor 1)

• ZUO1

• MPP11 (M-phase phosphoprotein 11)

• MPHOSPH11

Research interest in DNAJC2 has increased due to its associations with various diseases, particularly colorectal cancer and atherosclerotic conditions.

What applications are most suitable for DNAJC2 antibodies?

DNAJC2 antibodies have been validated for multiple laboratory applications:

Different antibodies may have specific optimization requirements depending on the sample type and experimental conditions.

What controls should be included when validating DNAJC2 antibodies?

When validating DNAJC2 antibodies, the following controls are essential:

• Positive controls: Cell lines with known high expression of DNAJC2 include HeLa, 293T (HEK-293T), and Jurkat cells . These have been consistently shown to express detectable levels of DNAJC2.

• Negative controls: Knockdown or knockout validation is ideal. Several studies have used DNAJC2 siRNA transfection in cell lines such as DLD-1 to validate antibody specificity .

• Recombinant protein: Using purified recombinant DNAJC2 protein as a positive control can help confirm antibody specificity .

• Loading controls: Standard loading controls such as β-actin should be used in Western blot applications to normalize expression levels .

Knockdown validation is particularly important as the calculated molecular weight of DNAJC2 is 72 kDa, but the observed molecular weight in Western blots is often 80/72 kDa, indicating potential post-translational modifications .

How should DNAJC2 antibodies be stored and handled?

Proper storage and handling of DNAJC2 antibodies is crucial for maintaining their activity:

• Storage temperature: Store at -20°C for long-term stability .

• Short-term storage: Some antibodies can be stored at 2-8°C for up to one month without detectable loss of activity .

• Avoid freeze-thaw cycles: Repeated freeze-thaw cycles can degrade antibody quality and should be avoided .

• Aliquoting: Upon receipt, aliquot antibodies to minimize freeze-thaw cycles .

• Buffer composition: Most DNAJC2 antibodies are formulated in PBS with glycerol (often 40-50%) and preservatives like sodium azide (0.02-0.05%) .

For optimal results, always refer to the specific manufacturer's recommendations, as formulations may vary between products.

What is the role of DNAJC2 in transcription-coupled nucleotide excision repair (TC-NER)?

Recent research has revealed that DNAJC2 plays a crucial role in TC-NER, which repairs UV-induced DNA damage on actively transcribed gene strands. Studies show that:

• DNAJC2 deficiency significantly reduces cell survival ability and transcription recovery rate following UV irradiation .

• As a cochaperone of HSC70, DNAJC2 interacts with CSB (Cockayne syndrome protein B) and promotes its degradation through the HSC70 chaperone-mediated autophagy (CMA) pathway .

• DNAJC2 knockout cells show significantly higher levels of cyclobutane pyrimidine dimers (CPDs, UV-induced DNA lesions) compared to control cells, particularly at 12-hour and 24-hour time points after UV exposure .

• The J domain of DNAJC2, which mediates its cochaperone activity, is essential for cellular response to UV-induced DNA damage .

Experimentally, DNAJC2's role in TC-NER can be assessed by measuring RNA synthesis recovery and strand-specific DNA repair using techniques such as PCR after UV irradiation, comparing wild-type cells to DNAJC2-deficient cells .

How is DNAJC2 expression dysregulated in colorectal cancer and what methods best detect this change?

DNAJC2 shows significant upregulation in colorectal cancer (CRC) and appears to play a role in tumor progression:

• Both mRNA and protein expression of DNAJC2 are significantly higher in CRC tissues compared to adjacent normal tissues .

• DNAJC2 expression levels correlate significantly with tumor size in CRC patients .

• Knockdown of DNAJC2 in CRC cell lines (such as DLD-1) reduces cell proliferation, as measured by CCK-8, EdU staining, and colony formation assays .

For optimal detection of DNAJC2 in CRC research:

• RT-qPCR: Best for quantifying mRNA expression differences between tumor and normal tissues. This method has successfully identified DNAJC2 upregulation in cohorts of CRC patients .

• Immunohistochemistry: Effective for visualizing protein expression within tissue context. Protocols typically use:

  • Primary antibody dilution: 1:400

  • Antigen retrieval methods

  • DAB (3,3'-diaminobenzidine) for visualization

  • Analysis of ≥5 randomly selected fields at magnifications of ×100 and ×400

• Western blotting: For protein quantification, using β-actin as a loading control. Typical protocol elements include:

  • Protein extraction using RIPA buffer

  • BCA protein assay for concentration determination

  • 10% SDS-PAGE for separation

  • Transfer to PVDF membranes

  • 10% BSA solution for blocking

  • Overnight primary antibody incubation at 4°C

What is the significance of elevated DNAJC2 autoantibodies in atherosclerotic diseases?

Elevated levels of autoantibodies against DNAJC2 (DNAJC2-Ab) have been identified as potential biomarkers for atherosclerotic diseases:

• DNAJC2-Ab levels are significantly higher in patients with transient ischemic attack (TIA), acute ischemic stroke (AIS), acute myocardial infarction (AMI), diabetes mellitus (DM), and chronic kidney disease (CKD) compared to healthy donors .

• Multivariate logistic regression analysis shows that DNAJC2-Ab levels have predictive odds ratios of:

  • 2.54 (95% CI: 1.36-4.74, p=0.0034) for TIA

  • 2.14 (95% CI: 1.39-3.30, p=0.0005) for AIS

• ROC analysis revealed the following AUC values:

  Condition	AUC Value
  TIA	0.6477
  AIS	0.6619
  AMI	0.6714
  DM	0.6765
  CKD type 1	0.8182
  CKD type 2	0.8232
  CKD type 3	0.7305

For detection of DNAJC2-Ab, researchers have used:

• SEREX (serological identification of antigens by recombinant cDNA expression cloning) for initial identification

• AlphaLISA (amplified luminescent proximity homogeneous assay-linked immunosorbent assay) for quantification in validation cohorts

• Western blotting with GST-DNAJC2 to confirm antibody presence in patient sera

The elevated levels of DNAJC2-Ab may be related to the expression of DnaJ family proteins in atherosclerotic plaques, suggesting their potential utility as predictive markers for atherosclerotic diseases .

How does miR-627-3p regulate DNAJC2 expression and what experimental approaches verify this relationship?

Research has established that DNAJC2 is negatively regulated by miR-627-3p, particularly in the context of colorectal cancer:

• miR-627-3p acts as a direct regulator of DNAJC2 expression .

• Experimental manipulation of miR-627-3p levels using mimics or inhibitors correspondingly affects DNAJC2 expression levels .

To investigate this regulatory relationship, researchers have employed several experimental approaches:

• miRNA mimics and inhibitors: The sequence for miR-627-3p mimics (5′-CCGATTCACCAACGA-3′) and control (5′-TTTCATACATTCCAGC-3′) can be transfected into cell lines to observe effects on DNAJC2 expression .

• Transfection methods: Lipofectamine is commonly used for the delivery of miRNA mimics and inhibitors into cells .

• Expression verification: Following transfection, DNAJC2 mRNA and protein levels should be assessed by RT-qPCR and Western blotting, respectively, to confirm the regulatory effect .

• Functional rescue experiments: To establish causation, researchers can perform rescue experiments where DNAJC2 is overexpressed in cells with miR-627-3p mimics to determine if it restores the phenotype .

This miRNA-mediated regulation represents a potential therapeutic target, as modulating miR-627-3p levels could potentially control DNAJC2 expression and its associated tumorigenic effects in colorectal cancer.

What are the optimal conditions for detecting phosphorylated forms of DNAJC2?

Detecting phosphorylated forms of DNAJC2 requires specific antibodies and optimized conditions:

• Phospho-DNAJC2/MPP11 (Ser47) antibodies have been developed specifically for detecting this modification .

For optimal detection of phosphorylated DNAJC2:

• Western blotting conditions:

  • Recommended dilution: 1:1000 for Phospho-DNAJC2/MPP11 (Ser47) antibodies

  • Expected molecular weight: 80 kDa for the phosphorylated form

  • Species reactivity: Human, Mouse, Rat, and Monkey samples have been validated

• Phosphatase inhibitors: When preparing cell or tissue lysates, it's crucial to include phosphatase inhibitors in the lysis buffer to preserve phosphorylation status.

• Positive controls: Cell lines treated with agents that affect the specific phosphorylation pathway should be used as controls.

• Sample preparation: Rapid processing of samples is essential to maintain phosphorylation states.

• Signal enhancement: For weak signals, consider using enhanced chemiluminescence detection systems or signal amplification methods.

The phosphorylation site at Ser47 was identified using PhosphoScan® LC-MS/MS platform for modification site discovery, suggesting that mass spectrometry-based approaches are also valuable for identifying novel phosphorylation sites on DNAJC2 .

How can researchers differentiate between the cytosolic chaperone and nuclear chromatin regulatory functions of DNAJC2?

Distinguishing between DNAJC2's dual functions requires specific experimental approaches:

• Subcellular fractionation:

  • Separate nuclear and cytoplasmic fractions using established protocols

  • Verify fractionation quality using markers like Lamin B (nuclear) and GAPDH (cytoplasmic)

  • Analyze DNAJC2 abundance in each fraction by Western blotting

• Immunofluorescence microscopy:

  • Use DNAJC2 antibodies validated for immunofluorescence

  • Co-stain with markers for ribosomes/ER (for chaperone function) and chromatin (for nuclear function)

  • Quantify colocalization coefficients

• Domain-specific functional studies:

  • The J domain (N-terminal region) is critical for chaperone function

  • Create J domain deletion mutants (DJ2-ΔJ) to specifically impair chaperone function while preserving nuclear functions

  • Verify differential effects on ribosome-associated activities versus chromatin modification

• Co-immunoprecipitation to identify binding partners:

  • For chaperone function: look for interaction with Hsp70-type chaperones HSPA14

  • For nuclear function: examine binding to histone H2A ubiquitinated at 'Lys-119' (H2AK119ub)

• Functional assays:

  • Chaperone function: nascent protein folding assays, protein aggregation assays

  • Nuclear function: chromatin immunoprecipitation (ChIP) to detect association with chromatin, gene expression analysis of polycomb-repressed genes

What cell lines and tissues show highest endogenous DNAJC2 expression for use as positive controls?

Based on research data, the following cell lines and tissues demonstrate consistent and high DNAJC2 expression:

Cell lines with high DNAJC2 expression:

• HeLa (human cervical cancer cell line)

• HEK-293T (human embryonic kidney cells)

• Jurkat (human T lymphocyte cell line)

• DLD-1 (colorectal adenocarcinoma cell line)

• HCT-116 (colorectal carcinoma cell line)

Tissue samples with notable DNAJC2 expression:

• Colorectal cancer tissues (significantly higher than adjacent normal tissues)

• Human placenta (shows robust staining in IHC)

• Human kidney (strong positive staining in IHC)

• Mouse liver, testis, kidney, and stomach (all show positive staining with DNAJC2 antibodies)

For experimental validation:

• Western blot detection: When using these positive controls, researchers should expect to observe bands at approximately 80/72 kDa (the calculated MW is 72 kDa, but observed MW can be 80 kDa due to post-translational modifications) .

• Loading amount recommendations:

  • For cell lysates: 20-30 μg total protein per lane is typically sufficient

  • For tissue lysates: 20 μg protein is recommended for optimal detection

• Band intensity quantification: ImageJ software (National Institutes of Health) is commonly used for densitometry analysis of Western blot bands .

What methodological approaches can overcome cross-reactivity issues with other DNAJ family members?

Cross-reactivity between DNAJ family members is a significant challenge due to their high sequence homology, particularly in the conserved J domain. To overcome this:

• Epitope selection and antibody validation:

  • Use antibodies raised against unique regions of DNAJC2 rather than the conserved J domain

  • The C-terminal region of DNAJC2 tends to be more unique and suitable for specific antibody generation

  • Verify specificity using knockout/knockdown controls, as cross-reactivity with DNAJA1 has been observed in some studies

• Immunoprecipitation followed by mass spectrometry:

  • Use IP to enrich for DNAJC2 and confirm identity by mass spectrometry

  • This approach can definitively identify the protein despite antibody cross-reactivity issues

• Blocking peptide competition assays:

  • Pre-incubate antibodies with specific blocking peptides corresponding to the immunogen

  • Compare results with and without the blocking peptide to confirm specificity

• Recombinant protein standards:

  • Include recombinant DNAJC2 and potentially cross-reactive family members as controls

  • Compare migration patterns and signal intensity

• Western blot optimization:

  • Use higher dilutions of primary antibody (1:1000-1:2000) to reduce non-specific binding

  • Increase washing stringency with higher concentrations of detergent in wash buffers

  • Extended blocking times (2 hours at room temperature) can reduce background

• Alternative detection methods:

  • RT-qPCR using primers specific to unique regions of DNAJC2 mRNA

  • RNA interference targeting unique sequences of DNAJC2

Research has shown that the 5'-terminal fragment of DNAJC2 contains a region highly conserved among DnaJ family members, and similarly, the 5'-terminal fragment of DNAJA1 was also isolated in some screening studies, indicating potential cross-reactivity issues that must be addressed methodologically .

What are the optimal immunohistochemistry protocols for DNAJC2 detection in different tissue types?

Optimized immunohistochemistry (IHC) protocols for DNAJC2 detection vary by tissue type, but several key parameters remain consistent:

General IHC Protocol for DNAJC2:

• Tissue preparation:

  • Fix tissues in formalin and embed in paraffin

  • Cut 4-5 μm thick sections and mount on positively charged slides

  • Deparaffinize in xylene and rehydrate through graded alcohols

• Antigen retrieval:

  • Heat-induced epitope retrieval is preferred

  • Use citrate buffer (pH 6.0) in a pressure cooker or microwave

  • For colorectal tissues: 10-20 minutes at 95-100°C has shown good results

• Peroxidase blocking:

  • 3% H₂O₂ for 5 minutes at room temperature

  • This step suppresses endogenous peroxidase activity to reduce background

• Blocking:

  • 10% normal goat serum and 5% BSA in TBS for 1 hour at room temperature

  • Thorough blocking is crucial for reducing non-specific binding

• Primary antibody:

  • Dilution ranges: 1:50-1:400 depending on tissue type and antibody

  • For colorectal tissues: 1:400 dilution works well

  • For kidney, placenta, and other tissues: 1:100 dilution is recommended

  • Incubation: Overnight at 4°C in a humidified chamber

• Secondary antibody and detection:

  • HRP-conjugated secondary antibody (1:100 dilution)

  • DAB substrate for visualization

  • Counterstain with hematoxylin

• Imaging and analysis:

  • Capture images using a digital light microscope

  • For each sample, examine ≥5 randomly selected fields

  • Use standard magnifications of ×100 and ×400

  • Analyze positive staining area using ImageJ 1.63 software

Tissue-specific considerations:

• Colorectal tissues: Extended antigen retrieval (15-20 min) may be needed due to dense tissue structure

• Kidney tissues: Require careful blocking due to high endogenous peroxidase activity

• Placenta: Shows robust staining with minimal background when using 1:100 antibody dilution

• Liver and testis: May benefit from reduced primary antibody concentration (1:200) to minimize background

How can researchers troubleshoot and optimize Western blotting for DNAJC2 detection?

Western blotting for DNAJC2 requires careful optimization to achieve clear, specific detection. The following troubleshooting guide addresses common issues:

Sample Preparation Optimization:

• Use RIPA buffer supplemented with protease inhibitors for efficient extraction

• Determine protein concentration using BCA Protein Assay kit

• Load 20 μg protein per lane for optimal detection

• Include phosphatase inhibitors if studying phosphorylated forms of DNAJC2

Electrophoresis Considerations:

• Use 10% SDS-PAGE for optimal separation

• Expected molecular weight: calculated at 72 kDa, but often observed at 80/72 kDa due to post-translational modifications

• Run the gel at lower voltage (80-90V) for better resolution around the target molecular weight

Transfer Optimization:

• Transfer to PVDF membranes (rather than nitrocellulose) for better protein retention

• For proteins >70 kDa, extend transfer time or use wet transfer systems

• Add SDS (0.1%) to transfer buffer if signal is weak

Blocking Optimization:

• Use 10% BSA solution for blocking (2 hours at room temperature)

• Alternative: 5% non-fat dry milk in TBST if background is high

Antibody Incubation:

• Primary antibody dilutions: 1:500-1:2000 depending on the specific antibody

• Incubate with primary antibody overnight at 4°C for optimal binding

• Secondary antibody: Anti-rabbit IgG-HRP at 1:2000-1:10000 dilution

• Wash thoroughly with TBST (3 times, 10-15 minutes each) after each antibody incubation

Detection Troubleshooting:

• For weak signals: Use enhanced chemiluminescence systems, extend exposure time

• For high background: Increase antibody dilution, add 0.001% Tween-20 to TBST wash buffer

• Multiple bands: Validate with knockout/knockdown controls to identify specific band

• No signal: Verify protein transfer with Ponceau S staining

Controls and Validation:

• Positive control: HeLa, 293T, or Jurkat cell lysates

• Negative control: DNAJC2 knockdown samples

• Loading control: β-actin (cat. no. ab179467) or similar housekeeping proteins

• For phospho-DNAJC2 detection: Include both phosphorylated and non-phosphorylated controls

How does heat shock or cellular stress affect DNAJC2 expression and detection?

As a member of the heat shock protein family, DNAJC2 expression and localization can be significantly affected by cellular stress conditions, which researchers must consider when designing experiments:

Effects of Cellular Stress on DNAJC2:

• Expression level changes:

  • Heat shock may increase DNAJC2 expression as part of the cellular stress response

  • This can affect baseline measurements and experimental outcomes if stress conditions aren't controlled

• Subcellular localization shifts:

  • Under stress conditions, DNAJC2 may redistribute between its cytosolic chaperone and nuclear regulatory roles

  • This can impact fractionation studies and immunofluorescence analysis

• Post-translational modifications:

  • Cellular stress often triggers phosphorylation of DNAJC2, particularly at Ser47

  • This affects detection with non-phospho-specific antibodies and may cause band shifts in Western blots

Methodological Considerations:

• Experimental design:

  • Include proper controls for stress conditions

  • Document and standardize temperature, cell density, and handling conditions

  • Allow recovery periods after passaging cells before experiments

• Detection strategy:

  • For stress studies, use antibodies that recognize both phosphorylated and non-phosphorylated forms

  • Consider using phospho-specific antibodies (like Phospho-DNAJC2/MPP11 (Ser47) ) alongside total DNAJC2 antibodies

• Sample preparation:

  • Rapid processing is crucial to maintain stress-induced modifications

  • Use phosphatase inhibitors in lysis buffers to preserve phosphorylation states

  • Consider using specialized extraction methods for stress-induced protein aggregates

• Interpreting results:

  • Account for stress-induced changes when comparing results across experiments

  • Document cell culture conditions thoroughly

  • Consider time-course experiments to track stress-related changes in DNAJC2