DNAJC1 Antibody

What is DNAJC1 and what are its known functions in cellular biology?

DNAJC1 is a member of the DNAJ family of proteins, which constitute the largest and most diverse family of co-chaperones that work with HCP70 and HSP90. The DNAJ family is involved in various cellular activities including protein translation, folding/unfolding/refolding, translocation, and degradation . DNAJC1 specifically:

• Functions as a DNAJ-like heat shock protein that binds the molecular chaperone BiP

• Contains two SANT domains that bind serpin alpha1-antichymotrypsin and inter-alpha trypsin inhibitor heavy chain 4

• Is a membrane protein encoded by the DNAJC1 gene

• Also known by aliases including HTJ1, DNAJL1, ERdj1, and MTJ1

• Has a molecular weight of approximately 64 kDa

Recent research has implicated DNAJC1 in pathways relevant to autoimmune diseases, neurodegenerative diseases, and cancer progression .

How is DNAJC1 expressed in normal versus disease states?

Research demonstrates that DNAJC1 expression varies significantly between normal and disease states:

• In hepatocellular carcinoma (HCC): DNAJC1 is highly expressed and significantly associated with patient prognosis. Bioinformatic analysis and experimental verification have demonstrated its upregulation in HCC tissues compared to normal liver tissues .

• In glioblastoma (GBM): DNAJC1 is frequently overexpressed and associated with clinical characteristics including WHO grade, IDH status, chromosome 1p/19q codeletion, and histological type .

• Across various cancers: Pan-cancer analysis shows DNAJC1 is remarkably elevated in most tumor tissues compared to corresponding normal tissues .

Expression can be validated through various techniques including Western blotting, qRT-PCR, and immunohistochemistry, with antibodies specifically targeting DNAJC1.

What types of DNAJC1 antibodies are available and how do they differ?

DNAJC1 antibodies come in several types, each with specific characteristics:

When selecting an antibody, researchers should consider:

• The specific application (Western blot, IHC, IF, IP)

• Species of interest (human, mouse, rat)

• Required specificity (monoclonal vs. polyclonal)

• Need for conjugation or special tags

How can I verify the specificity of my DNAJC1 antibody?

Establishing antibody specificity is crucial for reliable results. Recommended validation strategies include:

• Positive and negative controls:

  • Use cell lines known to express DNAJC1 (e.g., HEK-293, HeLa, Jurkat cells)

  • Include a negative control using DNAJC1 knockout cell lines when available

• Molecular weight verification:

  • DNAJC1 should appear at approximately 64 kDa on Western blots

  • Some antibodies may detect DNAJC1 at approximately 46 kDa in specific cell lysates

• Knockdown/knockout validation:

  • Perform siRNA knockdown experiments as demonstrated in HCC studies

  • Confirm reduced signal intensity corresponding to decreased DNAJC1 expression

• Cross-reactivity assessment:

  • Test antibody against related DNAJ family proteins to ensure specificity

  • Consider multiple antibodies targeting different epitopes of DNAJC1

What are the optimal conditions for Western blot detection of DNAJC1?

For successful Western blot detection of DNAJC1, consider these protocol recommendations:

• Sample preparation:

  • Use cell lysates from appropriate cell lines (HEK293, HepG2, Huh7, MHCC97H)

  • Include protease inhibitors to prevent degradation

• SDS-PAGE conditions:

  • Use 7.5-10% SDS-PAGE gels for optimal resolution

  • Load 30 µg of whole cell lysate per lane

• Antibody dilutions:

  • Primary antibody: 1:500-1:8000 depending on the specific antibody

  • Most DNAJC1 antibodies work optimally at 1:1000-1:2000 dilution

• Detection methods:

  • Enhanced chemiluminescence (ECL) provides adequate sensitivity

  • For stronger signal, consider using PVDF membrane over nitrocellulose

• Controls:

  • Include appropriate loading controls (e.g., GAPDH, β-actin)

  • Consider including a positive control lysate known to express DNAJC1

A representative Western blot shows detection of DNAJC1 at approximately 64 kDa in various cell lysates, with antibody dilution of 1:1000 .

How should immunohistochemistry protocols be optimized for DNAJC1 detection?

For optimal immunohistochemistry (IHC) detection of DNAJC1:

• Antigen retrieval:

  • Use TE buffer pH 9.0 for optimal retrieval

  • Alternative: citrate buffer pH 6.0 may be used if needed

• Antibody dilution:

  • Recommended dilution range: 1:20-1:500 depending on the antibody

  • Start with 1:50 and optimize based on signal-to-noise ratio

• Blocking:

  • Use 5-10% normal serum (species-dependent on secondary antibody)

  • Include 0.1-0.3% Triton X-100 for improved antibody penetration

• Detection systems:

  • DAB (3,3'-diaminobenzidine) works well for chromogenic detection

  • Fluorescent secondary antibodies can be used for co-localization studies

• Controls:

  • Include positive control tissues (e.g., liver cancer, breast cancer)

  • Use isotype controls to assess non-specific binding

DNAJC1 has been successfully detected in human liver cancer and breast cancer tissues using these methods .

How does DNAJC1 contribute to hepatocellular carcinoma progression?

Research has revealed multiple mechanisms through which DNAJC1 influences HCC progression:

• Cellular proliferation and survival:

  • Knockdown of DNAJC1 significantly reduces HCC cell proliferation as demonstrated by CCK-8 assays and colony formation assays

  • DNAJC1 knockdown increases cell inhibition rate at 24h, 48h, 72h, and 96h compared to control groups

• Apoptosis regulation:

  • DNAJC1 knockdown promotes apoptosis in HCC cells through the p53 signaling pathway

  • Western blot analysis shows increased expression of pro-apoptotic proteins (p21, p53, p-p53, Bax) and decreased anti-apoptotic proteins (Bcl-2, PARP) after DNAJC1 knockdown

• Migration and invasion:

  • Wound healing assays demonstrate that DNAJC1 knockdown significantly reduces migration capacity of HCC cells

  • Transwell migration and invasion assays confirm that suppression of DNAJC1 inhibits both migration and invasion abilities

• Signaling pathway modulation:

  • DNAJC1 appears to function through p53 and EMT signaling pathways

  • Knockdown affects expression of E-cadherin, MMP9, Vimentin, Snai1, and N-cadherin proteins

These findings suggest DNAJC1 as a potential therapeutic target for HCC treatment.

What is the role of DNAJC1 in glioblastoma development?

Recent studies have established DNAJC1's significance in glioblastoma (GBM):

• Expression pattern:

  • DNAJC1 is frequently overexpressed in GBM specimens compared to normal brain tissue

  • Expression is significantly associated with WHO grade, IDH status, chromosome 1p/19q codeletion, and histological type

• Prognostic value:

  • Kaplan-Meier and ROC analyses identify DNAJC1 as a negative prognostic predictor

  • DNAJC1 shows promise as a diagnostic biomarker for GBM patients

• Functional impact:

  • Silencing DNAJC1 impedes GBM cell proliferation and migration

  • DNAJC1 knockdown induces cell cycle arrest and enhances apoptosis

• Mechanistic insights:

  • DNAJC1 stimulates extracellular matrix reorganization

  • Triggers epithelial-mesenchymal transition (EMT)

  • Initiates immunosuppressive macrophage infiltration

These findings establish DNAJC1 as a pivotal player in GBM pathogenesis and suggest its potential as both a diagnostic and therapeutic target.

How can DNAJC1 antibodies be used in co-immunoprecipitation studies?

Co-immunoprecipitation (Co-IP) with DNAJC1 antibodies can reveal protein-protein interactions:

• Protocol recommendations:

  • Use antibodies specifically validated for IP applications

  • Recommended dilution: 1:200-1:1000 for IP applications

  • Cell lysate amount: 1.0-3.0 mg of total protein lysate per IP reaction

• Known interactions to investigate:

  • Molecular chaperone BiP (binding immunoglobulin protein)

  • Components of the p53 signaling pathway

  • EMT-related proteins (E-cadherin, Vimentin, Snai1)

• Controls and validation:

  • Include IgG control to assess non-specific binding

  • Confirm by reverse Co-IP when possible

  • Validate interactions with orthogonal methods (e.g., proximity ligation assay)

What are common challenges when working with DNAJC1 antibodies and how can they be overcome?

Researchers may encounter several challenges when working with DNAJC1 antibodies:

• Background signal:

  • Increase blocking time/concentration (5% BSA or milk)

  • Optimize antibody dilution and incubation conditions

  • Consider using monoclonal antibodies for higher specificity

• Inconsistent results between applications:

  • Verify antibody is validated for your specific application

  • Different epitopes may be accessible in different applications

  • Use antibodies that specifically target conserved regions for cross-species studies

• Cross-reactivity with other DNAJ family members:

  • Select antibodies raised against unique regions of DNAJC1

  • Validate with knockout/knockdown controls

  • Consider using multiple antibodies targeting different epitopes

• Storage and handling issues:

  • Store antibodies at recommended temperatures (-20°C to -80°C)

  • Limit freeze-thaw cycles (2-3 maximum)

  • Aliquot antibodies upon receipt to avoid repeated freeze-thaw cycles

• Signal detection problems:

  • For weak signals, try longer exposure times or more sensitive detection methods

  • For Western blots, consider membrane transfer optimization (time/voltage/buffer)

  • For IHC/IF, optimize antigen retrieval methods (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

How can I design experiments to study DNAJC1 function using antibody-based approaches?

To investigate DNAJC1 function using antibody-based approaches:

• Expression analysis across disease states:

  • Compare DNAJC1 expression in normal vs. disease tissues using IHC/IF

  • Quantify expression levels via Western blot in different cell lines and patient samples

  • Correlate expression with clinical data (survival, disease stage) as done in HCC and GBM studies

• Subcellular localization studies:

  • Use immunofluorescence with organelle markers to determine DNAJC1 localization

  • Monitor changes in localization under stress conditions or drug treatments

  • Combine with cell fractionation and Western blot for biochemical validation

• Functional knockdown studies:

  • Design siRNA experiments similar to those used in HCC studies

  • Use DNAJC1 antibodies to confirm knockdown efficiency

  • Assess phenotypic changes (proliferation, migration, apoptosis)

• Pathway analysis:

  • Combine with antibodies against p53 pathway components or EMT markers

  • Monitor changes in related proteins after DNAJC1 manipulation

  • Use phospho-specific antibodies to assess activation states of signaling pathways

• Clinical correlation studies:

  • Create tissue microarrays for high-throughput analysis

  • Score DNAJC1 expression and correlate with patient outcomes

  • Combine with other markers for improved prognostic value

These experimental approaches have successfully revealed DNAJC1's role in cancer progression and provide a framework for further investigations.