DNAJC10 Antibody

What types of DNAJC10 antibodies are available for research?

Multiple types of DNAJC10 antibodies are available for research applications, varying in their target epitopes, host species, and clonality. The most common varieties include:

• C-terminal targeted antibodies: These recognize epitopes within the C-terminal region of DNAJC10 and are available as polyclonal antibodies raised in rabbits

• N-terminal targeted antibodies: These recognize epitopes within the N-terminal region and are available for detecting specific amino acid sequences (AA 35-84, AA 56-82)

• Monoclonal antibodies: Available from mouse hosts (e.g., clone 3C4) targeting specific regions (AA 688-793)

• Polyclonal antibodies: Offer broader epitope recognition, available from rabbit hosts, and typically detect endogenous levels of total DNAJC10

When selecting an antibody, researchers should consider the specific application, required reactivity (human, mouse, rat, etc.), and the region of interest within the DNAJC10 protein.

What experimental applications are DNAJC10 antibodies suitable for?

DNAJC10 antibodies have been validated for multiple research applications, including:

• Western Blotting (WB): The most common application for detecting DNAJC10 protein expression levels in cell and tissue lysates

• Immunohistochemistry (IHC): For visualizing DNAJC10 expression patterns in tissue sections

• Immunofluorescence (IF): For subcellular localization studies and co-localization with other proteins

• ELISA: For quantitative detection of DNAJC10 in solution

• Immunoprecipitation: For studying protein-protein interactions of DNAJC10 with other components of the ERAD complex

Most DNAJC10 antibodies are available unconjugated, allowing researchers flexibility in secondary detection methods .

How can I optimize Western Blotting protocols for DNAJC10 detection?

For optimal Western Blot detection of DNAJC10, consider the following methodological recommendations:

• Sample preparation: Use RIPA buffer supplemented with protease inhibitors for efficient extraction of DNAJC10 from the ER membrane

• Protein loading: Load 20-40 μg of total protein per lane

• Gel percentage: Use 8-10% SDS-PAGE gels (DNAJC10 has a molecular weight of approximately 91 kDa)

• Transfer conditions: Transfer to PVDF membranes at 100V for 90 minutes in cold transfer buffer containing 20% methanol

• Blocking: Block membranes with 5% non-fat dry milk in TBST for 1 hour at room temperature

• Primary antibody: Dilute DNAJC10 antibody (typically 1:1000-1:2000) in blocking buffer and incubate overnight at 4°C

• Washing: Perform 3-5 five-minute washes with TBST

• Secondary antibody: Use HRP-conjugated secondary antibody (anti-rabbit or anti-mouse, depending on primary) at 1:5000 dilution

• Detection: Use enhanced chemiluminescence (ECL) for visualization

For challenging samples, consider using peptide affinity-purified antibodies, which offer improved specificity, such as those purified using SulfoLink™ Coupling Resin .

What controls should I include when using DNAJC10 antibodies in my experiments?

Proper controls are essential for ensuring experimental validity when working with DNAJC10 antibodies:

• Positive control: Include lysates from cells known to express DNAJC10 (e.g., HeLa cells for human studies, NIH/3T3 for mouse studies)

• Negative control: Consider CRISPR/Cas9 knockout cell lines as negative controls, as demonstrated in leukemia studies

• Loading control: Use housekeeping proteins like GAPDH, β-actin, or α-tubulin to normalize expression levels

• Antibody specificity control: Pre-absorption with the immunizing peptide to confirm specificity

• siRNA/shRNA knockdown: Include samples with DNAJC10 knockdown to validate antibody specificity, as shown in AML studies

• Isotype control: For IHC/IF applications, include an isotype-matched control antibody (e.g., rabbit IgG for rabbit polyclonal antibodies)

These controls help ensure that observed signals are specifically attributable to DNAJC10 and not to non-specific binding or technical artifacts.

How do I select the appropriate DNAJC10 antibody for cross-species studies?

When planning cross-species studies involving DNAJC10, consider the following approach:

• Sequence homology analysis: Compare DNAJC10 sequences across target species to identify conserved regions

• Epitope selection: Choose antibodies targeting highly conserved epitopes

• Validated reactivity: Select antibodies with experimentally confirmed cross-reactivity to your species of interest

• Multiple antibody validation: Test several antibodies targeting different epitopes to ensure consistent results

Based on available data, several DNAJC10 antibodies show cross-reactivity across multiple species, including human, mouse, rat, zebrafish, and even more diverse species like bovine, sheep, and xenopus . For the broadest cross-reactivity, consider antibodies targeting the N-terminal region, which shows high conservation across species.

How can DNAJC10 antibodies be used to study its role in hematological malignancies?

DNAJC10 antibodies can be instrumental in studying its role in hematological malignancies through several methodological approaches:

• Expression profiling: Use Western blotting and IHC to measure DNAJC10 expression levels across different leukemia subtypes and compare with normal hematopoietic cells. Recent studies have shown that DNAJC10 is frequently upregulated in various types of acute myeloid leukemia (AML) and in leukemia stem cell (LSC)-enriched populations .

• Functional studies: Combine DNAJC10 antibodies with knockdown/knockout approaches to correlate protein levels with phenotypic changes:

  • In vitro: Measure changes in cell proliferation, apoptosis, and colony formation

  • In vivo: Assess leukemia development in xenograft models following DNAJC10 modulation

• Pathway analysis: Use co-immunoprecipitation with DNAJC10 antibodies to identify interaction partners in the PERK-EIF2α-ATF4 branch of the unfolded protein response (UPR)

• Prognostic evaluation: Correlate DNAJC10 expression levels (detected by IHC) with patient outcomes to validate its potential as a prognostic biomarker in AML

• Therapy response: Monitor changes in DNAJC10 expression following treatment with chemotherapeutic agents like daunorubicin (DNR) and cytarabine (Ara-C), as deficiency of DNAJC10 has been shown to increase sensitivity to these drugs

These approaches can help elucidate the role of DNAJC10 as a potential oncogene in AML and develop therapeutic strategies targeting this protein.

What methods can be used to investigate the contrasting roles of DNAJC10 in different cancer types?

DNAJC10 exhibits intriguing contrasting roles across cancer types, functioning as an oncogene in AML but showing anti-oncogenic properties in breast cancer, neuroblastoma, prostate, and colon cancers . To investigate these contrasting roles, researchers can employ the following methodological approaches:

• Comparative expression analysis:

  • Use Western blotting and IHC with DNAJC10 antibodies to compare expression patterns across multiple cancer types

  • Correlate with clinical parameters to identify cancer-specific patterns

• Isogenic cell line models:

  • Generate DNAJC10 knockout and overexpression models across different cancer cell lines

  • Use DNAJC10 antibodies to confirm manipulation and measure downstream effects

• Domain-specific functional analysis:

  • Use antibodies targeting different domains of DNAJC10 to investigate domain-specific functions

  • Combine with truncation constructs to identify cancer-specific functional regions

• Context-dependent interactome mapping:

  • Perform co-immunoprecipitation with DNAJC10 antibodies in different cancer types

  • Identify cancer-specific interaction partners that may explain divergent functions

• UPR pathway analysis:

  • Measure changes in UPR components (particularly the PERK-EIF2α-ATF4 branch) following DNAJC10 modulation

  • Compare pathway activation patterns across cancer types

• In vivo validation:

  • Develop xenograft or transgenic models with tissue-specific DNAJC10 manipulation

  • Use antibodies to monitor protein expression and correlate with tumor progression

Recent studies have shown that while DNAJC10 promotes leukemia development in AML models , its downregulation is associated with poor survival in breast cancer patients , highlighting the importance of context-specific analysis.

How can I use DNAJC10 antibodies to study the unfolded protein response in cancer?

DNAJC10 is intimately involved in the unfolded protein response (UPR), particularly through its interaction with the PERK-EIF2α-ATF4 branch. To study this relationship in cancer, consider these methodological approaches:

• Stress induction experiments:

  • Treat cancer cells with UPR inducers (tunicamycin, thapsigargin, or DTT)

  • Use DNAJC10 antibodies to monitor changes in protein expression, localization, and post-translational modifications

• Co-localization studies:

  • Perform dual immunofluorescence with DNAJC10 antibodies and markers for UPR components

  • Analyze changes in co-localization patterns following stress induction

• Pathway component analysis:

  • Use Western blotting with antibodies against DNAJC10 and UPR markers (PERK, phospho-EIF2α, ATF4)

  • Correlate expression patterns to identify regulatory relationships

• Intervention studies:

  • Combine DNAJC10 knockdown/knockout with UPR pathway inhibitors (e.g., GSK2606414 for PERK inhibition)

  • Use antibodies to confirm intervention effects and measure downstream consequences

• Patient sample analysis:

  • Perform multiplex IHC on cancer tissue microarrays using DNAJC10 antibodies and UPR markers

  • Correlate expression patterns with clinical outcomes

Research has shown that inhibition of DNAJC10 specifically induces endoplasmic reticulum stress and promotes activation of the PERK-EIF2α-ATF4 branch of UPR. Furthermore, blocking PERK can rescue the loss of function of DNAJC10 both in vitro and in vivo, suggesting a mechanistic link between DNAJC10 and this UPR branch .

How can I design experiments to investigate DNAJC10's role in chemotherapy resistance?

Recent research has identified DNAJC10 as a potential factor in chemotherapy sensitivity, particularly in AML . To design experiments investigating this relationship, consider this methodological framework:

• Baseline expression analysis:

  • Use DNAJC10 antibodies to measure protein expression across cell lines with varying chemotherapy sensitivity

  • Correlate expression levels with established IC50 values for relevant chemotherapeutics

• Manipulation studies:

  • Generate DNAJC10 knockdown/knockout and overexpression models

  • Validate protein modulation using Western blotting with specific antibodies

  • Treat with increasing concentrations of chemotherapeutic agents (e.g., daunorubicin and cytarabine for AML)

  • Measure cell viability, apoptosis, and DNA damage responses

• Pathway interrogation:

  • Analyze UPR pathway activation in response to combined DNAJC10 modulation and chemotherapy

  • Use Western blotting to measure changes in PERK, phospho-EIF2α, and ATF4 levels

• In vivo validation:

  • Develop xenograft models with DNAJC10-modulated cells

  • Treat with chemotherapy regimens and monitor tumor response

  • Use IHC with DNAJC10 antibodies to confirm continued expression/knockdown during treatment

• Clinical correlation:

  • Analyze patient samples pre- and post-treatment using DNAJC10 antibodies

  • Correlate expression changes with treatment response and survival outcomes

Studies have demonstrated that deficiency of DNAJC10 significantly increased sensitivity of AML cells to daunorubicin and cytarabine, suggesting its potential as a therapeutic target for improving chemotherapy effectiveness .

What approaches can be used to investigate the prognostic value of DNAJC10 in different cancers?

To investigate DNAJC10's prognostic value across cancer types, consider these methodological approaches:

• Tissue microarray analysis:

  • Develop cancer-specific tissue microarrays with adequate sample numbers and clinical follow-up data

  • Perform IHC using validated DNAJC10 antibodies

  • Establish scoring criteria (e.g., H-score, Allred score) for quantification

  • Correlate expression with survival outcomes using Kaplan-Meier analysis and Cox regression

• Multi-cohort validation:

  • Analyze multiple independent patient cohorts

  • Use standardized staining and scoring protocols with the same DNAJC10 antibody clone

  • Perform meta-analysis to establish robust prognostic associations

• Multivariate analysis:

  • Include established prognostic factors in your models

  • Determine if DNAJC10 expression provides independent prognostic information

• Subgroup analysis:

  • Stratify patients by molecular subtypes, treatment regimens, or other clinicopathological parameters

  • Determine if DNAJC10's prognostic value differs across subgroups

• Comparative cancer analysis:

  • Apply the same methodologies across different cancer types

  • Identify cancer-specific prognostic patterns

How can I design experiments to study DNAJC10's interaction with the ERAD complex?

DNAJC10 functions as a component of the ER-associated degradation (ERAD) complex. To study these interactions, consider the following experimental design:

• Co-immunoprecipitation (Co-IP) approach:

  • Use DNAJC10 antibodies to pull down protein complexes

  • Analyze co-precipitated proteins by mass spectrometry or Western blotting for known ERAD components

  • Perform reciprocal Co-IPs to confirm interactions

  • Include appropriate controls (IgG control, lysate input)

• Proximity labeling techniques:

  • Generate DNAJC10 fusion constructs with BioID or APEX2

  • Express in relevant cell types and activate labeling

  • Purify biotinylated proteins and identify by mass spectrometry

  • Validate key interactions using DNAJC10 antibodies

• Domain mapping experiments:

  • Generate truncation constructs of DNAJC10

  • Perform Co-IPs with domain-specific antibodies

  • Identify regions required for ERAD complex interactions

• Functional ERAD assays:

  • Measure degradation of known ERAD substrates in DNAJC10 knockdown/knockout cells

  • Use pulse-chase experiments to track protein degradation kinetics

  • Validate DNAJC10 expression/depletion using specific antibodies

• Subcellular localization studies:

  • Perform immunofluorescence with DNAJC10 antibodies and markers for ERAD components

  • Analyze co-localization under normal and ER stress conditions

Previous studies have established DNAJC10's role in recognizing and degrading misfolded proteins as part of the ERAD complex , making these interaction studies particularly valuable for understanding its molecular functions.

What are common challenges when using DNAJC10 antibodies and how can they be addressed?

Researchers may encounter several challenges when working with DNAJC10 antibodies. Here are common issues and recommended solutions:

• Nonspecific binding:

  • Issue: Multiple bands observed in Western blots

  • Solution: Use antibodies purified by peptide affinity chromatography (e.g., using SulfoLink™ Coupling Resin)

  • Alternative: Increase blocking stringency with 5% BSA instead of milk

  • Validation: Compare pattern with DNAJC10 knockdown samples

• Low signal intensity:

  • Issue: Weak or undetectable DNAJC10 signal

  • Solution: Optimize protein extraction using specialized ER protein extraction buffers

  • Alternative: Increase antibody concentration or incubation time

  • Enhancement: Use signal amplification systems like tyramide signal amplification for IHC/IF

• Inconsistent results across applications:

  • Issue: Antibody works for Western blot but not IHC

  • Solution: Verify application-specific validation of the selected antibody

  • Alternative: Try antibodies targeting different epitopes of DNAJC10

• Cross-reactivity concerns:

  • Issue: Uncertain specificity in cross-species applications

  • Solution: Validate antibody in each species using positive and negative controls

  • Recommendation: Select antibodies with documented cross-reactivity to your species of interest

• High background in immunohistochemistry:

  • Issue: Non-specific staining masking specific signal

  • Solution: Optimize antigen retrieval methods (citrate vs. EDTA buffer)

  • Alternative: Use more dilute antibody solutions with longer incubation times

  • Enhancement: Consider biotin-free detection systems to reduce background

Proper antibody validation, including the use of DNAJC10 knockout controls as demonstrated in recent leukemia studies , can help address many of these challenges.

How do I quantitatively assess DNAJC10 expression levels across different experimental conditions?

For rigorous quantitative assessment of DNAJC10 expression, consider these methodological approaches:

• Western blot quantification:

  • Use fluorescent secondary antibodies for wider dynamic range

  • Include a standard curve of recombinant DNAJC10 protein

  • Normalize to multiple loading controls (e.g., GAPDH, β-actin)

  • Use image analysis software with background subtraction

  • Present data as fold-change relative to control conditions

• Immunohistochemistry quantification:

  • Use digital pathology platforms for automated scoring

  • Establish H-score (intensity × percentage) for semi-quantitative analysis

  • Include calibration standards on each slide

  • Employ multiple independent scorers for validation

• Flow cytometry:

  • Optimize cell permeabilization for intracellular DNAJC10 detection

  • Use fluorochrome-conjugated secondary antibodies

  • Include isotype controls and DNAJC10 knockdown samples

  • Report data as median fluorescence intensity (MFI)

• ELISA/AlphaLISA:

  • Develop sandwich ELISA using two antibodies targeting different DNAJC10 epitopes

  • Generate standard curves using recombinant DNAJC10

  • Validate using samples with known DNAJC10 expression levels

• qRT-PCR correlation:

  • Complement protein-level measurements with mRNA analysis

  • Correlate transcript and protein levels to identify post-transcriptional regulation

  • Use this approach to validate antibody-based quantification

When comparing DNAJC10 expression across cancer types or experimental conditions, standardized protocols and consistent analytical methods are essential for reliable quantitative assessment.