DNAJC10 Antibody, FITC conjugated

What is DNAJC10 and why is it important in cellular research?

DNAJC10, also known as ERdj5 or JPDI, functions as a crucial endoplasmic reticulum co-chaperone that plays a significant role in protein folding and translocation across the endoplasmic reticulum membrane. It's particularly important because it assists in proper folding of nascent polypeptides, maintaining cellular homeostasis and preventing accumulation of misfolded proteins that can lead to cellular stress and various diseases. DNAJC10 interacts with HSP70 chaperone machinery, enhancing activity and ensuring proteins are correctly folded before proceeding to functional destinations . Recent research has shown DNAJC10 is frequently up-regulated in various types of acute myeloid leukemia (AML) and in leukemia stem cell (LSC)-enriched cells, suggesting its importance in cancer research .

What detection methods are compatible with DNAJC10-FITC antibodies?

DNAJC10-FITC conjugated antibodies have been validated for multiple detection methods including:

• Flow cytometry - Optimal for quantifying expression levels in cell populations

• Immunofluorescence microscopy - For visualizing subcellular localization in the endoplasmic reticulum lumen

• Confocal microscopy - For high-resolution co-localization studies with other ER markers

• High-content imaging - For automated screening applications

When designing experiments, researchers should use positive controls (cells known to express DNAJC10) and negative controls (secondary antibody only) to validate staining specificity and optimize detection parameters .

How does FITC conjugation affect antibody performance compared to unconjugated versions?

FITC conjugation provides direct visualization capability but researchers should consider these methodological factors:

• FITC has excitation/emission peaks of approximately 495/519 nm, making it compatible with standard fluorescence filter sets

• The conjugation may slightly reduce antibody avidity compared to unconjugated versions

• FITC conjugated antibodies are more susceptible to photobleaching than some other fluorophores

• The conjugation ratio (FITC molecules per antibody) affects signal intensity and should be considered when comparing experimental results

• pH sensitivity of FITC may impact results in certain buffer conditions

To account for these factors, researchers should use consistent imaging parameters, incorporate anti-fade mounting media, and validate results with alternative detection methods when possible .

What are the optimal sample preparation techniques for DNAJC10-FITC antibody labeling?

For effective DNAJC10-FITC antibody labeling, sample preparation should follow these methodological guidelines:

• Cell fixation: 4% paraformaldehyde for 15-20 minutes maintains ER structure while preserving epitope accessibility

• Permeabilization: 0.1-0.2% Triton X-100 for 10 minutes provides optimal access to endoplasmic reticulum lumen antigens

• Blocking: 5% normal serum (from the species of secondary antibody if using indirect methods) with 1% BSA for 30-60 minutes minimizes non-specific binding

• Antibody dilution: Initial testing at 1:500-1:2000 dilution range is recommended based on validation data, with optimization for specific applications

• Incubation conditions: Overnight incubation at 4°C generally produces better signal-to-noise ratio than shorter incubations at room temperature

These parameters should be systematically optimized for each cell type and experimental condition to ensure reproducible results.

How can DNAJC10-FITC antibodies be used to study the unfolded protein response (UPR) in leukemia models?

To effectively study UPR in leukemia models using DNAJC10-FITC antibodies, researchers should implement this methodological framework:

• Baseline expression analysis: Establish DNAJC10 expression levels in normal vs. leukemic cells using flow cytometry with FITC-conjugated antibodies

• UPR induction verification: Confirm UPR activation using complementary markers (GRP78, CHOP) alongside DNAJC10

• Time-course analysis: Monitor DNAJC10 expression changes during UPR activation using:

  • Chemical inducers (tunicamycin, thapsigargin)

  • Physiological stressors (glucose deprivation, hypoxia)

  • Chemotherapeutic agents (daunorubicin, cytarabine)

• Co-localization studies: Use confocal microscopy to examine DNAJC10 interaction with PERK pathway components during stress

• Functional correlation: Integrate DNAJC10 expression data with survival analysis, as AML patients with high DNAJC10 expression show shorter disease-free survival after chemotherapy

This approach allows researchers to comprehensively characterize how DNAJC10 functions as an oncogene in MLL-AF9-induced AML via regulation of the PERK branch of the UPR .

What controls should be included when using DNAJC10-FITC antibodies?

A rigorous experimental design with DNAJC10-FITC antibodies requires these methodological controls:

• Positive control: Cells known to express DNAJC10 (such as THP-1 or U937 leukemia cell lines)

• Negative control: DNAJC10 knockout cells generated via CRISPR/Cas9 to confirm antibody specificity

• Isotype control: FITC-conjugated non-specific IgG1 (for monoclonal antibodies) or normal IgG (for polyclonal antibodies) at the same concentration

• Autofluorescence control: Unstained cells to establish baseline fluorescence

• Absorption control: Pre-incubation of antibody with recombinant DNAJC10 protein to demonstrate binding specificity

• Secondary antibody-only control: For indirect immunofluorescence protocols

• Wild-type vs. stressed cells: Comparing normal conditions with ER stress inducers to demonstrate functional regulation

These controls enable proper interpretation of results and troubleshooting of technical issues that may arise during experiments.

How can DNAJC10-FITC antibodies be optimized for multi-color flow cytometry?

For multi-color flow cytometry with DNAJC10-FITC antibodies, researchers should implement these methodological steps:

• Panel design considerations:

  • FITC emits in the 520nm range, so avoid fluorophores with significant spectral overlap (PE, GFP)

  • Pair with fluorophores like APC, PE-Cy7, or BV421 for clear separation

  • Reserve FITC for moderately expressed proteins; use brighter fluorophores for low-abundance targets

• Compensation controls:

  • Prepare single-color controls for each fluorophore

  • Include DNAJC10-FITC single-stained cells

  • Use compensation beads for channels where cellular expression is variable

• Staining protocol optimization:

  • Titrate antibody concentrations to determine optimal signal-to-noise ratio

  • Sequence antibody addition based on epitope accessibility

  • For intracellular DNAJC10 detection, use permeabilization reagents compatible with surface marker detection

• Data analysis approach:

  • Gate on live, single cells before analyzing DNAJC10 expression

  • Use fluorescence minus one (FMO) controls to set DNAJC10-positive gates

  • Consider correlation analysis between DNAJC10 and other UPR markers (GRP78)

This comprehensive approach enables accurate assessment of DNAJC10 expression in heterogeneous cell populations.

What are the methodological considerations for using DNAJC10 antibodies in patient-derived xenograft models?

When utilizing DNAJC10 antibodies in patient-derived xenograft (PDX) models, researchers should address these methodological considerations:

• Species cross-reactivity verification:

  • Confirm antibody reactivity with both human DNAJC10 (from patient-derived cells) and potential mouse DNAJC10 (from microenvironment)

  • Use species-specific blocking reagents if necessary

• Sample processing protocol:

  • Fresh tissue: Process within 1-2 hours of collection for optimal epitope preservation

  • Fixed tissue: Optimize antigen retrieval methods for FFPE samples

  • Bone marrow samples: Use gentle RBC lysis protocols to preserve DNAJC10 epitopes

• Experimental design framework:

  • Compare DNAJC10 expression between control and experimental groups

  • Track DNAJC10 expression during disease progression and treatment response

  • Correlate with clinical parameters and treatment outcomes

• Validation approaches:

  • Use multiple detection methods (flow cytometry, immunohistochemistry)

  • Confirm expression changes with protein quantification (Western blot)

  • Correlate with functional assays measuring UPR activation

Research has shown that mice transplanted with DNAJC10-KO cells displayed significantly decreased leukemia burden compared with wild-type controls, making this approach valuable for studying DNAJC10's role in disease progression .

How can DNAJC10 expression be quantified accurately using FITC-conjugated antibodies?

For accurate quantification of DNAJC10 expression using FITC-conjugated antibodies, implement this methodological framework:

• Flow cytometry quantification:

  • Use calibration beads with known FITC molecules to establish standard curves

  • Report results as molecules of equivalent soluble fluorochrome (MESF)

  • Apply compensation to correct for spectral overlap in multi-color panels

  • Calculate median fluorescence intensity (MFI) and fold change relative to controls

• Image-based quantification:

  • Capture images using consistent exposure settings

  • Apply background subtraction using non-specific regions

  • Use integrated density measurements (area × mean intensity)

  • Normalize to cell number or nuclear staining

• Western blot correlation:

  • Validate flow cytometry or imaging results with Western blot quantification

  • Expected molecular weight of DNAJC10 is approximately 91 kDa

  • Use housekeeping proteins for normalization

• Statistical analysis:

  • Apply appropriate statistical tests based on data distribution

  • Use multiple biological replicates (n≥3) to account for variability

  • Consider hierarchical analysis for patient-derived samples

This systematic approach ensures reliable quantification of DNAJC10 expression across different experimental platforms .

What are common sources of background when using DNAJC10-FITC antibodies and how can they be minimized?

When working with DNAJC10-FITC antibodies, researchers may encounter these background sources and should implement the corresponding methodological solutions:

• Non-specific binding:

  • Problem: Antibody binding to Fc receptors or hydrophobic interactions

  • Solution: Include 5-10% serum from the host species of secondary antibody and 1% BSA in blocking buffer; use Fc receptor blocking reagents for flow cytometry

• Autofluorescence:

  • Problem: Cellular components naturally fluoresce in the FITC channel

  • Solution: Include unstained controls; use Sudan Black B (0.1-0.3%) to quench autofluorescence; consider spectral unmixing during analysis

• Fixation artifacts:

  • Problem: Excessive fixation can create aldehyde-induced fluorescence

  • Solution: Optimize fixation time; use freshly prepared paraformaldehyde; include 0.1M glycine wash step to quench free aldehydes

• Insufficient washing:

  • Problem: Residual unbound antibody contributing to background

  • Solution: Increase number and volume of washing steps; use gentle agitation during washes

• Photobleaching effects:

  • Problem: FITC signal fading during analysis

  • Solution: Use anti-fade mounting media; minimize exposure to light; acquire images systematically from control to experimental groups

Implementing these targeted solutions will significantly improve signal-to-noise ratio when working with DNAJC10-FITC antibodies .

How should differences in DNAJC10 expression between normal and cancer cells be interpreted?

When interpreting DNAJC10 expression differences between normal and cancer cells, researchers should consider these methodological frameworks:

• Baseline expression contextualization:

  • DNAJC10 is frequently up-regulated in various types of acute myeloid leukemia (AML)

  • Expression is particularly elevated in leukemia stem cell (LSC)-enriched populations

  • DNAJC10 is not highly expressed in normal hematopoietic stem cells (HSC) and progenitor cells

• Functional correlation approach:

  • High DNAJC10 expression correlates with decreased disease-free survival in AML patients

  • Expression increases in response to chemotherapy (daunorubicin and cytarabine)

  • DNAJC10 knockout significantly reduces leukemia cell growth and prolongs survival in xenograft models

• Pathway integration framework:

  • Interpret DNAJC10 expression in context of UPR activation markers (GRP78, CHOP)

  • Correlate with PERK-EIF2α-ATF4 pathway activation status

  • Consider relationship with pro-apoptotic (BAX) and pro-survival UPR components

• Therapeutic relevance analysis:

  • Higher DNAJC10 expression correlates with chemotherapy resistance

  • DNAJC10 deficiency enhances sensitivity to daunorubicin and cytarabine

  • Expression changes during treatment may indicate developing resistance mechanisms

This interpretive framework places DNAJC10 expression changes within a clinically and biologically relevant context.

How can DNAJC10-FITC antibody signals be distinguished from non-specific fluorescence?

To distinguish specific DNAJC10-FITC signals from non-specific fluorescence, implement these methodological approaches:

• Control implementation strategy:

  • Use DNAJC10 knockout or knockdown cells as negative controls

  • Include isotype-matched FITC-conjugated irrelevant antibodies

  • Compare with secondary antibody-only controls for indirect detection methods

• Signal validation techniques:

  • Confirm subcellular localization matches expected endoplasmic reticulum lumen pattern

  • Verify signal reduction after competition with recombinant DNAJC10 protein

  • Compare staining pattern with alternative DNAJC10 antibody clones or detection methods

• Quantitative analysis approach:

  • Establish signal threshold based on negative controls

  • Apply consistent gating strategy across all samples

  • Use signal-to-noise ratio rather than absolute intensity for comparisons

• Advanced verification methods:

  • Demonstrate signal increase in cells overexpressing DNAJC10

  • Show signal augmentation under conditions known to upregulate DNAJC10 (ER stress)

  • Perform co-localization analysis with other ER markers

This comprehensive strategy ensures that observed signals represent genuine DNAJC10 expression rather than technical artifacts .

How can DNAJC10-FITC antibodies be used to study endoplasmic reticulum stress in acute myeloid leukemia?

DNAJC10-FITC antibodies can be effectively employed to study ER stress in AML using these methodological approaches:

• Baseline expression profiling:

  • Compare DNAJC10 expression across AML subtypes using flow cytometry

  • Correlate expression with clinical parameters and patient outcomes

  • Stratify samples based on leukemia stem cell markers to identify stem cell-specific patterns

• Stress induction monitoring:

  • Track DNAJC10 expression changes during:

    • Chemotherapy treatment (daunorubicin, cytarabine)

    • Pharmacological ER stress induction (tunicamycin, thapsigargin)

    • Physiological stress conditions (glucose deprivation, hypoxia)

  • Correlate with UPR activation markers (GRP78, CHOP, PERK phosphorylation)

• Functional manipulation experiments:

  • Compare wild-type and DNAJC10-knockout cells for:

    • Cell viability under stress conditions

    • Drug sensitivity profiles

    • Colony formation capacity

    • Engraftment potential in xenograft models

• Mechanistic pathway analysis:

  • Use multi-parameter flow cytometry to correlate DNAJC10 with:

    • PERK-EIF2α-ATF4 pathway activation

    • Pro-survival vs. pro-apoptotic UPR balance

    • Cell cycle distribution and apoptosis markers

Research has demonstrated that DNAJC10 deficiency leads to activation of the pro-apoptotic PERK branch of UPR and enhances chemosensitivity, providing a valuable framework for therapeutic investigations .

What experimental designs are most effective for investigating DNAJC10's role in chemotherapy resistance?

To effectively investigate DNAJC10's role in chemotherapy resistance, researchers should implement these methodological approaches:

• Expression correlation studies:

  • Analyze DNAJC10 expression in paired pre-treatment and relapsed patient samples

  • Correlate baseline expression with treatment response

  • Monitor expression changes during treatment and at relapse

  • Data shows AML patients with high DNAJC10 expression tend to have shorter disease-free survival after "DNR + Ara-C" treatment

• In vitro sensitivity profiling:

  • Compare IC50 values for chemotherapeutic agents in:

    • Wild-type vs. DNAJC10-knockout cells

    • DNAJC10-overexpressing vs. control cells

    • Baseline conditions vs. ER stress induction

  • DNAJC10-KO cells show decreased IC50 for daunorubicin and cytarabine compared to wild-type

• Mechanism exploration design:

  • Track UPR pathway activation during drug treatment

  • Combine DNAJC10 modulation with PERK inhibition (GSK2606414)

  • Monitor pro-apoptotic proteins (CHOP, GADD34, BAX) after treatment

  • Assess effects on protein misfolding and aggregation

• In vivo resistance models:

  • Establish PDX models with varying DNAJC10 expression levels

  • Monitor treatment response and resistance development

  • Analyze DNAJC10 expression in residual disease after treatment

  • Test combination strategies targeting DNAJC10 and standard chemotherapy

This comprehensive experimental framework addresses the finding that up-regulated DNAJC10 expression is closely related to daunorubicin and cytarabine resistance in AML patients .

How can researchers use DNAJC10 antibodies to identify potential therapeutic targets in the UPR pathway?

To identify potential therapeutic targets in the UPR pathway using DNAJC10 antibodies, researchers should implement this methodological framework:

• Interaction partner identification:

  • Use DNAJC10 antibodies for co-immunoprecipitation followed by mass spectrometry

  • Perform proximity ligation assays to confirm interactions in situ

  • Map interaction domains through deletion mutant analysis

  • DNAJC10 interacts with HSP70 chaperone machinery and components of the ERAD complex

• Pathway modulation screening:

  • Combine DNAJC10 knockdown with chemical inhibitors of UPR branches

  • Screen for synthetic lethality with PERK, IRE1, or ATF6 inhibitors

  • Blocking PERK by GSK2606414 (PERKi) or shRNA rescues the loss of function of DNAJC10 both in vitro and in vivo

• Drug sensitivity profiling:

  • Test how DNAJC10 modulation affects response to:

    • Proteasome inhibitors (bortezomib, carfilzomib)

    • HSP inhibitors (17-AAG, ganetespib)

    • ER stress modulators (salubrinal, ISRIB)

  • DNAJC10 deficiency remarkably enhances DNR and Ara-C sensitivity in AML cells

• Combination therapy design:

  • Develop rational combinations targeting:

    • DNAJC10 expression or function

    • Downstream effectors in pro-survival UPR branches

    • Protein quality control mechanisms

  • Test combinations in patient-derived models with varying DNAJC10 expression

This systematic approach leverages the finding that deficiency of DNAJC10 activates the PERK-EIF2α-CHOP axis and pro-apoptotic factors, potentially enhancing chemotherapy efficacy .

What are the optimal conditions for DNAJC10-FITC antibody validation in novel cell types?

For rigorous validation of DNAJC10-FITC antibodies in novel cell types, researchers should implement this methodological framework:

• Expression verification strategy:

  • Western blot validation (expected MW: 91 kDa)

  • RT-qPCR correlation with protein expression

  • Comparison with reference cell lines of known DNAJC10 expression

  • Validation across multiple antibody clones or vendors

• Dilution optimization protocol:

  • Perform titration series (1:500 to 1:2000) to identify optimal concentration

  • Evaluate signal-to-noise ratio across dilutions

  • Test multiple fixation/permeabilization combinations

  • Optimize incubation times and temperatures

• Specificity confirmation methods:

  • Generate transient knockdown controls (siRNA/shRNA)

  • Validate with genetic knockout references when available

  • Perform epitope-blocking experiments

  • Compare staining patterns across different detection platforms

• Application-specific validation:

  • For flow cytometry: optimize voltage settings and compensation

  • For microscopy: determine ideal exposure settings and filter combinations

  • For multiplexed assays: verify absence of interference with other fluorophores

This systematic validation approach ensures reliable and reproducible results when extending DNAJC10-FITC antibody applications to previously untested cell types .

How does DNAJC10 expression correlate with other UPR markers during cellular stress responses?

To characterize DNAJC10 correlation with other UPR markers during stress responses, researchers should implement this methodological framework:

• Temporal expression profiling:

  • Monitor expression kinetics of DNAJC10 alongside other UPR markers:

    • GRP78/BiP (general UPR indicator)

    • PERK, phospho-PERK (PERK pathway)

    • XBP1s (IRE1 pathway)

    • ATF6 (cleaved fragments)

  • Track at multiple timepoints (2h, 6h, 12h, 24h) after stress induction

  • Research shows Ara-C or DNR treatment significantly induces DNAJC10 and GRP78 up-regulated expression in a dose-dependent manner

• Pathway-specific correlation analysis:

  • Compare DNAJC10 with pro-survival UPR components

  • Correlate with pro-apoptotic UPR markers (CHOP, GADD34)

  • Analyze relationship with downstream effectors (BAX)

  • DNAJC10 deficiency activates the PERK-EIF2α-CHOP axis and pro-apoptotic factors

• Multi-parameter quantification approaches:

  • Use flow cytometry for single-cell correlation analysis

  • Implement immunofluorescence for spatial relationship assessment

  • Apply Western blotting for population-level protein quantification

  • Correlate with functional readouts (apoptosis, proliferation)

• Stress-type specific patterns:

  • Compare patterns across different stressors:

    • ER stress inducers (tunicamycin, thapsigargin)

    • Chemotherapeutic agents (daunorubicin, cytarabine)

    • Physiological stressors (hypoxia, nutrient deprivation)

This comprehensive approach provides mechanistic insights into how DNAJC10 functions within the broader UPR network during cellular stress responses .

How can DNAJC10-FITC antibodies be incorporated into high-throughput screening applications?

To effectively incorporate DNAJC10-FITC antibodies into high-throughput screening applications, researchers should implement this methodological framework:

• Assay miniaturization protocol:

  • Adapt staining protocols for 96/384-well format

  • Optimize antibody concentrations for microvolume applications

  • Develop automated liquid handling procedures

  • Establish consistent cell seeding densities for reproducibility

• Automated image acquisition parameters:

  • Define optimal exposure settings for FITC channel

  • Establish z-stack parameters for maximum signal capture

  • Program consistent field selection algorithms

  • Implement autofocus routines optimized for ER structures

• Analysis pipeline development:

  • Create cell segmentation algorithms based on nuclear and cytoplasmic markers

  • Develop DNAJC10 signal quantification methods:

    • Mean fluorescence intensity

    • Subcellular distribution patterns

    • Colocalization with ER markers

  • Establish normalization procedures for plate-to-plate comparison

• Application-specific optimization:

  • For drug screens: determine optimal treatment duration and concentration ranges

  • For genetic screens: establish positive/negative control thresholds

  • For patient sample analysis: develop standardization protocols

  • For all applications: implement quality control metrics and outlier detection