DNAJC10 Antibody, Biotin conjugated

What is DNAJC10 and why is it relevant for research?

DNAJC10 (DnaJ heat shock protein family member C10), also known as ERdj5, is an endoplasmic reticulum co-chaperone involved in protein folding and degradation pathways. It facilitates the removal of non-native disulfide bonds during protein folding and participates in ER-associated degradation (ERAD) by reducing incorrect disulfide bonds in misfolded glycoproteins. The protein contains a type III DnaJ domain, four thioredoxin-like domains, and a KDEL ER retention signal, with a calculated molecular weight of 91 kDa. Recent research has identified DNAJC10 as a potential prognostic biomarker in glioma, where higher expression correlates with poor prognosis and immune infiltration characteristics .

How do biotin-conjugated antibodies differ from other conjugates for DNAJC10 detection?

Biotin-conjugated antibodies utilize a small molecule (biotin) that non-covalently binds to avidin and streptavidin with exceptionally high affinity, making them excellent for detection in various immunological techniques . Unlike directly labeled antibodies (such as FITC or HRP conjugates), biotin-conjugated antibodies require a secondary detection system (typically streptavidin linked to a reporter), but offer significant advantages in signal amplification . For DNAJC10 detection specifically, biotin conjugation enables compatibility with streptavidin-based detection systems, enhancing sensitivity particularly in sandwich ELISA formats and immunohistochemistry applications.

What are the key molecular characteristics of DNAJC10 that might affect antibody recognition?

DNAJC10 presents several structural considerations that may affect antibody recognition:

The observed molecular weight (80-90 kDa) often differs from the calculated size (91 kDa), which is important to consider when validating antibody specificity in Western blot applications .

What is the optimal protocol for using DNAJC10 biotin-conjugated antibodies in ELISA applications?

For optimal ELISA performance with biotin-conjugated DNAJC10 antibodies:

• Coat wells with DNAJC10 antigen (typically recombinant protein)

• Add samples according to standard ELISA protocols

• Apply biotin-conjugated DNAJC10 antibody at dilutions ranging from 1:1000 to 1:5000

• Introduce streptavidin-conjugated reporter enzyme (typically HRP or alkaline phosphatase)

• Add appropriate substrate and measure signal

Critical optimization parameters include:

• Antibody dilution: Begin with manufacturer's recommended range (typically 1:1000-1:5000) and optimize for your specific experimental conditions

• Incubation times: Generally 1-2 hours at room temperature or overnight at 4°C

• Washing steps: Typically 3-5 washes with PBS-T between each step to minimize background

• Blocking: Use 1-5% BSA or similar blocking agent to reduce non-specific binding

How should researchers validate DNAJC10 antibody specificity for their particular experimental system?

A comprehensive validation approach for DNAJC10 antibodies should include:

• Positive control testing: Verify activity against cell lines known to express DNAJC10 (e.g., HeLa cells, HepG2 cells, mouse liver tissue)

• Western blot validation: Confirm single band at expected molecular weight (80-90 kDa); be aware that DNAJC10 may show bands that differ from theoretical calculations

• Immunohistochemistry controls: Include positive controls from verified tissues (e.g., human pancreas, human stomach, human testis)

• Knockout/knockdown validation: If possible, test antibody against DNAJC10 knockout or knockdown samples to confirm specificity

• Cross-reactivity assessment: Verify the antibody works with your species of interest - most DNAJC10 antibodies show reactivity with human, mouse, and rat samples

What dilution ranges are recommended for different applications of biotin-conjugated DNAJC10 antibodies?

Note: These ranges should be considered starting points; each antibody lot and experimental system may require specific optimization .

How can I optimize signal amplification when using biotin-conjugated DNAJC10 antibodies?

Biotin-conjugated antibodies offer several strategies for signal amplification:

• Biotin-streptavidin amplification systems: Utilize the multiple biotin-binding sites on streptavidin (four per molecule) for signal enhancement

  • Implementation: Use biotinylated secondary antibody followed by streptavidin-HRP or streptavidin-fluorophore

  • Expected outcome: 3-10 fold signal enhancement compared to direct detection

• Tyramide signal amplification (TSA): Combines biotin-streptavidin with HRP-catalyzed deposition of tyramide

  • Implementation: After streptavidin-HRP binding, add biotinylated tyramide followed by additional streptavidin-reporter

  • Expected outcome: Up to 100-fold signal enhancement for detection of low-abundance targets

• Biotin-SP conjugates: Use antibodies with extended spacer arms between the antibody and biotin

  • Implementation: Select biotin-SP conjugated antibodies specifically

  • Expected outcome: Increased sensitivity particularly with alkaline phosphatase-conjugated streptavidin due to improved accessibility of biotin to streptavidin binding sites

For DNAJC10 specifically, optimization may be necessary for cellular compartment targeting, as DNAJC10 is primarily localized to the endoplasmic reticulum lumen .

What are the common pitfalls when using biotin-conjugated DNAJC10 antibodies and how can they be addressed?

DNAJC10-specific consideration: The observed molecular weight (80-90 kDa) often differs from calculated size, which may cause confusion in data interpretation. This discrepancy is typically due to post-translational modifications or protein mobility factors in SDS-PAGE .

How does epitope accessibility affect DNAJC10 detection, and what methods can improve detection in different subcellular compartments?

DNAJC10's localization in the endoplasmic reticulum lumen presents specific challenges for antibody accessibility:

• Fixation optimization for immunocytochemistry/immunohistochemistry:

  • Paraformaldehyde (4%) provides good structural preservation while maintaining antigenicity

  • Methanol fixation may improve access to ER lumen proteins but can disrupt some epitopes

  • Combination fixation (PFA followed by methanol) may provide optimal results for DNAJC10

• Permeabilization strategies:

  • Standard protocols (0.1-0.5% Triton X-100) may be insufficient for ER lumen proteins

  • Digitonin (25-50 μg/ml) provides selective permeabilization of plasma membrane while keeping ER intact

  • Saponin (0.025-0.1%) offers reversible permeabilization that may better preserve ER structure

• Antigen retrieval methods for tissue sections:

  • Heat-induced epitope retrieval with TE buffer (pH 9.0) is specifically recommended for DNAJC10 detection

  • Alternative: Citrate buffer (pH 6.0) may be used but typically yields lower signal intensity

• Sample preparation for Western blotting:

  • Specialized lysis buffers containing mild detergents (1% Triton X-100 or 0.5% NP-40) better solubilize ER membrane proteins

  • Include reducing agents to maintain native protein conformation and epitope accessibility

How can DNAJC10 antibodies be used to investigate ER stress responses and protein quality control mechanisms?

DNAJC10 functions as an ER co-chaperone involved in protein folding and degradation, making it a valuable marker for studying ER stress responses:

• Co-immunoprecipitation applications:

  • Biotin-conjugated DNAJC10 antibodies can be used to pull down protein complexes

  • This approach can identify interaction partners within the ERAD machinery

  • Protocol modification: Use gentler lysis conditions (150-300 mM NaCl, 0.5-1% NP-40) to preserve protein-protein interactions

• Dual immunostaining strategies:

  • Combine DNAJC10 detection with other ER stress markers (BiP/GRP78, CHOP, XBP1)

  • Implementation: Use spectrally distinct detection systems for biotin-conjugated DNAJC10 antibody and other markers

  • Expected outcome: Co-localization patterns reveal stress-induced changes in DNAJC10 distribution

• Quantitative analysis approaches:

  • Western blotting with biotin-conjugated antibodies can quantify DNAJC10 upregulation during ER stress

  • RT-qPCR can complement protein data to assess transcriptional changes

  • Flow cytometry using permeabilized cells can measure DNAJC10 at the single-cell level

• Functional assays:

  • Monitor disulfide bond reduction activity in the presence of ER stressors

  • Assess ERAD substrate degradation rates in relation to DNAJC10 expression levels

What insights can DNAJC10 antibody studies provide about its role in cancer biology, particularly in glioma?

Recent research has identified DNAJC10 as potentially significant in cancer biology, particularly glioma:

How should researchers interpret discrepancies between predicted and observed molecular weights when using DNAJC10 antibodies in Western blot applications?

Western blotting with DNAJC10 antibodies frequently shows discrepancies between predicted (91 kDa) and observed (80-90 kDa) molecular weights:

• Common causes of molecular weight discrepancies:

  Factor	Explanation	Interpretation Approach
  Post-translational modifications	Phosphorylation, glycosylation, or other modifications	Compare to literature reports; use phosphatase or glycosidase treatments to confirm
  Protein isoforms	Alternative splicing generates multiple isoforms (37 kDa/86 kDa/91 kDa reported)	Run positive control samples; compare to transcript data
  Protein mobility factors	Charged residues affect migration in SDS-PAGE	Consider protein properties beyond just molecular weight
  Sample preparation	Reducing conditions affect disulfide-rich proteins like DNAJC10	Compare reducing vs. non-reducing conditions

• Validation approaches:

  • Positive control testing with verified cell lines (HeLa, HepG2, mouse liver tissue)

  • Comparison across different antibody clones targeting distinct epitopes

  • Mass spectrometry confirmation of band identity in critical applications

  • Knockdown/knockout validation to confirm specificity

• Reporting standards:

  • Always report both predicted and observed molecular weights

  • Document complete experimental conditions that may affect mobility

  • Consider including positive control samples when publishing novel findings

How can DNAJC10 antibodies be integrated into multi-parameter analyses of the unfolded protein response pathway?

Advanced research into unfolded protein response (UPR) pathways can benefit from incorporating DNAJC10 detection:

• Multiplex immunofluorescence strategies:

  • Biotin-conjugated DNAJC10 antibodies can be combined with antibodies against other UPR components

  • Implementation: Use streptavidin conjugated to spectrally distinct fluorophores alongside directly labeled antibodies

  • Analysis: Quantify colocalization coefficients and expression correlation at subcellular level

• Proximity ligation assays (PLA):

  • Detect protein-protein interactions between DNAJC10 and UPR components in situ

  • Approach: Combine biotin-conjugated DNAJC10 antibody with antibodies against potential interaction partners

  • Expected outcome: Visualization of specific interaction points within cells

• Single-cell analysis integration:

  • Combine flow cytometry using DNAJC10 antibodies with single-cell RNA-seq

  • Implementation: Index sorting of cells followed by transcriptome analysis

  • Advantage: Correlates protein-level DNAJC10 expression with genome-wide transcriptional profiles

• CRISPR-based functional screening:

  • Use DNAJC10 antibodies to validate knockouts/knockdowns in CRISPR screens

  • Approach: High-throughput immunostaining of edited cells to confirm protein depletion

  • Application: Identify functional interactions within UPR pathway

What methodological advances could address current limitations in studying DNAJC10's role in ER-associated degradation (ERAD)?

Current ERAD research faces several technical challenges that novel approaches could address:

• Live-cell imaging of ERAD dynamics:

  • Challenge: Traditional fixed-cell immunostaining provides only static snapshots

  • Solution: Develop cell lines expressing tagged ERAD substrates combined with DNAJC10 antibody-based detection after fixation at defined timepoints

  • Expected outcome: Temporal resolution of DNAJC10 involvement in ERAD process

• Improved isolation of ERAD complexes:

  • Challenge: Transient nature of ERAD complexes makes them difficult to isolate

  • Solution: Crosslinking approaches followed by immunoprecipitation with biotin-conjugated DNAJC10 antibodies

  • Advantage: Captures otherwise unstable protein interactions within the ERAD machinery

• Super-resolution microscopy applications:

  • Challenge: Conventional microscopy lacks resolution to visualize ERAD microdomains

  • Solution: STORM/PALM techniques using biotin-conjugated DNAJC10 antibodies with streptavidin-fluorophores

  • Expected outcome: Nanoscale visualization of DNAJC10 distribution relative to ERAD components

• Domain-specific functional analysis:

  • Challenge: Understanding contribution of specific DNAJC10 domains to ERAD function

  • Solution: Generate domain-specific antibodies or use epitope-tagged domain constructs

  • Application: Map functional domains through sophisticated immunolocalization and interaction studies

How should researchers approach conflicting data between DNAJC10 antibody-based studies and other methodologies?

When faced with conflicting data between antibody-based detection and other methods:

• Systematic validation framework:

  • Verify antibody specificity with multiple controls (western blot, immunoprecipitation, knockdown validation)

  • Test multiple antibody clones targeting different epitopes

  • Compare commercial antibodies from different vendors

• Technology-specific considerations:

  Method	Common Issues	Resolution Approach
  Antibody vs. mRNA data	Post-transcriptional regulation	Perform temporal studies to capture delay between transcription and translation
  Immunostaining vs. biochemical assays	Fixation artifacts	Test multiple fixation protocols; validate with live-cell approaches when possible
  Different antibody clones yielding different results	Epitope accessibility or specificity	Map epitopes; test under varying conditions; validate with non-antibody methods

• Integrated methodology approach:

  • Combine antibody-based detection with orthogonal methods (mass spectrometry, CRISPR validation, reporter systems)

  • Weight evidence based on methodological strengths and limitations

  • Consider biological context and known regulatory mechanisms that might explain discrepancies

• Reporting standards for conflicting data:

  • Transparently document all methodological details

  • Present conflicting data alongside possible explanations

  • Discuss limitations of each approach to guide future research