DNAJC15 Antibody, Biotin conjugated

What is DNAJC15 and what are its primary biological functions?

DNAJC15 (DnaJ homolog subfamily C member 15) is a member of the DnaJ family of chaperones that contains a transmembrane domain and a unique N-terminal domain not found in other family members. It functions primarily as a negative regulator of the mitochondrial respiratory chain by preventing mitochondrial hyperpolarization and restricting ATP generation . DNAJC15 acts as an endogenous brake for mitochondrial respiration, particularly in CD8 T cells, by interfering with the formation of electron transport chain (ETC) respiratory supercomplexes . Additionally, it serves as an import component of the TIM23 translocase complex and stimulates the ATPase activity of HSPA9 .

How does the biotin conjugation affect the functionality of DNAJC15 antibody?

Biotin conjugation to the DNAJC15 antibody provides significant advantages for detection sensitivity without compromising antibody specificity. The biotin-avidin system offers high affinity binding that amplifies detection signals in techniques like ELISA, where this antibody is primarily utilized . The small biotin molecule (244 Da) minimally impacts the antibody's binding properties to the DNAJC15 epitope while providing a strong interaction with streptavidin-linked detection systems. This conjugation enables greater flexibility in experimental design, allowing researchers to employ various detection strategies using the same primary antibody.

What are the key aliases and alternative nomenclature for DNAJC15 in the scientific literature?

When conducting literature searches, researchers should be aware of multiple designations for this protein. DNAJC15 is also known as: MCJ (Methylation-controlled J protein), DNAJD1, GIG22 (Cell growth-inhibiting gene 22 protein), and HSD18 . The UniProt ID for human DNAJC15 is Q9Y5T4, and the gene ID is 29103 . Understanding these alternative names is critical for comprehensive literature searches and avoiding duplicate or fragmented research efforts, especially when exploring DNAJC15's role in different physiological contexts or disease models.

How does DNAJC15 regulate T cell metabolism and immune function?

DNAJC15/MCJ functions as a critical metabolic checkpoint in CD8 T cells by regulating the transition between different metabolic states. In CD8 T cells, DNAJC15 interferes with the formation of electron transport chain respiratory supercomplexes, effectively limiting mitochondrial respiration . Loss of MCJ in CD8 T cells leads to enhanced mitochondrial metabolism, with metabolic profiling revealing increased oxidative phosphorylation and subcellular ATP accumulation .

Interestingly, this metabolic alteration selectively increases the secretion, but not the expression, of IFNγ. Studies have shown that MCJ knockout CD8 T cells secrete significantly higher levels of IFNγ compared to wild-type cells when activated with anti-CD3 and anti-CD28 antibodies . This selective enhancement of cytokine secretion without affecting expression levels suggests that DNAJC15 regulates post-translational processes related to cytokine trafficking and release. Additionally, MCJ assists in adapting effector CD8 T cell metabolism during the contraction phase, making memory CD8 cells lacking MCJ superior in providing protection against influenza virus infection .

What experimental approaches can resolve contradictions in DNAJC15 expression data between different tissue types?

Reconciling contradictory DNAJC15 expression data requires a multi-faceted approach:

• Epigenetic analysis: The dnajc15 gene is known to be regulated by methylation-induced suppression . Researchers should employ bisulfite sequencing or methylation-specific PCR to assess the methylation status of the DNAJC15 promoter across different tissues.

• Tissue-specific knockout models: Generate conditional knockout models to study tissue-specific functions of DNAJC15 rather than relying on global expression data.

• Single-cell techniques: Implement single-cell RNA sequencing to identify cell subpopulations with differential DNAJC15 expression within heterogeneous tissues.

• Standardized protein detection: Use the same DNAJC15 antibody with validated specificity across all tissue types, and confirm results with at least two detection methods (e.g., Western blot and immunohistochemistry).

• Metabolic state consideration: Document the metabolic state of tissues during sample collection, as glycolysis has been shown to induce MCJ expression in CD8+ T cells .

How does DNAJC15 interact with mitochondrial Complex I and what are the implications for cellular metabolism?

DNAJC15 interacts with NDUFA9, a component of Complex I of the electron transport chain, influencing mitochondrial efficiency and oxidative damage under stress conditions . This interaction appears to regulate Complex I activity in a tissue-specific manner. In CD8 T cells, MCJ deficiency results in reduced Complex II activity compared to wild-type cells, suggesting a functional uncoupling of Complex II from the rest of the electron transport chain .

Metabolic flux analyses using [13C, 15N]-glutamine revealed that MCJ knockout CD8 T cells produce significantly higher levels of newly synthesized succinate compared to wild-type cells, yet do not show increased levels of fumarate (the product of Complex II) . This indicates that Complex II becomes uncoupled from the rest of the ETC in MCJ-deficient cells, creating a unique metabolic configuration that affects cellular energy production and utilization.

The biochemical consequence of this interaction extends beyond energy production to influence immune cell function, suggesting that DNAJC15 serves as a molecular switch that coordinates mitochondrial metabolism with cellular effector functions.

What are the optimal protocols for using DNAJC15 Antibody, Biotin conjugated in ELISA applications?

For optimal ELISA applications with DNAJC15 Antibody, Biotin conjugated:

Standard Protocol:

• Coating: Adsorb recombinant DNAJC15 protein or tissue/cell lysate (2-10 μg/ml) in carbonate buffer (pH 9.6) overnight at 4°C.

• Blocking: Block with 3% BSA in PBS for 2 hours at room temperature.

• Primary antibody: Apply DNAJC15 Antibody at experimentally determined optimal concentration (starting recommendation: 1:1000 to 1:2000 dilution) .

• Detection: Use streptavidin-HRP (1:5000) for 1 hour followed by TMB substrate.

• Signal measurement: Read absorbance at 450 nm with 570 nm reference wavelength.

Critical Parameters:

• The antibody recognizes human DNAJC15 with highest specificity and has been purified using Protein G chromatography to >95% purity .

• Store antibody in aliquots at -20°C with 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin-300 to prevent repeated freeze/thaw cycles .

• Optimal dilutions must be determined empirically by each laboratory for their specific application .

What controls are essential when studying DNAJC15 function in mitochondrial respiration experiments?

When investigating DNAJC15's role in mitochondrial respiration, these controls are essential:

• Positive Expression Control: Include lysates from 293T cells transfected with DNAJC15 alongside non-transfected cells when validating antibody specificity .

• Methylation Inhibitor Control: When studying regulation of DNAJC15 expression, include 5-azacytidine (1 μM) treatment as a DNA methyltransferase inhibitor control, which has been shown to increase MCJ protein expression .

• Functional Respiratory Controls:

  • Measure Complex II activity independently of other complexes

  • Track metabolite flux using labeled substrates (e.g., [13C, 15N]-glutamine for succinate production)

  • Monitor both upstream substrates and downstream products (e.g., succinate and fumarate levels)

• Temporal Controls: For secretion studies, collect supernatants at multiple time points (2h, 4h, 6h) to capture the kinetics of protein release .

• Metabolic State Control: Document and standardize the glycolytic state of cells, as glycolysis has been shown to influence MCJ expression levels .

How can researchers effectively validate DNAJC15 antibody specificity for their experimental systems?

To ensure rigorous validation of DNAJC15 antibody specificity:

• Overexpression System: Compare antibody reactivity between cells transfected with DNAJC15 and non-transfected controls. Western blot should show a band at the predicted molecular weight of 16 kDa in transfected cells with minimal background in controls .

• Genetic Knockout Validation: Test the antibody in DNAJC15 knockout models or siRNA-depleted samples to confirm absence of signal.

• Epitope Competition: Pre-incubate the antibody with recombinant DNAJC15 protein (59-150AA) which represents the immunogen used to generate the antibody .

• Multiple Detection Methods: Confirm findings using alternative methods beyond the primary application (e.g., if using for ELISA, validate with Western blot).

• Cross-Reactivity Assessment: Test against closely related DnaJ family proteins to ensure specificity for DNAJC15.

• Isotype Control: Include rabbit IgG isotype control at the same concentration to identify non-specific binding .

How should researchers design experiments to investigate the relationship between DNAJC15 expression and mitochondrial function in different cell types?

A comprehensive experimental design should include:

• Baseline Characterization:

  • Quantify native DNAJC15 expression levels across cell types using validated antibodies

  • Measure baseline mitochondrial parameters (membrane potential, oxygen consumption, ATP production)

  • Analyze methylation status of the dnajc15 promoter region

• Manipulation Studies:

  • Generate DNAJC15 knockdown and overexpression models in each cell type

  • Use 5-azacytidine treatment to reverse methylation-based silencing

  • Implement metabolic switches (glycolysis induction/inhibition) to modulate expression

• Functional Assessment:

  Parameter	DNAJC15 Normal	DNAJC15 Knockdown	DNAJC15 Overexpression
  Complex I activity	Control value	Expected increase	Expected decrease
  Complex II coupling	Control value	Potential uncoupling	Enhanced coupling
  ATP production	Control value	Expected increase	Expected decrease
  Mitochondrial membrane potential	Control value	Expected increase	Expected decrease
  Cellular function (e.g., IFNγ secretion in T cells)	Control value	Expected increase	Expected decrease

• Colocalization Studies: Use high-resolution microscopy to track DNAJC15 interactions with respiratory complex components under different cellular conditions.

What are the key considerations when interpreting contradictory data on DNAJC15's role in different disease models?

When reconciling conflicting findings about DNAJC15's role in disease:

• Context Dependency: DNAJC15 was first identified in ovarian cancer cell lines and tumors as a gene negatively regulated by methylation . Its loss has been associated with chemoresistance in human breast cancer . Recognize that DNAJC15's function may be highly contextual and tissue-specific.

• Metabolic State Analysis: Document the predominant metabolic pathway (glycolysis vs. oxidative phosphorylation) in each disease model, as DNAJC15 expression is linked to glycolytic activity .

• Temporal Considerations: Determine whether observations represent acute responses or chronic adaptations, as DNAJC15 serves different roles during effector T cell activation versus memory cell development .

• Pathway Integration: Consider DNAJC15's position within larger signaling networks rather than in isolation. Its effects may be amplified or negated depending on the status of other pathway components.

• Genetic Background Effects: Document strain differences in animal models or genetic variation in human studies that might influence DNAJC15 function or expression.

• Methodological Differences: Standardize experimental approaches, including antibody selection, detection methods, and functional readouts to reduce technical variability as a source of apparent contradiction.

What emerging technologies could advance our understanding of DNAJC15's role in mitochondrial dynamics and cellular metabolism?

Several cutting-edge approaches show promise for elucidating DNAJC15 function:

• Cryo-EM Structural Analysis: Determine the atomic structure of DNAJC15 within the mitochondrial membrane to understand its physical interaction with respiratory complexes.

• Mitochondrial-Targeted CRISPR Screens: Employ mitochondrial-targeted CRISPR-Cas9 systems to identify synthetic lethal or synthetic viable interactions with DNAJC15.

• Real-time Metabolic Imaging: Utilize genetically-encoded metabolic sensors to visualize how DNAJC15 affects subcellular ATP distribution and consumption in live cells.

• Single-Organelle Metabolomics: Apply newly developed techniques for analyzing the metabolome of individual mitochondria to understand heterogeneity in DNAJC15 effects.

• Tissue-Specific Conditional Models: Develop inducible, tissue-specific DNAJC15 knockout or overexpression models to dissect its function in complex physiological systems.

• Multi-omics Integration: Combine transcriptomics, proteomics, and metabolomics data with machine learning approaches to identify patterns in DNAJC15-dependent cellular responses across different conditions and cell types.

These technologies would address current knowledge gaps regarding how DNAJC15 influences mitochondrial architecture and regulates the delicate balance between energy production and cellular function in health and disease.