CISD1 Antibody

What is CISD1 protein and why is it significant for research?

CISD1, also known as mitoNEET, belongs to an evolutionarily conserved family of proteins characterized by the presence of a unique 39 amino acid CDGSH domain. It is a single-pass type III membrane protein localized to the outer mitochondrial membrane . The significance of CISD1 lies in its role in:

• Regulating mitochondrial function and iron homeostasis

• Participating in cellular redox reactions via its iron-sulfur (2Fe-2S) cluster

• Potential involvement in neurodegenerative disorders, particularly Parkinson's disease

• Contributing to cell survival mechanisms and protection against oxidative damage

Recent research has highlighted CISD1's downstream involvement in the pathophysiological cascade initiated by PINK1 and partially PRKN loss of function, implicating it as a potential therapeutic target for Parkinson's disease .

What types of CISD1 antibodies are currently available for research applications?

Based on available research data, several types of CISD1 antibodies have been developed for various experimental applications:

These antibodies vary in their specificity, sensitivity, and optimal applications, making it essential to select the appropriate antibody based on experimental needs and target organisms .

How should CISD1 antibodies be stored to maintain optimal reactivity?

Proper storage of CISD1 antibodies is crucial for maintaining their reactivity and specificity over time. According to manufacturer recommendations:

• Store at -20°C for long-term preservation

• Antibodies are typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Stability is generally guaranteed for one year after shipment when properly stored

• Aliquoting is not necessary for -20°C storage according to manufacturer guidelines

• Some preparations (20μl sizes) may contain 0.1% BSA as a stabilizer

Repeated freeze-thaw cycles should be avoided to prevent protein degradation and loss of antibody activity. Before use, thaw antibodies completely and mix gently to ensure homogeneity.

What are the recommended dilutions for different applications of CISD1 antibodies?

The appropriate dilution of CISD1 antibodies varies significantly depending on the application and the specific antibody being used. Based on manufacturer recommendations:

It is recommended to titrate these antibodies in each testing system to obtain optimal results, as sensitivity can be sample-dependent .

How can researchers validate the specificity of CISD1 antibodies for their experimental models?

Validating antibody specificity is crucial for ensuring reliable research results. For CISD1 antibodies, several validation approaches are recommended:

• Positive controls: Use tissues/cells known to express CISD1, such as:

  • For 68030-1-Ig: LNCaP cells, HeLa cells, HEK-293 cells, brain tissues from various species

  • For 16006-1-AP: Mouse/rat kidney and skeletal muscle tissues, HepG2 cells

  • For CAB10317: Mouse/rat kidney

• Knockout validation: Utilize CISD1 knockout models as negative controls. As described in the literature, Cisd1 KO and wildtype (WT) MEFs have been used to identify correct bands in Western blot analysis .

• siRNA knockdown: Employ specific siRNA-mediated knockdown of CISD1 to confirm antibody specificity, as demonstrated in previous studies where siRNA knockdown of either CISD1 or CISD2 was used to validate antibody specificity .

• Molecular weight verification: Confirm that the observed molecular weight matches the expected size:

  • Calculated molecular weight: 12 kDa (108 amino acids)

  • Observed molecular weight: 14-17 kDa

  • Note that CISD1 forms homodimers that may be visible even on reducing gels

• Cross-reactivity assessment: Be aware that some antibodies may recognize both CISD1 and its homolog CISD2. Subcellular fractionation can help distinguish between mitochondrial CISD1 and ER-localized CISD2 .

How can researchers differentiate between CISD1 monomers and dimers in experimental analyses?

The differentiation between CISD1 monomers and dimers is a critical aspect of advanced CISD1 research, as dimerization patterns may be altered in pathological conditions:

• Western Blot Analysis:

  • CISD1 forms homodimers with high stringency even on reducing gels

  • Monomeric CISD1 typically appears at 14-17 kDa

  • Dimeric forms will appear at approximately 28-34 kDa

  • Calculate the dimer/monomer ratio to quantify dimerization levels, which may be altered in disease states (e.g., increased in PINK1 mutant neurons)

• Split Luciferase Assay:

  • A dynamic approach to investigate CISD1 dimerization in living cells

  • Fusion of CISD1 with small subunits of NanoLuc luciferase allows quantification of dimerization by measuring luminescence

  • This technique has revealed that iron-depleted CISD1 (e.g., C83S mutant) exhibits increased dimerization compared to wild-type

• In silico Analysis:

  • Calculation of molecular hydrophobicity potential and angle of rotation between CISD1 monomers

  • Analysis of surface-surface contact between monomers

  • This approach has shown larger surface-surface contact between iron-sulfur cluster-deficient CISD1 monomers compared to wild-type

• Iron-sulfur Cluster Manipulation:

  • The H87C mutation results in more stable 2Fe/2S complex coordination

  • The C83S mutation abolishes the ability of CISD1 to bind the 2Fe/2S complex

  • These mutations can be used experimentally to modulate dimerization propensity

What are the key considerations when investigating CISD1's role in Parkinson's disease using antibody-based approaches?

Recent research has implicated CISD1 in Parkinson's disease (PD) pathophysiology, particularly in relation to the PINK1/Parkin pathway. When investigating this connection using antibody-based approaches, researchers should consider:

• Patient-derived Models:

  • iPSCs from PD patients carrying specific mutations (e.g., p.Q456X mutation in PINK1) can be differentiated into neuronal models

  • Compare with isogenic gene-corrected controls for more reliable results

  • Ensure proper ethical approval and informed consent for human samples

• Dimerization Analysis:

  • Assess CISD1 dimerization status, as increased dimer/monomer ratio has been observed in mutant PINK1 neurons

  • Be aware of patient-specific variations that may influence dimerization patterns

  • Pool multiple differentiation experiments to obtain statistically significant results

• Cellular Localization:

  • Use subcellular fractionation to distinguish between mitochondrial CISD1 and ER-localized CISD2

  • Employ co-localization studies with mitochondrial markers in immunofluorescence experiments

  • Remember CISD1 is specifically located in the mitochondrial outer membrane

• Iron-sulfur Cluster Status:

  • Consider that iron-sulfur cluster loss in CISD1 may mediate PINK1-related pathology

  • Develop approaches to assess cluster integrity in experimental models

  • Understand that cluster status affects dimerization propensity

• Differentiation Protocols:

  • For neuronal models, follow established protocols (e.g., NESC conversion and midbrain-specific neuron generation)

  • Maintain appropriate culture conditions: N2B27 medium with specified supplements

  • Allow sufficient differentiation time (typically 28 days) for mature neuronal phenotypes

How should researchers optimize Western blot protocols for detecting both monomeric and dimeric forms of CISD1?

Detecting both monomeric and dimeric forms of CISD1 in Western blot requires careful optimization of experimental conditions:

• Sample Preparation:

  • Use appropriate lysis buffers that preserve protein-protein interactions

  • Consider non-denaturing conditions if native dimers are to be preserved

  • For reducing conditions, be aware that CISD1 dimers may still be visible due to their high stability

• Gel Selection and Electrophoresis:

  • Use gradient gels (e.g., 4-20%) to effectively separate both monomeric (14-17 kDa) and dimeric (28-34 kDa) forms

  • Optimize running conditions to ensure good separation of proteins in the lower molecular weight range

  • Consider native PAGE for analysis of natural dimerization state without denaturation

• Antibody Selection:

  • Choose antibodies validated for detecting both monomeric and dimeric forms

  • The 68030-1-Ig monoclonal antibody (1:5000-1:50000 dilution) has been successfully used for detecting CISD1 forms

  • The 16006-1-AP polyclonal antibody (1:5000-1:50000 dilution) may provide broader epitope recognition

• Controls and Validation:

  • Include Cisd1 KO samples as negative controls

  • Use recombinant CISD1 protein as a positive control

  • Consider including samples with altered dimerization (e.g., C83S mutant with increased dimerization)

• Quantification:

  • Calculate the dimer/monomer ratio to assess dimerization status

  • Use appropriate normalization (e.g., total CISD1 levels)

  • Be aware that dimerization may vary between individual samples, requiring multiple biological replicates

How can researchers investigate the relationship between CISD1's iron-sulfur cluster status and its function?

The iron-sulfur cluster of CISD1 is critical for its function, and investigating this relationship requires specialized approaches:

• Site-directed Mutagenesis:

  • Generate specific CISD1 mutants with altered cluster coordination:

    • H87C mutation: Creates more stable 2Fe/2S complex coordination

    • C83S mutation: Abolishes the ability to bind the 2Fe/2S complex

  • Express these mutants in appropriate cell models to assess functional consequences

• Spectroscopic Techniques:

  • UV-visible absorption spectroscopy to monitor the characteristic absorbance features of the 2Fe-2S cluster

  • Electron paramagnetic resonance (EPR) spectroscopy to assess the redox state of the iron-sulfur cluster

  • Resonance Raman spectroscopy to characterize metal-ligand interactions

• Functional Assays:

  • Measure mitochondrial function parameters (respiration, membrane potential) in models with altered CISD1 cluster status

  • Assess cellular responses to oxidative stress and iron homeostasis perturbations

  • Investigate dimerization patterns in relation to cluster integrity using split luciferase assays

• Structural Biology Approaches:

  • X-ray crystallography of wild-type and mutant CISD1 proteins

  • Cryo-electron microscopy to visualize protein conformations

  • In silico modeling to calculate surface-surface interactions between monomers with different cluster states

• Cellular Iron Measurements:

  • Quantify mitochondrial iron levels in systems with normal vs. cluster-deficient CISD1

  • Use fluorescent probes specific for mitochondrial iron

  • Correlate iron levels with CISD1 function and dimerization status

What methodological approaches are recommended for investigating CISD1 in induced pluripotent stem cell (iPSC)-derived neuronal models?

The use of iPSC-derived neuronal models for CISD1 research, particularly in the context of Parkinson's disease, requires specialized methodological approaches:

• iPSC Source and Differentiation:

  • Obtain iPSCs from patients with relevant mutations (e.g., PINK1 mutations) and matching controls

  • Generate isogenic gene-corrected controls for more precise comparisons

  • Convert iPSCs into neuroepithelial stem cells (NESCs) using established protocols

  • Further differentiate NESCs into midbrain-specific neurons using appropriate medium supplementation

• Differentiation Protocol:

  • Culture NESCs in N2B27 medium supplemented with CHIR-99021, purmorphamine, and ascorbic acid

  • For neuronal differentiation, modify medium to include purmorphamine, ascorbic acid, BDNF, GDNF, TGF-β3, and dbcAMP

  • After 6 days, remove purmorphamine and continue culture until day 28

• Validation of Neuronal Identity:

  • Confirm neuronal differentiation using markers such as TUJ1, MAP2, and NEUN

  • Verify dopaminergic phenotype using TH (tyrosine hydroxylase) immunostaining

  • Assess neuronal functionality using electrophysiological measurements

• CISD1 Analysis:

  • Perform immunoblotting to assess CISD1 expression levels and dimerization status

  • Use subcellular fractionation to distinguish between CISD1 and CISD2

  • Employ immunofluorescence to visualize CISD1 localization in neuronal structures

• Functional Assessments:

  • Measure mitochondrial function parameters (respiration, membrane potential)

  • Assess neuronal vulnerability to stressors

  • Investigate PINK1/Parkin pathway activation in response to mitochondrial stress

  • Evaluate the effects of manipulating CISD1 levels or function on neuronal survival

How can researchers reconcile contradictory findings regarding CISD1 expression and function across different experimental models?

Contradictory findings regarding CISD1 are not uncommon in the literature, and reconciling these discrepancies requires careful methodological consideration:

• Antibody Selection and Validation:

  • Be aware that some antibodies may recognize both CISD1 and its homolog CISD2

  • Validate antibody specificity using knockout models or siRNA knockdown

  • Consider using multiple antibodies targeting different epitopes to confirm findings

• Model System Considerations:

  • Different cell types may express varying levels of CISD1 and its interacting partners

  • Species differences may affect CISD1 function and regulation

  • Primary cells versus cell lines may show different CISD1 behaviors

  • Consider tissue-specific contexts when interpreting results

• Experimental Conditions:

  • Iron availability can significantly affect CISD1's cluster status and function

  • Oxidative stress levels may alter CISD1 dimerization and activity

  • Cell culture conditions (media composition, oxygen levels) can influence experimental outcomes

• Genetic Background Effects:

  • Patient-specific factors may influence CISD1 behavior, as observed in different PINK1 mutant lines

  • Consider genetic modifiers that may vary between experimental models

  • Use isogenic controls whenever possible to minimize background effects

• Technical Approaches:

  • Complement protein-level analyses with mRNA expression studies

  • Utilize multiple technical approaches to study the same phenomenon

  • Perform time-course experiments to capture dynamic changes in CISD1 behavior

  • Develop mathematical models to integrate diverse datasets and identify key variables affecting CISD1 function

What emerging technologies might enhance the specificity and utility of CISD1 antibodies in research?

Several emerging technologies hold promise for enhancing CISD1 antibody research:

• Nanobodies and Single-Domain Antibodies:

  • Smaller size allows better access to restricted epitopes

  • Enhanced penetration of cellular compartments for live-cell imaging

  • Potential for higher specificity to distinguish between CISD1 and CISD2

• Conformation-Specific Antibodies:

  • Development of antibodies that specifically recognize either the apo (iron-sulfur cluster-free) or holo (iron-sulfur cluster-bound) forms of CISD1

  • Would enable direct monitoring of CISD1's functional status in various conditions

  • Could help resolve the relationship between cluster status and dimerization

• Proximity Labeling Combined with Antibody Detection:

  • Use of APEX2 or BioID fused to CISD1 to identify proximal proteins

  • Combination with specific antibodies to map the CISD1 interactome

  • Could reveal context-specific interactions in different cell types or disease states

• Super-Resolution Microscopy:

  • Application of techniques like STORM or PALM with CISD1 antibodies

  • Would enable visualization of CISD1's precise localization at the mitochondrial outer membrane

  • Could reveal potential microdomains or clustering behaviors

• Multiparametric Antibody-Based Assays:

  • Development of multiplexed assays to simultaneously detect CISD1, its post-translational modifications, interaction partners, and conformational states

  • Would provide more comprehensive view of CISD1 biology in health and disease

How might CISD1 antibodies be utilized to explore therapeutic approaches for Parkinson's disease?

Given the emerging role of CISD1 in Parkinson's disease pathophysiology, antibody-based approaches could facilitate therapeutic development:

• Target Validation:

  • Use CISD1 antibodies to confirm altered expression, localization, or dimerization in patient samples

  • Validate CISD1 as a downstream effector in the PINK1/Parkin pathway

  • Map the relationship between CISD1 dysfunction and disease progression

• Compound Screening:

  • Develop high-content screening assays using CISD1 antibodies to identify compounds that:

    • Stabilize the iron-sulfur cluster

    • Modulate dimerization

    • Restore normal CISD1 function in disease models

  • Use immunofluorescence-based readouts to assess CISD1 localization and mitochondrial morphology

• Biomarker Development:

  • Investigate whether altered CISD1 dimerization could serve as a disease biomarker

  • Develop sensitive assays to detect CISD1 status in accessible samples

  • Correlate CISD1 alterations with clinical parameters and disease progression

• Therapeutic Monitoring:

  • Use CISD1 antibodies to assess the efficacy of experimental therapies in:

    • Restoring proper CISD1 dimerization levels

    • Maintaining iron-sulfur cluster integrity

    • Re-establishing normal mitochondrial function

• iPSC-Based Personalized Medicine:

  • Employ CISD1 antibodies in patient-derived iPSC neuronal models

  • Screen for compounds that normalize CISD1 function in individual patients

  • Develop personalized therapeutic approaches based on specific CISD1 alterations