CCS1 Antibody
Description
Definition and Target Specificity of CCS1 Antibody
The CCS1 antibody is a polyclonal immunoglobulin targeting the Copper Chaperone for Superoxide Dismutase (CCS1), a critical factor in activating copper-zinc superoxide dismutase (SOD1). CCS1 facilitates copper insertion into SOD1 and stabilizes its structure through disulfide bond formation . Key characteristics of the CCS1 antibody include:
| Property | Details |
|---|---|
| Target | Copper Chaperone for Superoxide Dismutase (CCS1) |
| Host Species | Rabbit |
| Clonality | Polyclonal |
| Reactivity | Human, Mouse |
| Molecular Weight | 34 kDa (observed); 29 kDa (calculated) |
| Applications | Western Blot (WB), Immunohistochemistry (IHC), Immunoprecipitation (IP) |
This antibody is widely used to study CCS1 expression, localization, and interaction with SOD1 in cellular models and tissues .
Mitochondrial Import and Redox Regulation
CCS1 contains a structural disulfide bond (C27–C64) in its N-terminal domain, essential for mitochondrial import. Mutations at these cysteines (e.g., C27S/C64S) disrupt CCS1 localization, reducing mitochondrial SOD1 levels and activity . Key findings include:
-
C27/C64 Double Mutants:
-
Redox State:
Disease Relevance
-
Amyotrophic Lateral Sclerosis (ALS): CCS1 overexpression accelerates disease progression in SOD1-linked ALS models by disrupting disulfide oxidation in mutant SOD1 .
-
S-Acylation: CCS1 undergoes S-acylation in human spinal cords, with altered levels observed in ALS patients .
Domain-Specific Mutagenesis
| Mutation | Mitochondrial Import Efficiency | SOD1 Activation |
|---|---|---|
| Wild-Type CCS1 | 100% | Full activity |
| C27S/C64S | <10% | Impaired |
| C229S/C231S | Normal | No activity |
Data derived from in vitro import assays and SOD1 activity measurements .
Key Functional Domains
-
Domain I (Residues 2–74): Mediates mitochondrial targeting via C27/C64 disulfide .
-
C-Terminal Domain: Binds SOD1 and facilitates copper transfer (C229/C231 critical for SOD1 activation) .
Experimental Workflows
-
Western Blot: Detects CCS1 at 34 kDa in human/mouse tissues .
-
Immunohistochemistry: Localizes CCS1 in mitochondrial and cytosolic compartments .
-
S-Acylation Assays: Acyl-RAC and ABE methods quantify post-translational modifications in ALS models .
Case Study: ALS Mechanisms
-
CCS1 overexpression in G93A SOD1 mice reduces detergent-insoluble SOD1 aggregates but accelerates disease onset, suggesting dual roles in SOD1 stability and toxicity .
Clinical and Therapeutic Implications
Product Specs
Constituents: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Target Background
Q&A
What experimental applications are most suitable for CCS1 antibody in copper homeostasis studies?
The CCS1 antibody (E-1) is widely used to investigate copper trafficking mechanisms, particularly its role in delivering copper to superoxide dismutase 1 (SOD1). Key applications include:
-
Western blotting (WB): Detect CCS expression levels in tissues like kidney, liver, and Purkinje cells, where oxidative stress mechanisms are critical .
-
Immunoprecipitation (IP): Study protein-protein interactions between CCS and SOD1 to map copper transfer pathways .
-
Immunofluorescence (IF): Localize CCS in cellular compartments, such as the cytoplasm or mitochondria, to assess spatial regulation of copper delivery .
-
Enzyme-linked immunosorbent assay (ELISA): Quantify CCS-SOD1 binding affinity under varying redox conditions .
For methodological rigor, combine CCS1 antibody with SOD1 activity assays to correlate protein interaction data with enzymatic function .
How should researchers validate CCS1 antibody specificity in heterogeneous samples?
Validation requires a multi-tiered approach:
-
Knockout controls: Use CCS−/− cell lines or tissues to confirm absence of non-specific bands in WB .
-
Cross-reactivity testing: Screen for off-target binding using protein arrays or immunoprecipitation-mass spectrometry (IP-MS), as polyspecificity can lead to artifacts in complex samples .
-
Orthogonal validation: Compare results with alternative antibodies or CRISPR-Cas9-mediated CCS knockdown models .
Table 1: CCS1 Antibody Validation Workflow
Which tissue types require optimized protocols for CCS1 antibody applications?
CCS expression varies significantly across tissues:
-
Kidney and liver: High baseline CCS levels enable standard WB protocols .
-
Central nervous system (CNS): Low CCS abundance in neurons necessitates signal amplification (e.g., tyramide-based IF) .
-
Blood cells: Pre-absorption with erythrocyte lysates reduces non-specific binding .
For CNS studies, combine CCS1 antibody with oxidative stress markers like 8-OHdG to contextualize SOD1-related neuroprotection .
How can researchers reconcile contradictory data on CCS overexpression in ALS models?
The G93A SOD1/CCS mouse model shows accelerated disease progression despite reduced SOD1 aggregation . Key methodological considerations:
Table 2: Contrasting Effects of CCS Overexpression in ALS Models
| Parameter | G93A SOD1 (Control) | G93A SOD1 + CCS Overexpression |
|---|---|---|
| Survival time | 242 days | 36 days |
| SOD1 disulfide oxidation | 60% oxidized | 22% oxidized |
| Detergent-insoluble SOD1 | High | Low |
Resolution strategy:
-
Use disulfide stability assays (e.g., AMS alkylation) to differentiate folded vs. misfolded SOD1 .
-
Employ compartment-specific CCS knockdown to isolate cytoplasmic vs. mitochondrial effects.
-
Integrate transcriptomic data to identify CCS-mediated pathways beyond copper chaperone activity .
What mechanisms link CCS1-dependent disulfide oxidation to neurodegenerative disease?
CCS catalyzes two critical SOD1 maturation steps: copper insertion and disulfide bond formation. Dysregulation causes:
-
Copper misallocation: Free copper ions generate hydroxyl radicals via Fenton chemistry, exacerbating neuronal damage .
-
Disulfide instability: Reduced SOD1 (Cys57–Cys146) aggregates in mitochondria, impairing electron transport chain function .
Experimental design:
-
Apply redox biosensors (e.g., roGFP2) to quantify CCS’s impact on SOD1 redox status in live neurons .
-
Combine CCS1 antibody with cryo-EM to resolve SOD1-CCS complex structures under oxidative stress .
How can polyreactivity risks be mitigated in CCS1 antibody-based assays?
Polyreactivity remains a major challenge in immunochemical studies:
Table 3: De-risking Strategies for Antibody Polyreactivity
For CCS1 studies, pre-incubate antibodies with heparin sulfate to neutralize charge-based non-specific interactions .
