ckb-1 Antibody
Description
Introduction to CKB-1 Antibody
The CKB-1 Antibody (Catalog No. CPTC-CKB-1) is a mouse monoclonal IgG1 antibody targeting creatine kinase B (CKB), a cytoplasmic enzyme critical for energy metabolism in tissues with high energy demands, such as muscle, brain, and heart . CKB catalyzes the reversible transfer of phosphate between ATP and creatine, enabling rapid ATP regeneration during energy fluctuations . Dysregulation of CKB has been linked to neurodegenerative diseases, muscular dystrophy, and cancer .
Performance in Assays
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Western Blot: Detects a band of ~43 kDa (predicted size) in recombinant CKB preparations .
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ELISA: Exhibits high binding affinity to CKB-coated plates .
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IHC: Not suitable for immunohistochemistry due to lack of specificity in tissue sections .
Role in Disease Pathology
CKB downregulation is associated with aggressive prostate cancer and poor prognosis . For example:
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IHC Studies: Anti-CKB antibodies (e.g., Abcam #ab108388) reveal reduced CKB staining in tumor vs. normal prostate tissue, correlating with epithelial-mesenchymal transition (EMT) suppression .
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WB Validation: CKB-1 detects CKB in recombinant proteins but lacks cross-reactivity with muscle-type creatine kinase (CKM) .
Comparative Analysis with Other CKB Antibodies
| Antibody (Source) | Host/Isotype | Reactivity | Applications | Citations |
|---|---|---|---|---|
| CPTC-CKB-1 (5) | Mouse IgG1 | Human | WB, ELISA | N/A |
| ab151579 (10) | Rabbit IgG | Human, Mouse | WB, IHC, ICC/IF | 3 pubs |
| 15137-1-AP (7) | Rabbit IgG | Human, Mouse, Rat | WB, IP, IHC, ELISA | 5 pubs |
| 66764-1-Ig (8) | Mouse IgG2a | Human, Mouse | WB, IHC, ELISA | 2 pubs |
Key Differences:
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CPTC-CKB-1 is mouse-derived and restricted to WB/ELISA, whereas rabbit polyclonal antibodies (e.g., ab151579, 15137-1-AP) enable broader applications (IHC, IF) .
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Monoclonal vs. Polyclonal: Monoclonal antibodies (CPTC-CKB-1, 66764-1-Ig) offer higher specificity, while polyclonal antibodies (ab151579) may detect multiple epitopes .
Therapeutic Relevance
CKB antibodies are critical for studying energy metabolism in:
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Neurodegenerative Diseases: CKB deficiency correlates with impaired ATP regeneration in brain tissues .
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Cancer Biology: CKB suppression in tumors may disrupt EMT pathways, influencing metastasis .
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Muscle Disorders: Dysregulated CKB activity contributes to muscular dystrophy and heart failure .
Optimized Protocols
Product Specs
Q&A
Basic Research Questions
How should researchers validate CKB-1 antibody specificity in experimental models?
Validation requires a multi-step approach:
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Knockout controls: Use CRISPR/Cas9-mediated CKB knockout cell lines (e.g., NCI-H82-shCKB) to confirm antibody specificity in Western blot (WB) and immunofluorescence (IF) .
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Cross-reactivity checks: Verify absence of signal in tissues/cells lacking CKB expression (e.g., Gboxin-resistant SF126 cells) .
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Dual antibody validation: Compare results with antibodies from different vendors (e.g., Proteintech #18713-1-AP vs. Assay Genie CAB12631) to rule out batch variability .
What are optimal dilution protocols for CKB-1 antibodies in tissue-specific applications?
Dilution ranges vary by tissue type and antigen retrieval methods:
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IHC: 1:50–1:500 with TE buffer (pH 9.0) for brain or tumor tissues .
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IF/ICC: 1:50–1:500 with Triton-X permeabilization for subcellular localization studies .
| Application | Tissue Type | Optimal Dilution | Antigen Retrieval |
|---|---|---|---|
| IHC | Human gliomas | 1:200 | Citrate buffer (pH 6.0) |
| WB | Mouse colon | 1:2,000 | RIPA buffer |
Advanced Research Questions
How does CKB knockdown affect cellular responses to OXPHOS inhibitors?
CKB silencing induces resistance to F1F0 ATP synthase inhibitors (e.g., Gboxin, Oligomycin A) but increases sensitivity to complex III inhibitors (e.g., Antimycin A):
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Mechanism: CKB maintains ATP flux via creatine-phosphate shuttle; its loss redirects energy dependency to non-ATP synthase pathways .
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Experimental design:
| Inhibitor Class | CKB-KO Cell Response | Key Data Source |
|---|---|---|
| F1F0 ATP synthase | Resistance (↑ IC50) | Figure 1G-H |
| Complex III (Antimycin A) | Hypersensitivity | Figure 1M |
How to resolve contradictions in CKB’s role in cancer progression?
CKB exhibits dual roles depending on cellular context:
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Pro-metastatic: In prostate cancer, CKB silencing promotes AKT/mTORC2-driven EMT .
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Anti-metastatic: In gliomas, high CKB correlates with energy-dependent invasion .
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Resolution strategy:
| Cancer Type | CKB Expression | Functional Outcome | Reference |
|---|---|---|---|
| Prostate | Low | ↑ EMT, ↑ Metastasis | |
| Glioblastoma | High | ↑ OXPHOS dependency |
What methodological pitfalls arise in co-IP studies of CKB-protein interactions?
Key challenges include:
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False positives: CKB binds nonspecifically to charged molecules (e.g., ATP). Use ATP-competitive elution buffers in co-IP protocols .
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Domain masking: The C-terminal 84aa domain of CKB mediates AKT sequestration, requiring mild detergents (e.g., 0.1% NP-40) to preserve interactions .
| Pitfall | Mitigation Strategy | Example Study |
|---|---|---|
| Nonspecific binding | ATP-blocked beads | Figure 4C |
| Transient interactions | Crosslinking (DSG) | Supplementary Figure S2 |
How to standardize CKB quantification across heterogeneous tumor samples?
Adopt a dual-index scoring system:
