CKB3 Antibody

What is the CKB3 Antibody and what target does it recognize?

The CPTC-CKB-3 is a mouse monoclonal antibody (IgG1 isotype) that specifically targets human Creatine Kinase B chain (CKB), which has a molecular weight of 42.9 kDa. This antibody was developed using recombinant full-length CKB protein as the immunogen and deposited to the Developmental Studies Hybridoma Bank (DSHB) by the Clinical Proteomics Technologies for Cancer initiative at the National Cancer Institute . The antibody recognizes the protein encoded by the CKB gene (Entrez Gene ID: 1152, UniProt ID: P12277) .

CKB plays a crucial role in cellular energy homeostasis, particularly in tissues with high and fluctuating energy demands such as brain, heart, and skeletal muscle. The specificity of this antibody for the B isoform makes it valuable for distinguishing between different creatine kinase subtypes in research contexts.

What are the validated applications for the CKB3 Antibody?

The CPTC-CKB-3 antibody has been characterized and validated primarily for:

• Enzyme-Linked Immunosorbent Assay (ELISA): The antibody demonstrates high binding specificity in ELISA applications with recommended working dilutions of approximately 1:160,000 .

• Western Blot: The antibody effectively detects denatured CKB protein in western blot applications, allowing for size-based protein identification and semi-quantitative analysis .

Current validation data indicates that this antibody specifically reacts with human CKB, though cross-reactivity with other species has not been extensively characterized. Researchers should perform validation studies when applying this antibody to novel experimental systems or applications beyond those mentioned above.

How should the CKB3 Antibody be stored to maintain optimal activity?

For optimal preservation of antibody activity, follow these evidence-based storage recommendations:

• Short-term storage (up to two weeks): 4°C is appropriate for immediate use applications .

• Long-term storage: Divide the antibody solution into small aliquots (minimum 20 μl) and store at -20°C or -80°C .

• Avoid freeze-thaw cycles as they can significantly degrade antibody quality and reduce binding efficacy .

• Upon reconstitution from lyophilized form, the antibody can be stored at 4°C for approximately 30 days, but freezer storage is recommended for periods beyond this .

These storage guidelines help maintain the structural integrity of the antibody and preserve its specific binding properties for experimental applications.

What is the recommended protocol for using CKB3 Antibody in Western Blot applications?

When utilizing CPTC-CKB-3 for Western Blot applications, consider implementing this methodological approach:

• Sample preparation:

  • Lyse cells in a buffer containing protease inhibitors

  • Denature proteins in sample buffer containing SDS and a reducing agent

  • Heat samples at 95°C for 5 minutes

• Gel electrophoresis:

  • Load 10-30 μg of total protein per lane

  • Include positive control (tissue/cells known to express CKB)

  • Include negative control (tissue/cells known not to express CKB)

• Transfer and blocking:

  • Transfer proteins to PVDF or nitrocellulose membrane

  • Block with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

• Primary antibody incubation:

  • Dilute CPTC-CKB-3 antibody appropriately (start with manufacturer's recommendation)

  • Incubate overnight at 4°C with gentle agitation

• Detection:

  • Use appropriate HRP-conjugated secondary anti-mouse antibody

  • Visualize using enhanced chemiluminescence

  • Expected band size: approximately 42.9 kDa

Optimization may be necessary for specific experimental systems, including adjustments to antibody concentration, incubation times, and detection methods.

How can I validate CKB3 Antibody specificity in my experimental system?

To ensure antibody specificity and validate experimental results, implement these methodological controls:

• Positive and negative tissue/cell controls:

  • Use tissues/cells with known CKB expression patterns

  • Brain tissue typically shows high CKB expression

  • Include tissues known to express other CK isoforms to check for cross-reactivity

• Knockdown/knockout validation:

  • Compare antibody signal in wild-type vs. CKB knockdown/knockout samples

  • siRNA or CRISPR-based approaches can create appropriate negative controls

• Orthogonal detection methods:

  • Confirm protein detection with a different antibody targeting another epitope of CKB

  • Verify using mass spectrometry or other protein identification techniques

• Epitope blocking:

  • Pre-incubate antibody with purified recombinant CKB protein

  • This should abolish specific signal if the antibody is truly CKB-specific

• Ligand-specific binding analysis:

  • Implement computational models to predict binding modes when comparing closely related targets

  • This approach is particularly valuable when discriminating between structurally similar proteins

These validation steps ensure reliable interpretation of experimental results and help identify potential cross-reactivity or non-specific binding.

What factors might affect CKB3 Antibody performance in experimental applications?

Several experimental variables can influence antibody performance and should be considered during experimental design:

Understanding these factors allows researchers to systematically optimize experimental conditions and troubleshoot potential issues when working with CKB3 antibody.

How do computational models enhance antibody specificity prediction and design?

Modern antibody research increasingly utilizes computational approaches to predict and design specificity profiles:

• Biophysics-informed modeling:

  • Computational models can disentangle different binding modes associated with specific ligands

  • These models enable prediction and generation of antibody variants with customized specificity profiles

• Machine learning integration:

  • High-throughput sequencing data combined with machine learning can identify patterns in antibody-antigen binding

  • This approach allows for predictions beyond experimentally observed sequences

• Multiple binding modes analysis:

  • Distinct binding modes can be identified and associated with particular ligands

  • This approach is especially valuable when discriminating between chemically similar epitopes

• Specificity profile design:

  • Computational approaches can optimize antibody sequences to:

    • Increase specificity for a particular target

    • Engineer cross-specificity for multiple desired targets

    • Reduce binding to undesired targets

For CKB3 antibody and related research, these computational techniques offer opportunities to enhance specificity for the CKB isoform while minimizing cross-reactivity with other creatine kinase variants.

What approaches can I use to address inconsistent CKB3 Antibody results across different experimental systems?

When facing variability in antibody performance across different experimental systems:

• Systematic validation:

  • Perform side-by-side comparisons using standardized protocols

  • Document lot-to-lot variations and antibody age/storage conditions

• Epitope mapping:

  • Determine if the recognized epitope might be differentially accessible across sample types

  • Consider if post-translational modifications might affect antibody binding in different cellular contexts

• Expression level normalization:

  • Use quantitative methods to account for different baseline expression levels

  • Implement appropriate loading controls and quantification methods

• Binding mode analysis:

  • Consider that different experimental conditions may favor different antibody binding modes

  • Analyze data using biophysical models that can account for multiple potential binding interactions

• Sample preparation standardization:

  • Ensure consistent protein extraction and sample handling procedures

  • Control for factors like protein denaturation, reduction status, and buffer composition

These approaches help identify the source of variability and establish more reliable experimental systems for antibody-based detection.

How can I optimize detection of low-abundance CKB expression?

For detecting CKB in samples with low expression levels:

• Signal amplification strategies:

  • Tyramide signal amplification (TSA) for immunohistochemistry/immunofluorescence

  • Polymeric HRP detection systems for western blotting

  • Extended substrate development time for ELISA

• Sample enrichment:

  • Immunoprecipitation to concentrate CKB before detection

  • Subcellular fractionation to isolate compartments with higher CKB concentration

• Sensitive detection methods:

  • Chemiluminescent substrates with enhanced sensitivity

  • Digital imaging systems with higher dynamic range

  • Fluorescent secondary antibodies with direct visualization

• Protocol optimization:

  • Extended primary antibody incubation (up to 48 hours at 4°C)

  • Reduced washing stringency (lower salt, shorter washing times)

  • Buffer composition adjustments to enhance binding kinetics

• Statistical approaches:

  • Multiple technical replicates

  • Appropriate controls for background subtraction

  • Quantitative image analysis with sophisticated software

These approaches can significantly improve the signal-to-noise ratio when detecting low-abundance CKB protein, enabling more reliable quantification and localization studies.

How do I interpret unexpected molecular weight bands when using CKB3 Antibody in Western Blot?

When encountering bands of unexpected molecular weight:

• Standard CKB band: The expected molecular weight of human CKB is approximately 42.9 kDa . Deviations may indicate:

  • Post-translational modifications (PTMs):

    • Phosphorylation (+80 Da per phosphate group)

    • Glycosylation (variable increase, often +2-5 kDa)

    • Ubiquitination (+8.5 kDa per ubiquitin)

  • Proteolytic processing:

    • Lower molecular weight bands may represent cleavage products

    • Verify with protease inhibitors in sample preparation

  • Protein isoforms:

    • Alternative splicing may generate variants

    • Compare with transcript data from RNA-seq if available

  • Non-specific binding:

    • Validate with knockout/knockdown controls

    • Perform peptide competition assays

• Methodological approach for band identification:

  • Run mass spectrometry on excised gel bands

  • Utilize antibodies targeting different CKB epitopes

  • Perform immunoprecipitation followed by western blotting

  • Compare results across different cell/tissue types

Systematic investigation of unexpected bands can provide valuable insights into protein processing, modification states, and potential antibody cross-reactivity.

What are the current limitations of using CKB3 Antibody in research applications?

Understanding the limitations of the CPTC-CKB-3 antibody is essential for appropriate experimental design and interpretation:

• Validation constraints:

  • Current characterization is primarily limited to ELISA and western blot applications

  • Performance in other applications (IHC, ICC, IP) requires additional validation

• Species reactivity:

  • Confirmed reactivity is limited to human CKB

  • Cross-reactivity with other species requires empirical testing

  • Sequence homology analysis can help predict potential cross-reactivity

• Epitope characterization:

  • The specific epitope recognized by CKB3 has not been mapped

  • This limits understanding of potential cross-reactivity with similar proteins

  • Sensitivity to conformation or denaturation state is not fully characterized

• Isoform specificity:

  • While designed for CKB specificity, cross-reactivity with other creatine kinase isoforms (CKM, CKMT1/2) should be empirically evaluated

  • This is particularly important in tissues expressing multiple CK isoforms

• Technical considerations:

  • Optimal working dilutions may vary between applications and experimental systems

  • Freeze-thaw cycles significantly impact antibody performance

  • Buffer composition effects on binding kinetics are not fully characterized

Researchers should conduct preliminary validation studies addressing these limitations when applying the antibody to novel experimental systems.

How can quantitative analysis of CKB expression be standardized across studies?

To ensure reproducibility and comparability of CKB quantification across different studies:

• Standardized reference materials:

  • Include recombinant CKB protein standards of known concentration

  • Develop and share calibration curves relating signal intensity to protein quantity

• Normalization strategies:

  • Utilize appropriate housekeeping proteins as loading controls

  • Consider multiple reference genes/proteins for robust normalization

  • Implement total protein normalization methods (e.g., Stain-Free technology, Ponceau staining)

• Quantification methodology:

  • Define linear range of detection for the antibody

  • Use digital image analysis with appropriate background subtraction

  • Report detailed acquisition parameters and analysis settings

• Statistical approaches:

  • Perform technical and biological replicates

  • Apply appropriate statistical tests for comparison

  • Report variability measures (standard deviation, coefficient of variation)

• Reporting standards:

  • Document antibody source, catalog number, and lot

  • Describe detailed protocols including blocking agents and detection methods

  • Share raw data when possible to enable meta-analysis

Implementing these standardization approaches facilitates meaningful comparison of CKB expression data across different studies, experimental systems, and laboratories.

How might advanced computational models enhance CKB3 antibody specificity for challenging research applications?

Emerging computational approaches offer promising avenues for enhancing antibody specificity:

• Biophysically interpretable models:

  • Can disentangle multiple binding contributions from a single experiment

  • Enable prediction of antibody sequences that discriminate closely related ligands

  • Could be applied to enhance CKB3 specificity against other creatine kinase isoforms

• Integration of high-throughput selection experiments:

  • Phage display experiments against multiple ligands can inform computational models

  • Machine learning techniques can identify sequence patterns associated with desired specificity profiles

  • This approach could optimize CKB3 variants for specific applications

• Custom specificity profile design:

  • Computational generation of antibody variants with tailored binding properties

  • Ability to design antibodies with specific high affinity for CKB or with cross-specificity for related targets

  • Potential to create CKB3 variants optimized for different experimental applications

These advanced computational approaches represent the cutting edge of antibody engineering, offering opportunities to develop next-generation CKB-targeting antibodies with enhanced specificity and performance characteristics.