CBP Tag Monoclonal Antibody

What is a CBP Tag and what is its amino acid sequence?

The CBP (Calmodulin Binding Peptide) tag is an affinity tag derived from muscle myosin light-chain kinase. It comprises 26 amino acid residues with the molecular weight of 4 kDa and the sequence KRRWKKNFIAVSAANRFKKISSSGAL . This tag can be added to either the N or C terminus of proteins of interest through DNA recombinant technology . The CBP tag has relatively high affinity for calmodulin (CaM), enabling efficient purification of tagged proteins using calmodulin affinity resin .

The CBP tag functions through specific binding to calmodulin in a calcium-dependent manner. CBP-tagged proteins can be purified from crude cell extracts through calmodulin affinity resin . The tag's relatively high affinity for calmodulin makes it an effective option for single-step or tandem affinity purification schemes .

The purification process typically follows this methodology:

• Express the CBP-tagged protein in an appropriate expression system

• Prepare cell lysate under conditions that preserve tag integrity

• Apply the lysate to a calmodulin-immobilized resin in the presence of calcium

• Wash to remove unbound proteins

• Elute the bound CBP-tagged protein using calcium chelators (e.g., EGTA)

This system provides a gentle purification method that often preserves protein activity and native conformation .

What is the formulation of CBP Tag Monoclonal Antibodies?

The standard formulation for CBP Tag Monoclonal Antibodies typically includes:

• Buffer: PBS, pH 7.4

• Protein stabilizer: 0.5% BSA

• Preservative: 0.02% sodium azide

• Cryoprotectant: 50% glycerol

This formulation maintains antibody stability during storage and prevents microbial contamination. Some variations exist between manufacturers, with some using mouse IgG1 in phosphate buffered saline (without Mg²⁺ and Ca²⁺) .

What are the primary applications of CBP Tag Monoclonal Antibodies?

Western blotting is the primary validated application for CBP Tag Monoclonal Antibodies . Some antibody variants are additionally validated for immunocytochemistry/immunofluorescence (ICC/IF) . The antibody specifically detects recombinant proteins tagged with the CBP sequence, allowing researchers to track expression, purification, and localization of their tagged proteins.

How can I optimize Western Blot protocols using CBP Tag Monoclonal Antibody?

Optimizing Western Blot protocols for CBP Tag detection requires careful consideration of several parameters:

Recommended Protocol:

• Sample Preparation:

  • Use fresh lysates with protease inhibitors to prevent tag degradation

  • Load 30 μg of whole cell extracts as demonstrated in validation studies

• Gel Electrophoresis and Transfer:

  • Use appropriate percentage gels based on your protein's molecular weight (accounting for the 4 kDa CBP tag)

  • Optimize transfer conditions for your specific protein size

• Antibody Incubation:

  • Primary antibody dilution: Start with 1:5000, with testing range from 1:1000-1:10000

  • Incubate overnight at 4°C for optimal sensitivity

  • Use appropriate HRP-conjugated secondary antibody against mouse IgG

• Detection:

  • Develop using ECL or enhanced chemiluminescence systems

  • Include proper controls: Non-transfected cells as negative control, known CBP-tagged protein as positive control

Western blot analysis from experimental validation shows clear detection of CBP recombinant protein at dilutions of 1:5000 and 1:10000, demonstrating the high sensitivity of these antibodies .

When encountering weak signals in CBP-tagged protein detection, implement these methodological approaches:

• Antibody Optimization:

  • Test concentration range from 1:1000 to 1:10000

  • Extend incubation time to overnight at 4°C

• Sample Enhancement:

  • Increase protein loading (30-60 μg for whole cell lysates)

  • Optimize lysis conditions to preserve tag integrity

  • Consider subcellular fractionation to concentrate tagged proteins

• Detection System Improvement:

  • Use high-sensitivity ECL substrates

  • Consider signal amplification systems (biotin-streptavidin)

  • Optimize exposure times based on signal intensity

• Expression Optimization:

  • Evaluate tag position (N-terminal vs. C-terminal)

  • Consider expression enhancers or stronger promoters

  • Optimize codon usage for your expression system

• Technical Adjustments:

  • Reduce blocking stringency (3% BSA instead of 5%)

  • Decrease washing stringency

  • Use PVDF membranes for increased protein binding

Western blot validation studies demonstrate detection at both 1:5000 and 1:10000 dilutions, suggesting good antibody sensitivity when protocols are properly optimized .

What considerations are important when designing CBP-tagged constructs for structural studies?

Designing CBP-tagged constructs for structural studies requires careful planning:

• Tag Placement:

  • Terminal placement (N or C) depends on the protein's structure and function

  • Consider flexible linkers (GGGGS)n between tag and protein to minimize structural interference

  • Domain boundaries must be precisely defined to avoid disrupting structural elements

• Cleavage Options:

  • Include protease recognition sites between tag and protein if tag removal is necessary

  • Common proteases include TEV, PreScission, and thrombin

  • Position cleavage sites to leave minimal or no residual amino acids

• Expression Considerations:

  • Mammalian expression may be necessary for complex or post-translationally modified proteins

  • CBP tag system has been validated with mammalian expression systems and showed no negative effect on expression level or protein stability

• Purification Strategy:

  • Consider whether single-step or multi-step purification is needed

  • For crystallography, >95% purity is typically required

  • Final buffer composition should be optimized for crystallization trials

• Structural Validation:

  • Verify that the tag does not interfere with structure using circular dichroism or limited proteolysis

  • Consider tag removal if it affects crystallization

Research findings demonstrate that the CBP tag system has enabled the production of diffraction quality crystals for certain target protein domains , indicating its compatibility with structural studies.

How can I validate the specificity of CBP Tag Monoclonal Antibody detection in my experimental system?

A comprehensive validation approach includes:

• Essential Controls:

  • Positive control: Purified recombinant CBP-tagged protein

  • Negative control: Non-transfected cells or lysates without CBP-tagged proteins

  • Peptide competition: Pre-incubation of antibody with excess CBP peptide should abolish specific signal

• Transfection Experiments:

  • Compare side-by-side: transfected cells expressing CBP-tagged protein versus empty vector controls

  • Western blot analysis should show band at expected molecular weight only in transfected samples

• Orthogonal Detection:

  • Confirm identity using mass spectrometry

  • Use antibody against another tag or the protein itself if available

  • Compare with calmodulin-based detection methods

• Size Verification:

  • Detected band should match predicted molecular weight (protein + 4 kDa CBP tag)

  • Use molecular weight markers to accurately estimate size

• Localization Analysis:

  • For fluorescent detection, subcellular localization should match expected distribution

  • Example: Immunofluorescence analysis comparing mock and transfected HEK-293T cells shows specific detection only in transfected cells

What are potential interference factors when using CBP Tag Monoclonal Antibody in complex biological samples?

Several factors can interfere with specific detection:

• Calcium-binding Proteins:

  • Endogenous calcium-binding proteins may cross-react

  • Use EGTA in buffers to reduce calcium-dependent interactions

• Proteolytic Degradation:

  • Protease activity can cleave the tag from protein

  • Use protease inhibitor cocktails during sample preparation

  • Process samples at 4°C to minimize degradation

• Epitope Masking:

  • Protein aggregation or folding may obscure the CBP tag

  • Optimize denaturation conditions for Western blotting

  • For native applications, ensure tag accessibility through appropriate design

• Post-translational Modifications:

  • Modifications near the tag may affect antibody recognition

  • Consider alternative tag placement if this is suspected

• Buffer Components:

  • High detergent concentrations may disrupt antibody binding

  • Certain chelators may affect calcium-dependent interactions

  • Optimize buffer composition for specific applications

Control for these interferences by including appropriate positive and negative controls in all experiments, and consider preliminary testing with simplified samples before moving to complex biological matrices.

How are CBP Tag Monoclonal Antibodies different from anti-CBP antibodies that detect the CREB-binding protein?

It's critical to distinguish between these antibodies to avoid experimental confusion:

The CREB-binding protein (also abbreviated as CBP) is an endogenous transcriptional co-activator with a molecular weight of approximately 300 kDa , which is entirely distinct from the 4 kDa CBP tag used in recombinant protein systems.

When ordering or using these antibodies, carefully examine product documentation to ensure you're selecting the correct antibody for your experimental purpose.