CRTC3 Antibody, Biotin conjugated

What is CRTC3 and why is it important in research?

CRTC3 (CREB-regulated transcription coactivator 3) is a critical coactivator that mediates CREB-dependent gene transcription. It plays significant roles in metabolic regulation, particularly in adipose tissue metabolism and energy homeostasis. The protein is encoded by the CRTC3 gene (also known as TORC3) and has been identified as a key regulator in multiple signaling pathways. Research into CRTC3 function has implications for understanding metabolic disorders, obesity, and insulin resistance. Detection and quantification of CRTC3 using specific antibodies allow researchers to investigate its expression patterns, subcellular localization, and interactions with other proteins across different experimental conditions and disease models.

What does biotin conjugation add to CRTC3 antibody functionality?

Biotin conjugation significantly enhances the utility of CRTC3 antibodies by exploiting the strong avidin-biotin binding system. This modification enables signal amplification through interaction with streptavidin-conjugated detection reagents, improving sensitivity in various immunoassays. The biotin molecule forms a covalent bond with the antibody while maintaining the antibody's specific binding capacity to CRTC3. This conjugation allows for multilevel signal amplification when used with streptavidin-HRP complexes, making detection more robust especially in samples with low target abundance. Additionally, biotin conjugation offers greater flexibility in experimental design as it permits more diverse detection strategies compared to directly labeled antibodies.

What are the key specifications to consider when selecting a CRTC3 biotinylated antibody?

When selecting a CRTC3 biotinylated antibody, researchers should evaluate several critical specifications:

Researchers should match these specifications to their experimental requirements, considering factors such as the cellular compartment being studied and the specific CRTC3 domain of interest.

What are the validated applications for CRTC3 biotinylated antibodies?

CRTC3 biotinylated antibodies have been validated for multiple research applications, each with specific methodological considerations:

• ELISA (Enzyme-Linked Immunosorbent Assay): All examined CRTC3 biotinylated antibodies have been validated for ELISA applications, making them suitable for quantitative detection of CRTC3 in solution.

• Western Blotting (WB): Several variants show strong performance in WB (recommended dilution range: 1:300-5000), allowing detection of denatured CRTC3 protein separated by gel electrophoresis.

• Immunohistochemistry (IHC): Both paraffin-embedded (IHC-P, recommended dilution 1:200-400) and frozen section (IHC-F, recommended dilution 1:100-500) protocols have been validated, enabling tissue localization studies.

• Immunocytochemistry (ICC): Some variants are suitable for cellular localization studies in cultured cells.

When transitioning between applications, optimization of antibody concentration is essential as the effective working dilution can vary significantly between techniques.

How should researchers optimize ELISA protocols using CRTC3 biotinylated antibodies?

ELISA optimization with CRTC3 biotinylated antibodies requires systematic adjustment of multiple parameters:

• Capture Antibody Selection: When using a sandwich ELISA format, pair the biotinylated CRTC3 antibody with a complementary capture antibody recognizing a different epitope (e.g., combine AA 151-260 with AA 373-480 targeted antibodies).

• Blocking Optimization: Use 1-5% BSA in TBS to minimize background signal. The buffer composition (0.01 M PBS, pH 7.4) used for antibody storage provides a starting point for optimization.

• Antibody Titration: Though manufacturers note "optimal dilutions should be determined by the end user," start with a dilution series (typically 1:500 to 1:5000) to identify the optimal signal-to-noise ratio.

• Detection System: Employ streptavidin-HRP systems that provide multilevel signal amplification with the biotin molecule, enhancing visualization and quantification sensitivity.

• Incubation Conditions: Standardize temperature (typically room temperature or 4°C) and duration (1-2 hours or overnight) to ensure reproducible results.

Each parameter should be systematically tested while keeping others constant to identify optimal conditions for specific experimental requirements.

What are the critical considerations for immunohistochemistry using CRTC3 biotinylated antibodies?

When performing immunohistochemistry with CRTC3 biotinylated antibodies, researchers should address several critical factors:

• Tissue Preparation: Different fixation protocols significantly impact epitope accessibility. For formalin-fixed paraffin-embedded (FFPE) tissues, appropriate antigen retrieval methods are essential due to potential epitope masking.

• Dilution Optimization: Start with the recommended dilution ranges (1:200-400 for IHC-P, 1:100-500 for IHC-F) and adjust based on signal intensity and background.

• Detection System Selection: While direct streptavidin-HRP systems work well, amplification systems like tyramide signal amplification (TSA) can further enhance sensitivity for low-abundance CRTC3 detection.

• Endogenous Biotin Blocking: Tissues with high endogenous biotin (liver, kidney) require specific blocking steps to prevent false-positive signals. Commercial avidin/biotin blocking kits should be incorporated into protocols.

• Controls: Include both positive controls (tissues known to express CRTC3) and negative controls (primary antibody omission and isotype controls) to validate staining specificity.

The subcellular localization pattern of CRTC3 should be carefully evaluated, as both nuclear and cytoplasmic staining may occur depending on cellular activation state.

How do different epitope targets of CRTC3 antibodies affect experimental outcomes?

The choice between different epitope-targeted CRTC3 antibodies (such as AA 151-260 versus AA 373-480) can significantly impact experimental results:

• Domain-Specific Function Analysis: The AA 151-260 region contains regulatory domains that may be modified post-translationally, while the AA 373-480 region encompasses potential protein-protein interaction sites. Antibodies targeting these different regions enable detection of domain-specific functions.

• Conformational Sensitivity: Certain epitopes may be masked in protein complexes or specific conformational states, resulting in differential detection efficiency depending on CRTC3's activation state or binding partners.

• Cross-Reactivity Profiles: Different epitope regions share varying degrees of homology with other CRTC family members (CRTC1, CRTC2), potentially affecting specificity. Researchers should select epitopes with minimal sequence homology to related proteins when specificity is paramount.

• Functional Interference: Antibodies binding to functionally critical domains may interfere with CRTC3 activity in certain assay formats, particularly in protein-protein interaction studies or cellular assays.

Researchers should select epitope targets based on their specific experimental questions and validate multiple antibodies targeting different regions when investigating novel CRTC3 functions.

What methodological approaches can validate the specificity of CRTC3 biotinylated antibodies?

Validating antibody specificity is crucial for reliable research outcomes. Several methodological approaches should be employed:

• Western Blot Validation: Confirm the antibody detects a protein of the expected molecular weight (~67 kDa for full-length CRTC3). Multiple bands may indicate isoforms, degradation products, or post-translational modifications.

• Knockout/Knockdown Controls: Compare antibody reactivity in wild-type samples versus CRTC3 knockout or siRNA-knockdown samples. Signal reduction in knockdown samples confirms specificity.

• Peptide Competition Assays: Pre-incubate the antibody with excess immunizing peptide before sample application. Specific signals should be eliminated or significantly reduced.

• Orthogonal Detection Methods: Compare results with alternative detection methods or antibodies targeting different CRTC3 epitopes. Concordant results increase confidence in specificity.

• Mass Spectrometry Validation: For ultimate confirmation, immunoprecipitate CRTC3 using the biotinylated antibody and identify the pulled-down proteins by mass spectrometry.

These validation steps should be systematically performed and documented before using the antibody in critical experiments.

How can CRTC3 biotinylated antibodies be integrated into multiplex immunoassays?

Integrating CRTC3 biotinylated antibodies into multiplex detection systems requires careful experimental design:

• Spectral Separation Strategy: When combined with fluorescent detection systems, pair the biotinylated CRTC3 antibody with a streptavidin conjugate carrying a fluorophore spectrally distinct from other detection channels (e.g., streptavidin-Cy5 if using FITC and TRITC for other targets).

• Sequential Detection Protocol: For chromogenic multiplexing, implement sequential detection using the biotin-streptavidin system first, followed by thorough washing and blocking of remaining biotin sites before subsequent detection steps.

• Cross-Reactivity Prevention: When using multiple rabbit-derived antibodies in the same assay, consider utilizing specialized blocking systems (e.g., monovalent Fab fragments) to prevent secondary antibody cross-reactivity.

• Signal Balancing: Adjust CRTC3 biotinylated antibody concentration independently of other antibodies in the panel to achieve balanced signal intensities across all targets.

• Automated Analysis Parameters: When using image analysis software, develop specific algorithms to distinguish CRTC3 signal from other targets based on subcellular localization patterns and signal intensity thresholds.

These methodological considerations enable researchers to study CRTC3 in the context of other proteins or signaling pathways simultaneously.

What are common issues when using CRTC3 biotinylated antibodies and how can they be resolved?

Researchers commonly encounter several technical challenges when working with CRTC3 biotinylated antibodies:

For optimal results, researchers should store antibodies according to manufacturer recommendations (aliquoted at -20°C, avoiding repeated freeze-thaw cycles) and carefully optimize each step of their specific experimental protocol.

How should researchers determine optimal antibody concentrations for different applications?

Determining the optimal concentration of CRTC3 biotinylated antibodies requires systematic titration across different applications:

• ELISA Optimization:

  • Create a standard curve with recombinant CRTC3 protein

  • Test antibody dilutions from 1:500 to 1:5000

  • Select the dilution that provides the best combination of sensitivity and dynamic range

• Western Blot Optimization:

  • Test dilutions across the recommended range (1:300-5000)

  • Begin with a moderate dilution (1:1000) and adjust based on signal strength

  • Consider extended incubation at 4°C (overnight) for lower antibody concentrations

• Immunohistochemistry Optimization:

  • Start with mid-range dilutions (1:200 for IHC-P, 1:200 for IHC-F)

  • Prepare a dilution series on control tissues known to express CRTC3

  • Evaluate both signal intensity and background at each dilution

• Cell-Type Specific Adjustments:

  • Cell types with high CRTC3 expression may require more dilute antibody

  • Tissues with high background (e.g., adipose tissue) may require more stringent washing

Document optimization results systematically, as optimal concentrations may vary between sample types and experimental conditions.

What controls are essential when working with CRTC3 biotinylated antibodies?

Implementing appropriate controls is critical for reliable interpretation of results with CRTC3 biotinylated antibodies:

• Positive Controls:

  • Cell lines with confirmed CRTC3 expression (based on literature)

  • Tissues known to express CRTC3 (e.g., adipose tissue)

  • Recombinant CRTC3 protein standards (for quantitative assays)

• Negative Controls:

  • Primary antibody omission control (tests detection system specificity)

  • Isotype control (rabbit IgG at matching concentration)

  • CRTC3 knockout or knockdown samples (when available)

• Procedural Controls:

  • Endogenous biotin blocking controls (samples with and without avidin/biotin blocking)

  • Antigen competition controls (antibody pre-adsorbed with immunizing peptide)

  • Cross-reactivity assessment (testing in species with predicted reactivity)

• Signal Validation Controls:

  • Independent antibody targeting different CRTC3 epitope

  • Correlation with mRNA expression data

  • Subcellular fractionation to confirm localization patterns

How can CRTC3 biotinylated antibodies be utilized in EMSA (Electrophoretic Mobility Shift Assay) applications?

CRTC3 biotinylated antibodies can be effectively incorporated into EMSA workflows to investigate CRTC3-DNA interactions:

• Supershift EMSA Protocol: Adding CRTC3 biotinylated antibody to the EMSA reaction mixture containing CRTC3-bound DNA complexes results in a "supershift" (further retardation of migration) of bands containing CRTC3, confirming its presence in specific DNA-protein complexes.

• Probe Design Considerations: When designing biotin-labeled DNA probes for CRTC3 binding studies, researchers should include known CREB binding elements (CRE) as CRTC3 functions as a CREB coactivator.

• Competitor Probe Strategy: Include both unlabeled competitive probes and mutation probes to validate binding specificity, following approaches similar to those documented for other transcription factors.

• Detection Optimization: The biotin-streptavidin HRP system provides multilevel signal amplification, making visualization more obvious compared to radioactive methods. This parallels the detection advantage of using biotinylated antibodies in other applications.

• Sample Preparation Considerations: Nuclear extracts from cells with activated CRTC3 (dephosphorylated, nuclear localized) should be prepared using buffers that preserve protein-protein interactions important for CRTC3 function.

These methodological approaches allow researchers to investigate CRTC3's role in transcriptional regulation at specific genomic loci.

What are methodological considerations for using CRTC3 biotinylated antibodies in chromatin immunoprecipitation (ChIP) studies?

Adapting CRTC3 biotinylated antibodies for ChIP applications requires specific methodological considerations:

• Crosslinking Optimization: As CRTC3 is a transcriptional coactivator that doesn't directly bind DNA, dual crosslinking protocols (combining formaldehyde with protein-specific crosslinkers like DSG) may improve capture of CRTC3-containing transcriptional complexes.

• Streptavidin Bead Selection: Rather than using protein A/G beads (standard for unconjugated antibodies), researchers should use streptavidin-coated magnetic beads that directly capture the biotinylated antibody-CRTC3-chromatin complex.

• Pre-Clearing Strategy: Given potential endogenous biotinylated proteins in nuclear extracts, thorough pre-clearing with streptavidin beads before adding biotinylated CRTC3 antibody is essential to reduce background.

• Sequential ChIP Approach: For studying CRTC3 co-occupancy with CREB or other transcription factors, sequential ChIP (first with anti-CREB, then with biotinylated anti-CRTC3) can elucidate co-regulatory mechanisms.

• Elution Considerations: Standard ChIP elution buffers may not efficiently disrupt biotin-streptavidin interactions. Consider using biotin elution buffers or more stringent conditions while ensuring chromatin integrity is maintained.

These methodological adaptations enable investigation of genomic loci where CRTC3 participates in transcriptional regulation, providing insights into its target genes and coregulatory networks.

How should researchers interpret post-translational modifications of CRTC3 when using epitope-specific biotinylated antibodies?

Interpreting post-translational modifications (PTMs) of CRTC3 requires careful consideration of epitope location and antibody specificity:

• Phosphorylation-Sensitive Detection: CRTC3 activity is regulated by phosphorylation-dependent nuclear-cytoplasmic shuttling. Antibodies targeting regions containing phosphorylation sites (e.g., near Ser162, Ser329, Ser370) may show differential detection depending on CRTC3's phosphorylation state.

• Comparative Profiling Approach: Researchers should compare results from multiple antibodies targeting different CRTC3 epitopes (e.g., AA 151-260 vs. AA 373-480) to identify potential PTM-dependent detection biases.

• Subcellular Localization Analysis: Active (dephosphorylated) CRTC3 translocates to the nucleus. Researchers should correlate subcellular staining patterns with known activation states and validate with fractionation studies.

• Validation with Phospho-Specific Tools: Results from general CRTC3 biotinylated antibodies should be compared with phospho-specific antibodies or phosphatase treatment experiments to confirm PTM-dependent observations.

• Functional Correlation: Changes in detected CRTC3 levels should be correlated with functional readouts (e.g., target gene expression) to distinguish between actual protein level changes and epitope masking due to PTMs.

Understanding these interpretation principles allows researchers to distinguish between changes in CRTC3 abundance versus changes in its modification state, providing deeper insights into its regulatory mechanisms.