CTBP2 Antibody

What is CTBP2 and what are its primary functions in cellular biology?

CTBP2 (C-terminal-binding protein 2) functions primarily as a transcriptional corepressor that targets diverse transcription regulators. The protein contains an NAD+ binding domain similar to NAD+-dependent 2-hydroxyacid dehydrogenases and operates by interacting with transcription factors and histone-modifying enzymes that modulate chromatin structure to control access to DNA .

CTBP2 exists in multiple isoforms with distinct functions:

• The main isoform acts as a transcriptional repressor

• Isoform 2 functions as a scaffold for specialized synapses, particularly in synaptic ribbons

CTBP2 has been implicated in:

• Brown adipose tissue (BAT) differentiation

• Cancer cell migration promotion

• Metabolic regulation related to diabetes and hepatic steatosis

How does CTBP2 differ from CTBP1 in structure and function?

CTBP1 and CTBP2 share approximately 80% sequence homology but exhibit distinct expression patterns and functions:

• Both proteins interact with transcription factors through a PLDLSL sequence motif

• Both are highly expressed in embryonic tissues, but CTBP1 shows higher expression in adult tissues and is more widely distributed in both embryonic and adult tissues

• Both bind to zinc finger-homeodomain transcription factors like δEF1 and enhance transcriptional repression

• CTBP2 has additional unique interaction partners including hFOG-2, Evi-1, AREB6, and ZEB

This distinction is critical when designing experiments that specifically target CTBP2 versus CTBP1, particularly when selecting antibodies with minimal cross-reactivity.

What are the validated applications for CTBP2 antibodies in research?

Based on the technical validation data from multiple manufacturers, CTBP2 antibodies have been successfully employed in the following applications:

When designing experiments, researchers should consider that different antibody clones may perform optimally in different applications. For instance, the EPR7611(B) clone (ab128871) has demonstrated exceptional performance across multiple applications .

What are the optimal conditions for Western blot detection of CTBP2?

For optimal Western blot detection of CTBP2, follow these methodological guidelines:

• Sample preparation:

  • Load 20-30μg of whole cell lysate per lane

  • Successfully detected in various cell lines including HeLa, SK-BR-3, HEK293, CACO-2, U20S, and tissue lysates from brain and lung

• Electrophoresis conditions:

  • 5-20% SDS-PAGE gel at 70V (stacking gel)/90V (resolving gel) for 2-3 hours

  • Transfer to nitrocellulose membrane at 150mA for 50-90 minutes

• Blocking and antibody incubation:

  • Block with 5% non-fat milk in TBS for 1.5 hours at room temperature

  • Incubate with primary antibody at dilutions between 1:1000-1:10000 (0.5μg/mL for PA1554) overnight at 4°C

  • Wash with TBS-0.1% Tween (3 times, 5 minutes each)

  • Incubate with HRP-conjugated secondary antibody (typically 1:5000-1:20000 dilution) for 1-1.5 hours at room temperature

• Detection:

  • Develop using enhanced chemiluminescence (ECL) detection systems

  • Expected band size is approximately 47-49 kDa

How should samples be prepared for immunohistochemical detection of CTBP2?

Proper sample preparation is critical for successful immunohistochemical detection of CTBP2:

For paraffin-embedded sections:

• Heat-mediated antigen retrieval:

  • Use citrate buffer (pH 6.0) for 20 minutes , or

  • Use Bond™ Epitope Retrieval Solution 2 (pH 9.0)

• Blocking:

  • Block with 10% goat serum

• Primary antibody incubation:

  • Incubate with CTBP2 antibody at dilutions ranging from 1:6000-1:12000 (0.011-0.021 μg/ml for ab128871) or 1μg/ml for PA1554

  • Incubate overnight at 4°C

• Secondary antibody and detection:

  • For chromogenic detection: Use biotinylated secondary antibody (e.g., goat anti-rabbit IgG) followed by Streptavidin-Biotin-Complex (SABC) with DAB as chromogen

  • For automated systems: Use detection systems like LeicaDS9800 (Bond™ Polymer Refine Detection)

For frozen sections:

• Block with 10% goat serum

• Follow similar antibody incubation procedures as for paraffin sections

• CTBP2 antibodies have been validated on various tissue types including human mammary cancer, rat intestine, rat brain, mouse intestine, and human ovarian carcinoma tissues

How can I address non-specific binding when using CTBP2 antibodies?

Non-specific binding is a common challenge when working with CTBP2 antibodies. Here are methodological approaches to minimize this issue:

• Optimize blocking conditions:

  • Extend blocking time to 2 hours with 5% non-fat milk or BSA

  • Consider adding 0.1-0.3% Triton X-100 to blocking buffer for better penetration

• Antibody dilution optimization:

  • Perform a dilution series experiment (e.g., 1:500, 1:1000, 1:2000, 1:5000, 1:10000)

  • Compare signal-to-noise ratio across dilutions to identify optimal concentration

• Stringent washing:

  • Increase washing duration and number of washes (e.g., 5 washes of 5-10 minutes each)

  • Use TBS-T with 0.1-0.3% Tween-20 for more stringent washing

• Include proper controls:

  • Negative controls: omit primary antibody, use isotype control (e.g., rabbit monoclonal IgG for ab128871)

  • Positive controls: cell lines with known CTBP2 expression (HeLa, MCF7, HEK293 cells)

  • Consider CTBP2 knockdown samples as specificity controls

• Pre-adsorption:

  • If cross-reactivity is suspected, pre-adsorb antibody with the immunizing peptide

What are the best strategies for distinguishing between CTBP1 and CTBP2 in experimental analysis?

Given the high homology (80%) between CTBP1 and CTBP2 , distinguishing between these proteins requires careful experimental design:

• Antibody selection:

  • Choose antibodies raised against regions with lowest sequence homology between CTBP1 and CTBP2

  • Validate antibody specificity using recombinant CTBP1 and CTBP2 proteins

  • Use monoclonal antibodies targeting unique epitopes (e.g., clone EPR7611(B) for CTBP2)

• Molecular techniques:

  • Employ siRNA/shRNA knockdown specific to either CTBP1 or CTBP2 to confirm antibody specificity

  • Use CRISPR-Cas9 knockout models as definitive controls

• Expression pattern analysis:

  • Leverage differential expression patterns (CTBP1 is more widely expressed in adult tissues)

  • Compare subcellular localization patterns which may differ between the proteins

• Functional analysis:

  • Design experiments that exploit the distinct interaction partners of CTBP2 (hFOG-2, Evi-1, AREB6, ZEB)

  • Examine differential regulation of target genes (e.g., Tiam1 is specifically regulated by CTBP2)

• Co-immunoprecipitation approaches:

  • Use antibodies against specific CTBP2 interaction partners to immunoprecipitate complexes

  • Perform reciprocal IP experiments to confirm specificity

How can I investigate the relationship between NADH/NAD+ levels and CTBP2 function?

CTBP2 contains an NAD+ binding domain that affects its function . To study this relationship:

• Modulation of cellular NADH/NAD+ ratio:

  • Use metabolic inhibitors to alter NADH/NAD+ ratios (e.g., lactate dehydrogenase inhibitors)

  • Employ hypoxic conditions to increase NADH levels

  • Use compounds like FK866 (NAMPT inhibitor) to deplete NAD+

• CTBP2 mutant studies:

  • Express wild-type CTBP2 alongside NADH-binding defective CTBP2 mutants

  • Compare their effects on target gene expression (wild-type CTBP2 showed 2.75-fold induction of Tiam1 mRNA expression compared to 1.5-fold with NADH-binding defective CTBP2)

• Fluorescence resonance energy transfer (FRET):

  • Design FRET biosensors to monitor CTBP2 conformational changes upon NADH binding

  • Study real-time changes in CTBP2 activity in response to metabolic fluctuations

• ChIP-seq analysis:

  • Compare CTBP2 chromatin occupancy under conditions with altered NADH/NAD+ ratios

  • CTBP2 is more frequently recruited to transcriptional start sites (TSS)

  • Analyze how metabolic changes affect CTBP2 recruitment patterns

• Transcriptional reporter assays:

  • Employ reporters for CTBP2-regulated genes (e.g., G6pc) under different NADH/NAD+ conditions

  • Forkhead response element (FHRE) luciferase reporter assays can measure CTBP2 repression activity

What methodologies are recommended for studying CTBP2's role in cancer cell migration?

CTBP2 has been implicated in promoting cancer cell migration . The following methodologies can be used to investigate this role:

• Cell migration assays:

  • Transwell migration assays to quantify migration potential

  • Wound healing (scratch) assays to monitor migration dynamics

  • Single-cell tracking using time-lapse microscopy for detailed migration analysis

  • CTBP2 knockdown reduced migration by 70% in some cancer cell lines

• Molecular manipulation:

  • siRNA/shRNA-mediated CTBP2 knockdown (demonstrated to reduce Tiam1 levels and inhibit migration)

  • CRISPR-Cas9 for CTBP2 knockout

  • Overexpression of wild-type and mutant CTBP2 constructs

• Mechanistic studies:

  • Investigate Tiam1 as a downstream mediator:

    • Simultaneous knockdown of CTBP2 and Tiam1 showed enhanced inhibition of migration (~90% reduction)

    • Measure Tiam1 protein and mRNA levels via immunoblotting and qRT-PCR

    • CTBP2 overexpression increased Tiam1 protein (3-fold) and mRNA (2-fold) levels

• Protein-protein interaction studies:

  • Co-immunoprecipitation to identify CTBP2 binding partners in migrating cells

  • Proximity ligation assays to visualize protein interactions in situ

  • Mass spectrometry to identify migration-specific CTBP2 complexes

• In vivo metastasis models:

  • Xenograft models with CTBP2-manipulated cancer cells

  • Bioluminescence imaging to track metastatic spread

  • Tissue analysis for CTBP2 and Tiam1 expression in primary and metastatic sites

How can researchers effectively investigate CTBP2's role in metabolic regulation?

CTBP2 plays a significant role in metabolic regulation, including effects on diabetes and hepatic steatosis . To study this function:

• Gene expression analysis:

  • qRT-PCR to measure expression of CTBP2-regulated metabolic genes (e.g., G6pc)

  • RNA-seq to identify global transcriptional changes upon CTBP2 manipulation

  • CTBP2 suppression increased expression of G6pc, and this effect was enhanced by forskolin (cAMP activator)

• Protein interaction studies:

  • ChIP-seq to map CTBP2 binding sites on metabolic gene promoters

  • CTBP2 is frequently recruited to transcriptional start sites (TSS)

  • Co-IP to identify metabolic transcription factor partners (e.g., FoxO1)

  • Simultaneous suppression of CTBP2 and FoxO1 prevented induction of metabolic genes

• Metabolic phenotyping:

  • Glucose production assays in hepatocytes with CTBP2 manipulation

  • Lipid accumulation measurement in hepatocytes

  • Insulin sensitivity assays

• In vivo metabolic models:

  • Diet-induced obesity models with CTBP2 modulation

  • Liver-specific CTBP2 knockout or overexpression

  • Glucose tolerance and insulin tolerance tests

  • Hepatic steatosis assessment

• Reporter assays:

  • Forkhead response element (FHRE) luciferase reporter assays

  • CTBP2 suppression activated FHRE luciferase reporter activity

  • G6pc promoter reporter assays under various metabolic conditions

What are the current approaches for studying CTBP2 isoform-specific functions?

CTBP2 exists in multiple isoforms with distinct functions, including a transcriptional repressor form and a synaptic ribbon component (isoform 2) . To study isoform-specific functions:

• Isoform-specific antibodies:

  • Select antibodies targeting unique regions of specific isoforms

  • Validate isoform specificity using overexpression systems

  • Use appropriate positive control tissues (e.g., retinal tissue for synaptic ribbon isoform)

• Genetic manipulation strategies:

  • Design isoform-specific siRNAs/shRNAs targeting unique exons

  • Create isoform-specific CRISPR-Cas9 knockout models

  • Develop transgenic models expressing single CTBP2 isoforms

• Subcellular localization analysis:

  • Perform immunofluorescence with isoform-specific antibodies

  • Use confocal microscopy to distinguish nuclear (transcriptional repressor) versus synaptic (ribbon component) localization

  • Combine with synaptic markers for co-localization studies

• Functional assays:

  • Transcriptional reporter assays for repressor isoform

  • Electrophysiology for synaptic ribbon isoform

  • Calcium imaging to assess synaptic function

• Isoform-specific interactome analysis:

  • BioID or proximity labeling to identify isoform-specific protein interactions

  • Mass spectrometry to characterize unique protein complexes

  • Yeast two-hybrid screening with isoform-specific bait constructs

How can advanced imaging techniques be applied to CTBP2 research?

Advanced imaging techniques offer powerful tools for investigating CTBP2 function and dynamics:

• Super-resolution microscopy:

  • STORM or PALM imaging to resolve CTBP2 localization at synaptic ribbons beyond diffraction limit

  • SIM microscopy to visualize CTBP2 distribution in nuclear transcriptional complexes

  • Quantitative analysis of CTBP2 clustering in different cellular compartments

• Live-cell imaging approaches:

  • FRAP (Fluorescence Recovery After Photobleaching) to study CTBP2 dynamics

  • Single-molecule tracking to monitor CTBP2 movement and binding kinetics

  • Optogenetic manipulation of CTBP2 activity with temporal precision

• Multi-color imaging strategies:

  • Simultaneous visualization of CTBP2 with interaction partners

  • Triple labeling has been demonstrated with CTBP2, tubulin beta, and nuclear staining

  • Co-localization analysis with transcription factors or synaptic proteins

• Correlative light and electron microscopy (CLEM):

  • Combine fluorescence imaging of CTBP2 with ultrastructural context

  • Particularly valuable for studying synaptic ribbon structure and composition

• Functional imaging:

  • Combine CTBP2 visualization with calcium imaging in neurons

  • Simultaneous monitoring of CTBP2 localization and synaptic activity

  • FLIM-FRET to detect CTBP2 conformational changes upon NADH binding or protein interactions

What are the essential validation steps for CTBP2 antibodies in research applications?

Thorough validation is crucial for ensuring reliable results with CTBP2 antibodies:

• Positive and negative controls:

  • Positive controls: Cell lines with known CTBP2 expression (HeLa, A549, HEK293, MCF-7)

  • Negative controls: Primary antibody omission, isotype controls, pre-immune serum

• Multiple detection methods:

  • Validate antibody specificity across multiple applications (WB, IHC, IF)

  • Confirm expected molecular weight (47-49 kDa) in Western blots

  • Verify expected subcellular localization pattern

• Genetic validation:

  • siRNA/shRNA knockdown to confirm signal reduction

  • CRISPR-Cas9 knockout as definitive negative control

  • Overexpression to confirm increased signal intensity

• Cross-reactivity testing:

  • Test antibody against related proteins (especially CTBP1)

  • Evaluate reactivity across multiple species (human, mouse, rat)

  • Peptide competition assays to confirm epitope specificity

• Reproducibility assessment:

  • Test multiple antibody lots

  • Compare results using antibodies from different vendors or different clones

  • Document batch-to-batch variation

How can researchers address inconsistent results when using CTBP2 antibodies across different experimental systems?

Inconsistent results are a common challenge in antibody-based research. Here's a methodological approach to troubleshooting:

• Systematic comparison:

  • Create a standardized experimental pipeline to test antibodies

  • Use identical samples processed in parallel with different antibodies

  • Document all experimental variables (buffers, incubation times, temperatures)

• Antibody characterization:

  • Compare monoclonal versus polyclonal antibodies

  • Test different epitope regions (N-terminal vs. C-terminal)

  • Evaluate different clones (e.g., EPR7611(B) , clone 16 )

• Technical optimization:

  • Titrate antibody concentrations systematically

  • Optimize antigen retrieval methods for tissue samples

  • Test different fixation protocols (paraformaldehyde, methanol, acetone)

  • Vary incubation conditions (time, temperature)

• Sample preparation considerations:

  • Compare fresh versus frozen tissue

  • Test different lysis buffers for protein extraction

  • Evaluate the impact of phosphatase or protease inhibitors

• Data integration approach:

  • Combine results from multiple antibodies and techniques

  • Use orthogonal methods to confirm findings (e.g., mRNA analysis, mass spectrometry)

  • Document and report all validation steps in publications

Through this comprehensive approach to troubleshooting, researchers can identify sources of variability and establish robust protocols for consistent CTBP2 detection across experimental systems.