CHC2 Antibody

What is the CHC-2 antibody and what specific target does it recognize?

The CHC-2 antibody is a rabbit monoclonal antibody that specifically recognizes the BAD (BCL2-Associated Agonist of Cell Death) protein. This antibody has been purified using affinity chromatography and is formulated in phosphate buffered saline (pH 7.4) with 150mM NaCl, 0.02% sodium azide, 50% glycerol, and 0.4-0.5mg/ml BSA . BAD is a pro-apoptotic protein that functions as a regulator of programmed cell death, making this antibody valuable for studying apoptotic pathways in research settings.

What species reactivity has been validated for the CHC-2 antibody?

The CHC-2 antibody has been validated for reactivity with human, mouse, and rat species . Cross-reactivity with other species may be possible but would require validation by individual researchers. When selecting antibodies for experimental use, confirming species reactivity is essential as improper species matching can lead to experiment failure and wasted resources.

What applications is the CHC-2 antibody validated for?

The CHC-2 antibody has been validated for multiple applications including:

• Western Blot (WB) at dilutions of 1:1000-1:2000

• Immunohistochemistry (IHC) at dilutions of 1:50-1:200

• Immunocytochemistry/Immunofluorescence (ICC/IF) at dilutions of 1:50-1:200

• Immunoprecipitation (IP) at a dilution of 1:30

• Flow Cytometry at a dilution of 1:50

Each application requires specific optimization for your experimental system.

How should I design positive and negative controls when using CHC-2 antibody?

When designing controls for CHC-2 antibody experiments:

Positive Controls:

• Cell lines known to express BAD protein (check literature for expression levels)

• Tissue samples with confirmed BAD expression

• Recombinant BAD protein for Western blot applications

Negative Controls:

• For IHC/ICC: Omit primary antibody but include all other reagents

• BAD-knockout cell lines (if available)

• Isotype controls for flow cytometry applications

• Pre-absorption with immunizing peptide (if available)

Control design is critical for validating results and distinguishing specific from non-specific binding.

When should I choose monoclonal CHC-2 antibody over polyclonal alternatives?

The monoclonal CHC-2 antibody offers higher specificity but potentially lower sensitivity compared to polyclonal alternatives . Choose the monoclonal CHC-2 antibody when:

• Experimental goals require consistent lot-to-lot reproducibility

• You need to minimize background/non-specific binding

• The target epitope is well-exposed in your experimental conditions

• You're performing co-localization studies requiring precise epitope targeting

Polyclonal antibodies may provide higher sensitivity but at the cost of potentially increased background . The choice between monoclonal and polyclonal should be guided by your specific experimental requirements and the nature of your target protein's presentation in your samples.

What are the optimal protocols for using CHC-2 antibody in Western blotting?

For optimal Western blot results with CHC-2 antibody:

• Sample Preparation:

  • Use RIPA or other appropriate lysis buffer with protease inhibitors

  • Load 20-50 μg of total protein per lane

  • Include appropriate positive controls

• Running Conditions:

  • Use 10-12% SDS-PAGE gels for optimal separation

  • Transfer to PVDF or nitrocellulose membrane

• Antibody Application:

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

  • Incubate with CHC-2 primary antibody at 1:1000-1:2000 dilution overnight at 4°C

  • Wash thoroughly with TBST (3-5 times for 5 minutes each)

  • Incubate with appropriate anti-rabbit secondary antibody

  • Include a loading control antibody (such as anti-GAPDH or anti-β-actin)

• Detection:

  • Use chemiluminescent substrate appropriate for your detection system

  • Optimize exposure time to prevent signal saturation

How should I optimize CHC-2 antibody for immunohistochemistry applications?

For optimal IHC results with CHC-2 antibody:

• Tissue Preparation:

  • Fix tissues appropriately (10% neutral buffered formalin is standard)

  • Use antigen retrieval methods (heat-induced epitope retrieval in citrate buffer pH 6.0 or EDTA buffer pH 9.0)

  • Test multiple antigen retrieval conditions if initial results are suboptimal

• Antibody Optimization:

  • Begin with a dilution range of 1:50-1:200

  • Test multiple dilutions to determine optimal signal-to-noise ratio

  • Incubate at 4°C overnight for optimal sensitivity or 1-2 hours at room temperature

• Detection System:

  • Use appropriate HRP-conjugated secondary antibody

  • Consider signal amplification systems for low-abundance targets

  • Use DAB or other chromogen suitable for your experimental design

• Controls:

  • Include positive and negative controls in each experimental run

  • Consider tissue microarrays for antibody validation across multiple samples

How can I use CHC-2 antibody to investigate BAD protein post-translational modifications?

BAD protein function is heavily regulated by post-translational modifications, particularly phosphorylation. To investigate these modifications:

• Experimental Design:

  • Use phosphatase inhibitors in your sample preparation

  • Consider using phospho-specific antibodies alongside CHC-2

  • Design experiments to capture dynamic phosphorylation states

• Technical Approaches:

  • For Western blotting: Use Phos-tag gels to separate phosphorylated from non-phosphorylated forms

  • For IP: Perform CHC-2 immunoprecipitation followed by phospho-specific Western blotting

  • Consider mass spectrometry analysis of immunoprecipitated samples to identify novel modifications

• Data Analysis:

  • Compare band migration patterns with and without phosphatase treatment

  • Quantify relative levels of modified vs. unmodified protein

  • Correlate modifications with functional outcomes in your experimental system

How can I effectively use CHC-2 antibody in multiplex immunofluorescence studies?

For multiplex studies involving CHC-2 antibody:

• Panel Design:

  • Select additional antibodies raised in different host species to avoid cross-reactivity

  • Ensure spectral separation between fluorophores

  • Consider antibody order when using sequential staining protocols

• Optimization Steps:

  • Test each antibody individually before combining

  • Use CHC-2 at 1:50-1:200 dilution range for IF applications

  • Validate specificity with appropriate controls

  • Test different fixation methods if co-localization issues arise

• Data Collection and Analysis:

  • Use appropriate imaging controls (single-stained samples, no primary controls)

  • Employ spectral unmixing if fluorophore spectra overlap

  • Perform quantitative co-localization analysis using appropriate software

What are common reasons for CHC-2 antibody failure in specific applications?

When CHC-2 antibody performs well in one application (e.g., Western blot) but fails in others (e.g., IHC or IF), consider these potential issues:

The conformation of the BAD protein may differ between applications - CHC-2 may recognize a linear epitope (good for WB) but struggle with native conformations (needed for IP or IF) .

How can I validate the specificity of CHC-2 antibody results in my experimental system?

To thoroughly validate CHC-2 antibody specificity:

• Genetic Approaches:

  • Use BAD-knockout or knockdown systems as negative controls

  • Perform rescue experiments with BAD overexpression

  • Compare staining patterns with multiple antibodies targeting different BAD epitopes

• Biochemical Validation:

  • Perform peptide competition assays

  • Confirm expected molecular weight in Western blot

  • Verify subcellular localization is consistent with known BAD biology

• Reproducibility Assessment:

  • Test antibody across multiple lots if possible

  • Compare results across different experimental conditions

  • Validate key findings with orthogonal methods not relying on antibodies

How can I optimize CHC-2 antibody for proximity ligation assays to study BAD protein interactions?

Proximity Ligation Assay (PLA) optimization for CHC-2 antibody:

• Experimental Design:

  • Pair CHC-2 with antibodies against known BAD interaction partners (e.g., 14-3-3 proteins, Bcl-2, Bcl-xL)

  • Use antibodies raised in different host species

  • Include appropriate controls (single antibody controls, known non-interacting proteins)

• Protocol Optimization:

  • Test multiple fixation and permeabilization conditions

  • Use CHC-2 at 1:50-1:100 dilution for optimal signal

  • Adjust incubation times and temperatures for maximum sensitivity

• Analysis Considerations:

  • Quantify PLA signals per cell

  • Compare signal distribution with known BAD localization patterns

  • Correlate PLA results with functional outcomes (e.g., apoptosis measurements)

What analytical methods are appropriate for quantifying BAD expression using CHC-2 antibody?

For quantitative analysis using CHC-2 antibody:

• Western Blot Quantification:

  • Use standard curves with recombinant BAD protein

  • Normalize to appropriate loading controls

  • Employ digital imaging with linear dynamic range

  • Use analytical software for densitometry

• Flow Cytometry Quantification:

  • Use fluorescence quantitation beads for standardization

  • Report results as molecules of equivalent soluble fluorochrome (MESF)

  • Include appropriate isotype controls

  • Consider median fluorescence intensity rather than mean

• Image-Based Quantification:

  • Apply consistent thresholding algorithms

  • Use nuclear counterstains for cell identification

  • Measure intensity, area, and subcellular distribution parameters

  • Consider machine learning approaches for complex pattern recognition

This comprehensive analysis ensures reliable quantification of BAD expression across experimental conditions.