CBR4 Antibody

What is the optimal storage condition for CBR4 antibodies?

CBR4 antibodies should be stored at -20°C, where they typically remain stable for one year after shipment . Most commercially available antibodies are provided in PBS buffer (pH 7.3) containing preservatives such as 0.02% sodium azide and 50% glycerol . These additives help maintain antibody stability during freeze-thaw cycles. For antibodies that come in small volumes (20μl), manufacturers often include 0.1% BSA to prevent protein loss through adsorption to tube walls . While aliquoting is generally recommended for antibodies, some suppliers specifically note it is unnecessary for -20°C storage of CBR4 antibodies . Before use, allow the antibody to equilibrate to room temperature and mix gently by inverting the tube rather than vigorous vortexing.

What species reactivity should be expected from different CBR4 antibodies?

CBR4 antibodies demonstrate diverse species reactivity profiles depending on the manufacturer and clone. Rabbit polyclonal antibodies like ABIN7261179 show documented reactivity with human and rat samples . The more extensively characterized 13725-1-AP antibody from Proteintech has been validated for human, mouse, and rat reactivity . Similarly, the mouse monoclonal antibody (clone OTI3C10, catalog # M07359) also demonstrates cross-reactivity with human, mouse, and rat samples . Some antibodies offer broader species coverage, with certain polyclonal variants reported to react with samples from human, rat, mouse, cow, dog, rabbit, pig, guinea pig, and horse species . When selecting an antibody for your experiment, ensure the documented reactivity matches your target species to avoid false negatives.

What are the recommended dilutions for different applications of CBR4 antibodies?

Dilution requirements vary significantly based on both the antibody and the application technique:

Always optimize dilutions for your specific sample type and experimental conditions. Start with the manufacturer's recommended range and perform a dilution series to determine optimal signal-to-noise ratio for your specific application.

What positive controls are recommended for validating CBR4 antibody performance?

When validating CBR4 antibodies, specific cell lines and tissue samples have been documented as reliable positive controls. For Western blotting, BxPC-3 (pancreatic cancer) and HepG2 (liver cancer) cell lines consistently show CBR4 expression and are recommended as positive controls . For immunoprecipitation experiments, HepG2 cells have been validated as suitable positive controls . In immunohistochemistry applications, human hepatocirrhosis tissue samples have demonstrated positive CBR4 staining, particularly when using appropriate antigen retrieval methods . Human kidney tissue has also been documented as a useful positive control for IHC applications . Including these validated positive controls alongside experimental samples helps confirm antibody performance and troubleshoot potential technical issues.

What antigen retrieval methods are recommended for CBR4 antibody in IHC applications?

For optimal CBR4 detection in formalin-fixed paraffin-embedded (FFPE) tissue sections, heat-induced epitope retrieval (HIER) is recommended. The primary suggested method is Tris-EDTA (TE) buffer at pH 9.0 . This alkaline pH HIER method is particularly effective for unmasking CBR4 epitopes that may be cross-linked during formalin fixation. Alternatively, citrate buffer at pH 6.0 can be used if TE buffer retrieval yields suboptimal results . When using monoclonal antibodies like OTI3C10 (M07359), heat-induced epitope retrieval has been validated for human kidney tissue sections . Regardless of the buffer chosen, ensure consistent retrieval conditions (temperature, time, and buffer composition) when comparing multiple samples. Always include positive control tissues (such as human hepatocirrhosis or kidney) to confirm successful antigen retrieval and antibody performance.

How should I troubleshoot weak or non-specific signals when using CBR4 antibodies in Western blotting?

When encountering weak or non-specific signals with CBR4 antibodies in Western blotting, systematically address potential issues:

For weak signals:

• Increase antibody concentration while staying within recommended range (1:200-1:2000 for ABIN7261179 or 1:500-1:1000 for 13725-1-AP)

• Extend primary antibody incubation time (overnight at 4°C instead of 1-2 hours)

• Increase protein loading (CBR4 is observed at approximately 28 kDa, slightly higher than its calculated 25 kDa weight)

• Enhance detection sensitivity by using a more sensitive substrate (chemiluminescent vs. colorimetric)

• Confirm sample preparation preserves CBR4 integrity (include protease inhibitors)

For non-specific signals:

• Increase blocking time/concentration (5% BSA or milk in TBST)

• Extend washing steps (5-6 times for 5 minutes each with fresh TBST)

• Dilute primary antibody further while maintaining overnight incubation

• Use validated positive controls (BxPC-3 or HepG2 cells) to differentiate specific from non-specific bands

• Validate with a different CBR4 antibody to confirm true positive signals

For each troubleshooting step, change only one variable at a time to identify the source of the problem.

What are the considerations for using CBR4 antibodies in co-immunoprecipitation experiments?

When designing co-immunoprecipitation (co-IP) experiments with CBR4 antibodies, several methodological considerations are critical for success:

• Antibody selection: Use antibodies validated for IP applications, such as 13725-1-AP, which is recommended at 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

• Lysis buffer optimization:

  • Use a gentle lysis buffer (e.g., NP-40 or Triton X-100 based) to preserve protein-protein interactions

  • Include protease inhibitors and phosphatase inhibitors if investigating phosphorylation-dependent interactions

  • Maintain cold temperature (4°C) throughout sample preparation

• Pre-clearing strategy:

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Reserve 5-10% of pre-cleared lysate as input control

• IP controls:

  • Include IgG control matching the host species of the CBR4 antibody

  • Use known CBR4-expressing cells (HepG2) as positive control

  • Consider using CBR4-null or knockdown samples as negative controls

• Elution and detection:

  • Elute under mild conditions if planning to assess enzymatic activity

  • For Western blot detection, use a different CBR4 antibody from a different host species or targeting a different epitope to avoid detecting the IP antibody heavy chain

The co-IP protocol should be optimized for each specific protein-protein interaction being investigated.

How do monoclonal and polyclonal CBR4 antibodies compare in detecting post-translational modifications?

When investigating post-translational modifications (PTMs) of CBR4, the choice between monoclonal and polyclonal antibodies significantly impacts detection sensitivity and specificity:

Polyclonal CBR4 antibodies (such as ABIN7261179 and 13725-1-AP) :

• Recognize multiple epitopes across the CBR4 protein, increasing the likelihood of detecting the protein even if some epitopes are masked by PTMs

• May detect CBR4 regardless of certain modification states, providing higher sensitivity for total CBR4 detection

• Less suitable for specifically targeting modified forms unless raised against modification-specific peptides

• Show batch-to-batch variation that may affect consistency in PTM studies

Monoclonal CBR4 antibodies (such as OTI3C10) :

• Recognize a single epitope, providing higher specificity

• May fail to detect CBR4 if the target epitope is modified or masked by a PTM

• More consistent between experiments due to clonal production

• Better suited for detecting specific PTM forms when developed against modified epitopes

For comprehensive PTM analysis, a dual-approach strategy is recommended:

• Use polyclonal antibodies to capture total CBR4 protein

• Use monoclonal antibodies or modification-specific antibodies to detect specific PTM forms

• Confirm findings with mass spectrometry to identify the exact nature and location of modifications

When comparing data between antibody types, be aware that observed differences may reflect antibody characteristics rather than biological variation.

What approaches can resolve contradictory results obtained using different CBR4 antibodies?

When facing contradictory results from different CBR4 antibodies, employ a systematic reconciliation approach:

• Epitope mapping analysis:

  • Compare the immunogen sequences used to generate each antibody

  • ABIN7261179 uses recombinant fusion protein of human CBR4 (NP_116172.2)

  • 13725-1-AP uses CBR4 fusion protein Ag4678

  • OTI3C10 uses full-length human recombinant CBR4 (NP_116172) produced in E.coli

  • Different epitopes may explain detection discrepancies due to epitope masking or conformation differences

• Validation with orthogonal methods:

  • Perform mRNA analysis (RT-qPCR) to confirm CBR4 expression at transcript level

  • Use CRISPR/Cas9 knockout or siRNA knockdown to generate negative controls

  • Employ mass spectrometry to confirm protein identity

• Technical validation:

  • Test multiple antibody lots to rule out lot-to-lot variation

  • Use complementary application techniques (e.g., if WB and IHC results differ, add IF or ELISA)

  • Standardize experimental conditions across antibodies (same lysis buffer, same sample preparation)

• Reconciliation strategy:

  • Create a comparative table of results with different antibodies

  • Weight findings based on antibody validation quality (monoclonal vs polyclonal, validation depth)

  • Consider biological context (tissue-specific isoforms, interaction partners)

• Documentation:

  • Thoroughly document all antibody information (catalog number, lot, dilution, incubation conditions)

  • Explicitly acknowledge contradictory results in publications

  • Provide alternative interpretations based on antibody limitations

This systematic approach helps differentiate true biological findings from antibody-related technical artifacts.

How can I optimize CBR4 antibodies for multiplex immunofluorescence applications?

Optimizing CBR4 antibodies for multiplex immunofluorescence requires careful consideration of several factors:

• Antibody selection and validation:

  • Prioritize monoclonal antibodies (like OTI3C10) for higher specificity in multiplex settings

  • Validate each antibody individually before combining in multiplex panels

  • Test for cross-reactivity between secondary antibodies

• Fluorophore selection strategy:

  • Assign brighter fluorophores (Alexa Fluor 488, 555) to lower-abundance targets like CBR4

  • Reserve dimmer fluorophores for high-abundance targets

  • Ensure spectral separation between fluorophores to minimize bleed-through

• Staining protocol optimization:

  • For sequential staining with CBR4:

    • Start with heat-induced epitope retrieval using TE buffer pH 9.0

    • Block with 10% normal serum from secondary antibody host species

    • Dilute CBR4 antibodies according to validated ranges (1:20-1:200)

    • Extend incubation times (overnight at 4°C) to improve sensitivity

    • Include DAPI nuclear counterstain for cellular context

• Signal amplification strategies:

  • Consider tyramide signal amplification (TSA) for low-abundance CBR4 detection

  • Use quantum dots for improved photostability in extensive imaging sessions

  • Employ proximity ligation assay (PLA) to visualize CBR4 interactions with other proteins

• Imaging and analysis considerations:

  • Acquire single-color controls for spectral unmixing

  • Use consistent exposure settings between experimental groups

  • Perform automated quantification with appropriate thresholding

  • Include colocalization analysis when studying CBR4 interaction with other mitochondrial proteins

By systematically optimizing each step, researchers can successfully incorporate CBR4 antibodies into multiplex immunofluorescence panels to study its localization and interactions in complex biological systems.