ABR Antibody

What is the ABR protein and why is it targeted in research?

The ABR (Active BCR-Related) gene encodes the Active breakpoint cluster region-related protein, which plays significant roles in cellular signaling pathways. This protein is important in research due to its involvement in various cellular functions, including GTPase regulation. Detecting ABR expression patterns across different tissues and under various experimental conditions provides valuable insights into cell regulatory mechanisms . Methodologically, researchers should consider ABR's expression levels in their specific experimental models before antibody selection, as this will impact detection sensitivity requirements.

What applications are ABR antibodies validated for in research settings?

ABR antibodies have been validated for several key research applications, primarily Western Blotting (WB) and Immunohistochemistry with paraffin-embedded sections (IHC-p). These applications have been confirmed in human, mouse, and rat samples . When designing experiments, researchers should note that validation in one application doesn't necessarily translate to performance in another. For example, an antibody performing well in Western blot may not maintain specificity in immunofluorescence applications due to differences in protein conformation and epitope accessibility.

How should researchers interpret cross-reactivity data for ABR antibodies?

While some ABR antibodies claim "no cross-reactivity with other proteins," researchers should approach these claims with appropriate scientific skepticism . Cross-reactivity should be experimentally verified in the specific biological system being studied. Methodologically, this requires:

• Testing the antibody against known negative controls (tissues/cells not expressing the target)

• Comparing detection patterns against multiple antibodies targeting different epitopes of the same protein

• Validating specificity using genetic approaches (knockout/knockdown samples) rather than relying solely on orthogonal approaches

What validation strategies provide the highest scientific confidence for ABR antibody specificity?

Genetic approaches for antibody validation demonstrate superior scientific value compared to orthogonal approaches. Research indicates that 89% of antibodies validated using genetic strategies (knockout/knockdown controls) perform as expected, compared to 80% of those validated using orthogonal approaches for Western blot applications . For immunofluorescence applications, the difference is even more pronounced, with only 38% of orthogonally-validated antibodies confirming specificity when tested against knockout controls .

For rigorous ABR antibody validation, researchers should:

• Use CRISPR/Cas9 knockout cell lines as negative controls

• Compare staining patterns between wildtype and knockout samples

• Evaluate antibody performance across multiple applications rather than assuming cross-application reliability

• Document validation data comprehensively to support reproducibility

How can researchers address epitope-specific considerations when selecting ABR antibodies?

The epitope recognized by an ABR antibody significantly impacts its experimental utility. For the ABIN4886413 antibody, the epitope corresponds to amino acids 370-407 in the middle region of the human ABR protein (sequence: HPFPDHELEDMKMKISALKSEIQKEKANKGQSRAIERL) . This sequence differs from the mouse sequence by one amino acid. When designing experiments, researchers should:

• Analyze whether their experimental conditions might alter epitope accessibility (denaturation, fixation, etc.)

• Consider whether post-translational modifications occur near the epitope region that might affect binding

• Evaluate whether splice variants of ABR in their experimental system contain the epitope

• Determine if species-specific sequence variations may impact cross-species reactivity

What methodological approaches can resolve contradictory data when using ABR antibodies?

When faced with contradictory results using ABR antibodies, researchers should implement a systematic troubleshooting approach:

• Employ multiple antibodies targeting different epitopes of ABR

• Compare results across different detection techniques (WB, IHC, IF)

• Include genetic controls (siRNA knockdown or CRISPR knockout)

• Validate findings with orthogonal, non-antibody-based methods (e.g., mass spectrometry)

• Optimize experimental conditions specifically for the chosen antibody (buffer compositions, incubation times, blocking agents)

What are the optimal protocols for ABR antibody use in Western blotting?

For Western blot applications using ABR polyclonal antibodies, specific technical considerations include:

• Sample preparation: Use appropriate lysis buffers that preserve epitope integrity

• Protein denaturation: Optimize temperature and duration to ensure epitope exposure without degradation

• Blocking conditions: Use 5% non-fat milk or BSA in TBST, depending on whether phospho-specific detection is required

• Primary antibody incubation: Dilute according to manufacturer's recommendations (typically 1:500-1:2000) and incubate overnight at 4°C

• Washing: Perform stringent washing steps (4-5 times for 5 minutes each) to reduce background

• Detection method: Choose chemiluminescence for highest sensitivity or fluorescent detection for quantitative analysis

Always include appropriate positive and negative controls, including knockout samples if available, to confirm antibody specificity .

How should researchers optimize immunohistochemistry protocols for ABR antibody staining?

For IHC applications with ABR antibodies, protocol optimization should address:

• Fixation conditions: Overfixation can mask epitopes; a time-course optimization may be necessary

• Antigen retrieval: Test multiple methods (heat-induced in citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

• Blocking endogenous peroxidase: Use 3% hydrogen peroxide for 10 minutes before antibody incubation

• Antibody concentration: Titrate to determine optimal dilution that maximizes signal while minimizing background

• Detection system: Choose between polymer-based systems or avidin-biotin complexes based on sensitivity requirements

• Counterstaining: Adjust timing to ensure nuclear details remain visible without obscuring antibody signals

Validation should include isotype controls and absorption controls with the immunizing peptide to confirm staining specificity .

How can researchers quantitatively analyze ABR expression patterns across different tissues?

Quantitative analysis of ABR expression requires standardized approaches:

• Western blot quantification:

  • Normalize ABR signal to appropriate loading controls (β-actin, GAPDH)

  • Use digital image analysis software with linear dynamic range

  • Apply statistical analysis across multiple biological replicates (n≥3)

• IHC quantification:

  • Establish clear scoring criteria (intensity scales, percentage positive cells)

  • Employ digital pathology software for unbiased quantification

  • Use multiple independent observers for manual scoring

  • Calculate inter-observer reliability statistics

• Cross-validation:

  • Compare protein expression data with mRNA expression (qPCR, RNA-seq)

  • Correlate findings with publicly available databases (Human Protein Atlas, GTEx)

This multi-faceted approach minimizes bias and increases confidence in expression pattern interpretations .

What strategies can address antibody lot-to-lot variability in long-term research projects?

Antibody lot-to-lot variability presents significant challenges for longitudinal studies. Researchers working with ABR antibodies should implement:

• Bulk purchasing and aliquoting of a single lot for the duration of critical projects

• Comprehensive validation of each new lot against previous standards

• Maintenance of internal reference standards (positive control lysates)

• Documentation of lot numbers in all experimental records and publications

• Implementation of bridging studies when lot changes are unavoidable

Validation between lots should include side-by-side comparisons of signal intensity, background levels, and specificity using identical samples and protocols .

How can high-throughput screening approaches be combined with ABR antibody detection?

Integration of ABR antibody-based detection with high-throughput screening requires specialized methodological adaptations:

• Miniaturization of antibody-based assays for microplate formats

• Optimization of automated liquid handling parameters

• Development of image-based detection algorithms for quantitative analysis

• Implementation of quality control metrics for assay reproducibility

• Design of data normalization approaches to account for plate-to-plate variability

These technical considerations enable screening of compound libraries or genetic perturbations while monitoring ABR protein levels or modifications as biological readouts.

What considerations should guide ABR antibody selection for multiplex immunofluorescence applications?

Multiplex immunofluorescence with ABR antibodies requires careful planning:

• Host species selection: Choose ABR antibodies raised in different host species than other target antibodies

• Fluorophore compatibility: Select fluorophores with minimal spectral overlap

• Sequential staining protocols: Develop when antibodies from the same species must be used

• Epitope retention: Verify that multiplexing conditions don't compromise epitope recognition

• Signal amplification: Determine if tyramide signal amplification is needed for low-abundance targets

• Image acquisition parameters: Optimize exposure settings to capture all signals while avoiding bleed-through

Validation of multiplex panels should include comparison to single-stain controls to ensure antibody performance is not compromised in the multiplex context .