ADRA1B Antibody

What criteria should researchers use when selecting an ADRA1B antibody for their experiments?

When selecting an ADRA1B antibody, researchers should evaluate several critical parameters to ensure experimental success. First, consider the specific application requirements (WB, IHC, FC, or IF) as antibodies demonstrate variable performance across different techniques. The available ADRA1B antibodies show distinct application profiles - while some like the 22419-1-AP perform well in Western Blot (1:500-1:2000 dilution) and Flow Cytometry, others like 19776-1-AP are optimized for Immunohistochemistry (1:50-1:500 dilution) .

Second, assess species reactivity based on your experimental model. Current commercial antibodies demonstrate tested reactivity with human, mouse, and rat samples, though specific batch validation should be performed . For rigorous research, evaluate the immunogen used for antibody production - peptide-derived antibodies like 19776-1-AP or fusion protein-generated antibodies like 22419-1-AP may exhibit different epitope recognition characteristics and specificity profiles .

Finally, review published applications to determine real-world performance. The literature contains specific ADRA1B antibody applications in studies examining noradrenergic regulation of neural circuits and adrenoreceptor involvement in pulmonary artery smooth muscle cell proliferation .

How can I verify the specificity of my ADRA1B antibody?

Validating ADRA1B antibody specificity requires a multi-faceted approach beyond manufacturer specifications. Begin with positive control tissues known to express ADRA1B, such as brain, heart, and testis tissues as indicated in validation data . The expected molecular weight for ADRA1B is approximately 57 kDa (calculated), though observed molecular weights of 70 kDa have been reported in certain applications .

For definitive validation, implement knockout/knockdown controls where cells or tissues lacking ADRA1B expression serve as negative controls. Alternatively, pre-absorption tests using the immunizing peptide can confirm specificity. When evaluating Western blot results, compare the observed band pattern with the predicted size of 57 kDa while noting that post-translational modifications may cause the apparent molecular weight to differ from predicted values .

For immunohistochemical applications, compare staining patterns with established literature and consider dual-labeling approaches with antibodies targeting known ADRA1B-interacting proteins. When troubleshooting specificity issues, optimize antigen retrieval conditions - for example, 19776-1-AP performs optimally with TE buffer pH 9.0, though citrate buffer pH 6.0 provides an alternative method .

What are the optimal protocols for using ADRA1B antibodies in Western blot applications?

Successful Western blot detection of ADRA1B requires careful consideration of sample preparation, antibody dilution, and detection parameters. Begin with protein extraction from tissues with confirmed ADRA1B expression (brain, heart, or testis samples) . For membrane protein preparation, use specialized extraction buffers containing mild detergents to maintain membrane protein conformation.

When optimizing blotting conditions, be aware that the observed molecular weight of ADRA1B (approximately 70 kDa) may differ from the calculated value (57 kDa) due to post-translational modifications and membrane protein characteristics . For challenging samples or weak signals, consider implementing signal enhancement systems or extending exposure times while maintaining low background.

For quantitative Western blot analysis, verify linear detection range and use appropriate housekeeping proteins as loading controls. The documented WB protocol developed specifically for ADRA1B antibody 22419-1-AP provides a validated starting point for optimizing this application .

What considerations are important when using ADRA1B antibodies for immunohistochemistry?

Successful immunohistochemical detection of ADRA1B requires attention to tissue fixation, antigen retrieval, and antibody incubation parameters. Formalin-fixed paraffin-embedded tissues require appropriate antigen retrieval, with specific ADRA1B antibodies showing optimal performance under defined conditions. For instance, 19776-1-AP requires TE buffer pH 9.0 for optimal antigen retrieval, though citrate buffer pH 6.0 provides an alternative .

Antibody dilution ranges vary significantly between products, with 19776-1-AP recommended at 1:50-1:500 for IHC applications, while ab84405 has been validated at 1:100 dilution . Researchers should conduct serial dilution experiments to determine optimal concentration for their specific tissue samples. For human heart tissue, validated staining has been achieved with ab84405 at 1:100 dilution .

Detection sensitivity can be enhanced through amplification systems like polymer-HRP or avidin-biotin complexes, though these require careful optimization to avoid nonspecific background. When analyzing ADRA1B localization patterns, compare with published literature describing both membrane and nuclear distribution patterns, as nuclear ADRA1A-ADRA1B heterooligomers have been documented in cardiac myocytes .

For dual-labeling studies, select compatible detection systems and appropriate controls to exclude cross-reactivity. The published IHC protocol specifically developed for ADRA1B-Specific antibody 19776-1-AP provides a validated methodological framework .

How should ADRA1B antibodies be stored and handled to maintain optimal performance?

Proper storage and handling of ADRA1B antibodies is critical for maintaining reactivity and specificity over time. Current commercial ADRA1B antibodies are supplied in liquid form with PBS buffer containing 0.02% sodium azide and 50% glycerol at pH 7.3 . This formulation enables storage at -20°C without the need for aliquoting, as indicated by manufacturer stability data .

Long-term stability has been confirmed for up to one year after shipment when stored at -20°C . For smaller volume formats (20μl), products may contain 0.1% BSA as a stabilizer . When handling these antibodies, avoid repeated freeze-thaw cycles that can compromise antibody integrity.

During experimental procedures, maintain antibodies on ice when in use and return to -20°C promptly after experiments. When preparing working dilutions, use high-quality diluents containing appropriate blocking proteins to minimize nonspecific binding. For sensitive applications, prepare fresh working dilutions for each experiment rather than storing diluted antibody.

If diminished performance is observed over time, verify antibody integrity through positive control experiments before troubleshooting experimental conditions. The documented stability data supports continuous performance without the need for aliquoting, simplifying laboratory handling protocols .

How can ADRA1B antibodies be utilized for studying receptor heterodimerization and signaling complexes?

Investigating ADRA1B receptor heterodimerization and signaling complexes requires sophisticated immunological approaches beyond standard detection methods. Co-immunoprecipitation (Co-IP) using ADRA1B antibodies can identify interaction partners, with particular relevance to the documented ADRA1A-ADRA1B heterooligomers that regulate phenylephrine-stimulated ERK signaling in cardiac myocytes . When designing Co-IP experiments, select antibodies recognizing distinct epitopes for precipitation and detection to avoid steric hindrance.

Advanced microscopy techniques such as proximity ligation assay (PLA) or Förster resonance energy transfer (FRET) can visualize ADRA1B interactions with resolution below the diffraction limit. For these applications, compatible fluorophore-conjugated secondary antibodies or direct conjugates are required, with careful validation to maintain epitope recognition.

When investigating nuclear-cytoplasmic shuttling of ADRA1B, consider subcellular fractionation followed by immunoblotting, or live-cell imaging with tagged constructs validated against antibody staining patterns. The documented nuclear localization of ADRA1A-ADRA1B heterooligomers provides a biological context for these experimental approaches .

For functional studies of receptor complexes, combine antibody-based detection with pharmacological manipulation using selective agonists and antagonists. Correlation between observed molecular interactions and downstream signaling events (e.g., phosphatidylinositol-calcium second messenger system activation) establishes biological relevance of detected complexes .

What approaches can be used to investigate ADRA1B post-translational modifications using available antibodies?

Investigating ADRA1B post-translational modifications (PTMs) requires specialized experimental design integrating antibody-based detection with biochemical characterization. The discrepancy between calculated molecular weight (57 kDa) and observed molecular weight (70 kDa) for ADRA1B suggests significant post-translational processing , which can be systematically investigated.

For glycosylation analysis, compare migration patterns before and after treatment with glycosidases (PNGase F, Endo H) using Western blot detection with ADRA1B antibodies. Similarly, phosphorylation sites can be investigated through treatment with phosphatases followed by mobility shift analysis, or through specialized phospho-state specific antibodies when available.

Mass spectrometry approaches combined with immunoprecipitation using ADRA1B antibodies can comprehensively map PTM landscapes. For these applications, antibodies must efficiently capture the native receptor from detergent-solubilized preparations, with validation using known positive control tissues such as brain or heart .

For site-directed functional studies, combine site-specific mutagenesis of predicted modification sites with antibody detection to correlate structural changes with altered detection patterns. When analyzing tissue-specific modification patterns, compare ADRA1B migration profiles across validated positive tissues, including brain, heart, and testis samples .

How can ADRA1B antibodies be utilized in flow cytometry for receptor expression profiling?

Flow cytometric analysis of ADRA1B expression requires specific methodology for this G-protein coupled receptor. For intracellular staining, which has been validated for ADRA1B detection, implement appropriate fixation and permeabilization protocols to preserve epitope recognition while enabling antibody access to intracellular domains . The validated protocol using 22419-1-AP antibody recommends 0.80 μg per 10^6 cells in a 100 μl suspension for intracellular staining .

Cell type validation is essential, with PC-3 cells documented as positive controls for intracellular flow cytometry of ADRA1B . When analyzing receptor expression in primary cells or tissues, include appropriate isotype controls at matching concentrations to establish specific staining thresholds.

For quantitative expression analysis, consider calibration with beads containing known antibody binding sites or comparative staining with cell lines expressing defined receptor levels. When investigating receptor internalization or trafficking, combine surface and intracellular staining protocols with time-course analysis following agonist stimulation.

Multi-parameter flow cytometry enables correlation of ADRA1B expression with cell phenotype markers, activation states, or functional readouts such as calcium flux. For these complex panels, careful compensation and controls are required to address spectral overlap and ensure detection specificity.

What are common challenges in ADRA1B antibody applications and how can they be addressed?

Researchers frequently encounter several technical challenges when working with ADRA1B antibodies that require systematic troubleshooting approaches. For Western blot applications, weak or absent signals despite confirmed expression may indicate insufficient protein extraction efficiency for this membrane-bound receptor. Optimize extraction using specialized detergent combinations (RIPA buffer supplemented with deoxycholate or low concentrations of SDS) while maintaining protein structure integrity.

In immunohistochemical applications, high background staining can be addressed through systematic optimization of blocking conditions, antibody dilutions, and washing stringency. When using 19776-1-AP, verify that the recommended antigen retrieval methods (TE buffer pH 9.0 or alternative citrate buffer pH 6.0) have been correctly implemented . Poor signal in IHC may indicate epitope masking, requiring extended antigen retrieval times or alternative buffer systems.

For flow cytometry applications, cell autofluorescence can interfere with specific signal detection. Implement appropriate compensation controls and consider alternative fluorophores with emission spectra distinct from cellular autofluorescence profiles. Validate permeabilization conditions using known intracellular antigens as positive controls alongside ADRA1B detection.

When encountering discrepancies between different detection methods, compare epitope accessibility under various sample preparation conditions. For contradictory results between laboratories, detailed methodology comparison focusing on antibody source, experimental conditions, and biological material can identify critical variables affecting detection outcomes.

How should researchers interpret differences in ADRA1B detection patterns across different tissues and experimental systems?

When comparing staining patterns or band intensity across tissues, consider tissue-specific post-translational modifications that may affect epitope recognition or molecular weight. The documented difference between calculated (57 kDa) and observed (70 kDa) molecular weights suggests tissue-specific processing may influence detection patterns .

In heterologous expression systems, receptor overexpression may alter trafficking, processing, or complex formation compared to endogenous expression. Compare detection patterns between overexpression systems and tissues with endogenous expression to identify potential artifacts. For comprehensive expression analysis, combine multiple detection methods (e.g., Western blot, IHC, and qPCR) to build a complete picture of receptor expression and localization.

What controls should be implemented when using ADRA1B antibodies for publishable research?

Rigorous research with ADRA1B antibodies requires comprehensive controls addressing both technical variables and biological specificity. For primary antibody validation, positive controls should include tissues with documented ADRA1B expression, such as brain, heart, and testis samples . Negative controls should include tissues known to lack ADRA1B expression or knockdown/knockout samples when available.

Technical controls must address non-specific binding through secondary-only incubations (omitting primary antibody) and isotype controls matching the primary antibody host species and immunoglobulin class. For Western blot applications, molecular weight markers must confirm the observed band corresponds to the expected size range (57-70 kDa) .

Antibody specificity can be further validated through peptide competition assays, where pre-incubation with the immunizing peptide should abolish specific signals. For quantitative applications, include standard curves or reference samples to demonstrate detection linearity across the relevant concentration range.

When using multiple antibodies targeting different epitopes of ADRA1B, concordance between detection patterns strengthens result validity. For tissues with heterogeneous expression, correlation between protein detection methods and mRNA expression data provides additional validation. Publications should include detailed methodology including antibody catalog numbers, dilutions, incubation conditions, and all implemented controls for experimental reproducibility.

How are ADRA1B antibodies being utilized in studies of neural circuit regulation and neurological disorders?

ADRA1B antibodies have become instrumental in elucidating noradrenergic regulation of neural circuits, with published applications demonstrating specific utility in neurological research. Recent studies have employed these antibodies to investigate the structural basis for noradrenergic regulation in the mouse olfactory bulb, establishing receptor localization patterns in specific neural populations . This application enables correlation between receptor expression and functional circuit properties.

When designing neurological research using ADRA1B antibodies, consider combined electrophysiological recording with immunohistochemical characterization to correlate receptor expression with functional properties. The validated immunohistochemical protocols using 19776-1-AP (1:50-1:500 dilution) provide a starting point for detection in neural tissues .

For investigating receptor trafficking in response to neuronal activity, combine antibody-based detection with activity-dependent labeling such as immediate early gene expression. Time-course studies following pharmacological manipulation can reveal dynamic regulation of receptor expression or subcellular localization.

In neurological disorder models, comparative analysis of ADRA1B expression, localization, and complex formation between control and pathological conditions may identify disease-associated alterations. Recent studies have examined neurobehavioral alterations in offspring born to polycystic ovary syndrome model rats, demonstrating broader applications for ADRA1B investigation in complex neurological contexts .

What methodological approaches can researchers use to study ADRA1B involvement in cardiovascular physiology and pathology?

Cardiovascular research applications of ADRA1B antibodies require specialized approaches addressing tissue-specific expression patterns and functional roles. Immunohistochemical detection of ADRA1B in human heart tissue has been validated using antibodies such as ab84405 at 1:100 dilution , providing a foundation for expression studies in cardiac tissue.

For investigating the documented role of nuclear ADRA1A-ADRA1B heterooligomers in regulating phenylephrine-stimulated ERK signaling in cardiac myocytes , combine subcellular fractionation with co-immunoprecipitation using optimized extraction conditions that preserve protein-protein interactions. When analyzing signaling pathways, correlate receptor detection with downstream effector activation using phospho-specific antibodies targeting key pathway components.

In vascular research, ADRA1B has been implicated in norepinephrine-induced pulmonary artery smooth muscle cell proliferation via p38 signaling . For these applications, combine receptor expression analysis with proliferation assays and specific pathway inhibitors to establish causal relationships.

For translational research comparing healthy and pathological cardiac tissue, standardize tissue collection, fixation, and processing to minimize technical variables. When analyzing receptor expression in different cardiac regions or cell types, implement dual-labeling strategies with cell-type specific markers to resolve expression patterns at the cellular level.

What dilution ranges and validated protocols are available for different ADRA1B antibody applications?

Optimized protocols with validated dilution ranges have been established for major ADRA1B antibody applications across different experimental systems. For Western blot applications using 22419-1-AP, the recommended dilution range is 1:500-1:2000, with documented detection in mouse and rat brain tissue, mouse heart tissue, and mouse testis tissue . For ab84405, Western blot applications have been validated at 1:500 dilution using human fetal brain lysates .

Immunohistochemical applications show variable optimal dilution ranges depending on the specific antibody. The 19776-1-AP antibody is recommended at 1:50-1:500 for IHC, with validated detection in mouse brain tissue and human heart tissue . For ab84405, IHC-P applications have been validated at 1:100 dilution in human heart tissue . Abbexa's ADRA1B antibody recommends IHC-P dilutions of 1:10-1:50 .

Flow cytometry applications have been specifically validated for intracellular staining using 22419-1-AP at 0.80 μg per 10^6 cells in a 100 μl suspension, with documented positive detection in PC-3 cells . For immunofluorescence applications, methodological details from published studies provide guidance, with documented applications in visualization of noradrenergic regulation in mouse olfactory bulb .

Across all applications, researchers should implement titration experiments to determine optimal working concentrations for their specific experimental systems, as antibody performance may vary with tissue type, fixation method, and detection system. The availability of product-specific protocols for ADRA1B antibodies provides validated methodological frameworks for optimization .

What sample preparation considerations are critical for successful ADRA1B detection in different experimental systems?

Sample preparation methodology significantly impacts ADRA1B detection outcomes across experimental systems. For Western blot applications, effective extraction of this membrane-bound G-protein coupled receptor requires specialized buffers containing appropriate detergents. Standard RIPA buffer may be supplemented with additional detergents for improved extraction efficiency while maintaining antibody epitope recognition.

In immunohistochemical applications, fixation type and duration significantly impact epitope preservation and accessibility. Formalin-fixed paraffin-embedded tissues require specific antigen retrieval conditions, with ADRA1B antibodies showing optimal performance under defined pH and buffer compositions. For 19776-1-AP, TE buffer at pH 9.0 is recommended, with citrate buffer at pH 6.0 as an alternative .

For flow cytometry and immunofluorescence applications detecting intracellular ADRA1B, permeabilization conditions must balance membrane permeabilization with epitope preservation. Validated protocols typically employ mild detergents or alcohol-based permeabilization followed by appropriate blocking to minimize non-specific binding.

When working with tissues exhibiting variable ADRA1B expression, consider enrichment strategies such as laser capture microdissection or cell sorting to isolate specific cellular populations. For all sample types, include appropriate positive control tissues with documented ADRA1B expression (brain, heart, testis) to verify detection protocols .