ADRBK2 Antibody

What is ADRBK2/GRK3 and what are its key functional characteristics?

ADRBK2 (Adrenergic, Beta, Receptor Kinase 2), also known as GRK3 or BARK2, belongs to the G protein coupled receptor kinases (GRKs) family. These serine/threonine kinases regulate a diverse class of G-protein coupled receptors (GPCRs). ADRBK2 specifically phosphorylates the agonist-occupied form of beta-adrenergic and related GPCRs. The protein has been shown to play critical roles in various physiological and pathophysiological processes including:

• Chemotaxis

• Cell signaling

• Cell migration

• Inflammatory gene expression

• CXCL12/CXCR4 signaling regulation

The beta adrenergic receptor kinase 2 has 85% amino acid similarity with beta adrenergic receptor kinase 1, with the protein kinase catalytic domain having 95% similarity, suggesting a family of receptor kinases that may broadly regulate receptor function .

What are the molecular characteristics of ADRBK2 protein that guide antibody selection?

When selecting an ADRBK2 antibody, researchers should consider the following molecular characteristics:

This information is essential for validating antibody specificity and interpreting experimental results correctly.

What are the optimized application parameters for ADRBK2 antibodies in different experimental systems?

ADRBK2 antibodies have been validated for several applications, with specific recommended dilutions:

It is strongly recommended to titrate each antibody in your specific testing system to obtain optimal results, as performance may be sample-dependent .

How should researchers perform antigen retrieval for ADRBK2 immunohistochemistry applications?

For optimal antigen retrieval in IHC applications with ADRBK2 antibodies:

• Primary suggestion: Use TE buffer pH 9.0

• Alternative method: Use citrate buffer pH 6.0

These recommendations are based on successful detection in mouse brain tissue and human colon tissue . Optimization may be necessary for different tissue types or fixation methods.

How can researchers validate ADRBK2 antibody specificity to ensure reliable results?

A comprehensive validation approach for ADRBK2 antibodies includes:

• Testing with exogenously expressed proteins to confirm western blot results identity

• Verifying detection in known positive controls:

  • Cell lines: K-562 cells, HL-60 cells, U-87MG, SH-SY5Y

  • Tissues: Mouse brain, human colon, mouse lung, rat brain

• Performing dilution series to determine optimal concentrations

• Testing for cross-reactivity with other GRK family members

Research by Matthees et al. (2021) demonstrated that some GRK antibodies showed unspecific signals or cross-reactivity with other GRK family members, with one antibody (sc-365197 for GRK3) failing to detect its target at all . This emphasizes the critical importance of thorough validation.

How does cross-reactivity among GRK family members impact experimental design and data interpretation?

Cross-reactivity between GRK antibodies can significantly impact results interpretation. According to Matthees et al. (2021), they observed that:

• Some GRK2 antibodies detected overexpressed GRK3

• Some GRK6 antibodies detected GRK5

This cross-reactivity is particularly problematic when analyzing samples with varying expression ratios of these proteins. Researchers should consider:

• Using knockout or knockdown controls

• Employing multiple antibodies targeting different epitopes

• Complementing antibody-based detection with other methods (e.g., mass spectrometry)

• Accounting for potential cross-reactivity when interpreting results, especially in tissues where multiple GRK isoforms are expressed

How can researchers quantitatively compare different GRK isoform levels using antibody-based detection?

Matthees et al. (2021) describe STARPA (Simple Tag-guided Analysis of Relative Protein Abundance), a western blot-based, cost-effective method to compare protein levels obtained using different antibodies:

• Create expression constructs for all four ubiquitously expressed human GRKs

• Transfect HA-tagged GRK isoforms into ΔQ-GRK cells (which lack endogenous GRK expression)

• Analyze lysates for HA-antibody signals and determine optimal dilution factors for equal signals

• Create standardized samples with normalized signals

• Use these as references on the same gel as unknown samples

• Calculate relative amounts by normalizing to these standards

As shown in their study: "We created multiple dilution series ranging from 1:10 to 1:100, and they were analyzed by western blotting using an anti-HA antibody (Figure 4a). Quantification of several independently mixed dilutions and multiple analyses of them allowed the calculation of optimal standard dilutions (Figure 4b)."

This method enables researchers to compare relative levels of different GRK isoforms across cell lines or tissues despite using different antibodies.

What approaches exist for studying allele-specific expression of ADRBK2 using antibodies?

For researchers investigating allele-specific effects on ADRBK2 expression, two complementary approaches have been documented:

• Chromatin immunoprecipitation (ChIP) of acetylated histone H3 (ac-H3) at individual ADRBK2 regulatory alleles:

  • Uses anti-acetylated histone H3 antibody (Millipore 06–599)

  • Couples 25 μg antibody to 250 μL of sheep anti-rabbit IgG magnetic beads

  • Pre-clears chromatin with secondary antibody alone to reduce background

  • Incubates overnight with antibody-coupled beads

  • Performs PCR on selected regions to assess chromatin modifications

• Measuring allelic expression imbalances (AEI) among transcribed SNPs in ADRBK2:

  • Uses TaqMan allele-specific PCR for target SNPs

  • Compares Ct values for common and rare alleles in both input and ChIP-selected materials

  • Verifies discrimination between alleles by confirming at least 5 cycle difference between target and mismatch probes

These approaches are particularly valuable when studying rare variants associated with conditions like bipolar disorder.

How can ADRBK2 antibodies be utilized in research on β-adrenergic receptor systems in intestinal epithelial cells?

Recent research has identified an unexpected role for β2-adrenergic receptors (β2-ARs) in the apical membrane of intestinal epithelial cells, where they function in sugar sensing and glucose uptake stimulation . ADRBK2/GRK3 antibodies can be employed to investigate the regulation of these receptors:

• Localization studies:

  • Immunohistochemistry using β2-AR antibodies shows strong expression at the enterocyte apical membrane

  • Co-localization with SGLT1 (sodium/glucose cotransporter) can be demonstrated with dual immunofluorescence staining

• Regulatory mechanisms:

  • ADRBK2 antibodies can help investigate how GRK3 regulates β2-AR internalization and desensitization in response to glucose or catecholamines

  • Western blotting can assess GRK3 expression levels and phosphorylation state

• Specificity controls:

  • When studying β2-AR in intestinal systems, researchers should note: "Singh et al. (2009) reported the mRNA expression level for β2-AR in murine duodenal epithelial cells, as well as the strong enrichment of β2-ARs by Western blotting in the apical brush border membrane compared to the total cell lysate. Both β2-AR bands (monomer and dimer) were completely blocked in the Western blot using the immunising peptide, showing the specificity of their antibody."

This represents an emerging area where ADRBK2 antibodies could provide valuable insights into novel glucose sensing mechanisms.

What are the critical considerations when using ADRBK2 antibodies in neurological research models?

When applying ADRBK2 antibodies in neurological research:

How can researchers address non-specific bands or background in Western blots with ADRBK2 antibodies?

When encountering non-specific bands or high background in Western blots:

• Antibody selection considerations:
  In their comprehensive validation study, Matthees et al. (2021) found that "Most of the tested antibodies (GRK2: sc-13143, CS #3982; GRK3: CS #80362; GRK5: sc-518005, VPA00469KT; GRK6: CS #5878, PB9709) are able to detect the targeted protein, but some also strongly label background bands with similar protein size leading to difficult interpretation of the expression levels especially at endogenous levels (GRK5: VPA00469KT, GRK6: PB9709)."

• Technical optimization strategies:

  • Titrate antibody concentration to optimal dilution (typically 1:1000-1:4000 for Western blot)

  • Include positive and negative controls (e.g., overexpressed protein, knockout samples)

  • Increase blocking stringency (5% milk or BSA in TBST)

  • Extend washing steps with TBST

  • Consider using secondary antibodies at higher dilutions (e.g., 1:10,000)

• Molecular weight verification:

  • The calculated molecular weight of ADRBK2 is 80 kDa

  • Observed molecular weights may be 70 kDa and 90 kDa

  • Some researchers have reported detection at 39 kDa , which may represent degradation products or isoforms

What optimization approaches are recommended for ADRBK2 antibody application in challenging tissue types?

For challenging tissue types, consider these optimization approaches:

• Antigen retrieval methods:

  • For formalin-fixed tissues, test both TE buffer pH 9.0 and citrate buffer pH 6.0

  • For frozen tissues, optimize fixation time and temperature

• Signal amplification options:

  • Consider biotinylated secondary antibodies with streptavidin-HRP systems

  • Tyramide signal amplification can enhance sensitivity for low abundance targets

• Tissue-specific controls:
  For human Brodmann area 23 (as mentioned in customer queries in result #8):

  • Perform careful validation with positive and negative controls

  • Consider testing the antibody on frozen tissue sections first before proceeding with other applications

  • As noted in the FAQ response: "We have an innovator award program that if you test this antibody and show it works in human brodmann (1909) area 23 in IHC-frozen, you can get your next antibody for free."

• Compatibility verification:

  • Test antibody compatibility with your specific fixation method

  • For diagnostic applications, remember that research antibodies are not validated for this purpose: "The products we sell, including anti-GRK 3 antibody A06422, are only intended for research use. They would not be suitable for use in diagnostic work."