GHRHR Antibody, HRP conjugated

What are the primary research applications for GHRHR antibodies in growth hormone pathway studies?

GHRHR antibodies are versatile tools for investigating growth hormone regulation in multiple experimental contexts. Current evidence supports their application in:

• Western Blotting: Detection of GHRHR protein (~47-48 kDa) has been validated in various cell lines including HEK-293, LOVO, and pituitary cells . HRP-conjugated secondary antibodies allow for sensitive detection when using primary anti-GHRHR antibodies.

• Immunohistochemistry: Successful detection in paraffin-embedded pituitary tissue sections, where GHRHR shows cytoplasmic localization .

• Immunofluorescence: Detection in transfected cell lines and native tissues expressing GHRHR .

• ELISA: Quantitative measurement of GHRHR in biological samples .

When performing Western blot analysis, typical GHRHR detection requires antibody dilutions of 1:500-1:1000, with optimal results observed at concentrations of 3-4 μg/mL in most applications .

Where is GHRHR primarily expressed, and what are the considerations for antibody-based detection across different tissues?

GHRHR expression demonstrates tissue-specific patterns that researchers should consider when designing experiments:

For optimal detection in tissue sections, antigen retrieval is critical. GHRHR detection in pituitary tissue typically requires heat-induced epitope retrieval using basic pH buffers (pH 9.0) . For kidney tissue, both TE buffer (pH 9.0) and citrate buffer (pH 6.0) have shown efficacy, though results may vary based on fixation methods .

What controls should be included when using GHRHR antibodies in experimental protocols?

Rigorous control implementation is essential for valid GHRHR detection experiments:

• Positive controls: Transfected HEK293 cells expressing GHRHR show specific staining in cytoplasm and serve as excellent positive controls .

• Negative controls: Wild-type (non-transfected) cells demonstrate minimal background staining . Rat liver tissue has also been validated as a negative control for GHRHR expression .

• Peptide competition assays: Co-incubation with immunizing peptide has been shown to effectively neutralize specific GHRHR antibody binding, confirming specificity .

• Cross-reactivity controls: Testing against other G-protein coupled receptors to ensure specificity, particularly when detecting specific GHRHR isoforms.

Experimental validation shows that GHRHR antibody (1:500 dilution) exhibits specific 47 kDa bands in positive control tissues that are eliminated when pre-incubated with immunizing peptide .

How does HRP conjugation affect GHRHR antibody performance in different detection systems?

HRP conjugation provides several advantages in GHRHR detection systems:

• Direct conjugation: Primary anti-GHRHR antibodies directly conjugated with HRP eliminate the need for secondary antibodies, reducing background and cross-reactivity.

• Indirect detection: When using unconjugated primary GHRHR antibodies, Anti-Rabbit IgG VisUCyte™ HRP Polymer Antibodies have shown excellent results at standard concentrations .

• Signal amplification: HRP-based amplification systems increase sensitivity for detecting low abundance GHRHR in certain tissues.

• Colorimetric detection: DAB (3,3'-diaminobenzidine) substrate with HRP-conjugated antibodies produces brown precipitate for GHRHR localization, which contrasts well with hematoxylin counterstain (blue) .

For IHC applications, incubation with primary GHRHR antibody at 3 μg/mL for 1 hour at room temperature followed by HRP-conjugated secondary antibody has shown optimal results .

How do researchers address species-specific variations when using GHRHR antibodies across different experimental models?

GHRHR sequence conservation varies across species, requiring careful antibody selection:

The C-terminal intracellular region (392-404) of rat GHRHR exhibits 85% sequence identity with human GHRHR, making antibodies targeting this region potentially useful for cross-species applications . When studying porcine models, researchers have developed specific antibodies (e.g., AN98) through hybridoma techniques that do not cross-react with other species .

How can researchers distinguish between GHRHR splice variants using antibody-based approaches?

GHRHR exists in multiple splice variants that can be distinguished with strategic antibody selection:

• Main transcript (GHRHR): Forms through connection of exons 12 and 13; detected by most commercial antibodies .

• GHRHR SV1: Forms through connection of exons 12 and 14; may require variant-specific antibodies .

• GHRHR SV2: Contains all 14 exons; distinguishable by size in Western blot analysis .

Research has demonstrated that let-7e and miR-328-5p target different GHRHR splice variants, which affects GH synthesis through different signaling pathways . When designing experiments to distinguish these variants:

• Use antibodies targeting unique epitopes in the variant-specific regions

• Employ RT-PCR to confirm variant expression alongside protein detection

• Correlate antibody detection with functional assays to confirm biological activity of specific variants

What are common challenges in GHRHR antibody experiments and how can they be resolved?

Researchers frequently encounter several technical challenges when working with GHRHR antibodies:

For Western blots showing multiple bands, researchers should note that GHRHR can appear at several molecular weights: 47-48 kDa (main form), 44 kDa, and 65 kDa in rat anterior pituitary, or 52-55 kDa in human anterior pituitary preparations . Cross-linking experiments with radioactive ligands can help confirm which bands represent functional GHRHR.

How can GHRHR antibodies be used to investigate downstream signaling pathways?

GHRHR activates multiple signaling cascades that can be monitored using phospho-specific antibodies in conjunction with GHRHR detection:

• cAMP/PKA pathway: The primary signaling route activated by GHRHR involves adenylyl cyclase activation. Researchers can use phospho-CREB antibodies to monitor this pathway alongside GHRHR detection .

• NO/NOS signaling: Evidence indicates GHRHR also signals through nitric oxide pathways, which can be monitored with appropriate antibodies against phosphorylated NOS .

• GPR101 interaction: Recent research has demonstrated connection between GHRHR and GPR101 in growth regulation, suggesting multiplex studies with antibodies against both targets .

Experimental design should consider temporal aspects of signaling activation. For example, when investigating GHRHR-mediated signaling, researchers typically collect samples at multiple time points (5, 10, 15, 30 minutes) after GHRH stimulation to capture the full signaling profile .

How do GHRHR mutations affect antibody binding and experimental interpretation?

GHRHR mutations, particularly those causing isolated growth hormone deficiency (IGHD), can significantly impact antibody binding:

• p.Glu72 mutation*: This common founder mutation creates a premature termination codon resulting in a truncated protein that lacks most of the receptor . Antibodies targeting epitopes after this position will fail to detect the mutant protein.

• C-terminal mutations: Antibodies targeting the C-terminus (like those against the 392-404 region) will not detect truncated receptors but can be useful for distinguishing between full-length and truncated variants .

• Transmembrane domain mutations: Can alter receptor conformation and potentially mask antibody epitopes even when the sequence is present.

When studying patient samples with suspected GHRHR mutations, researchers should use antibodies targeting different regions of the receptor and correlate findings with genetic analysis. For the p.Glu72* mutation, antibodies targeting the N-terminal region would be required to detect the truncated protein .

What role do GHRHR antibodies play in developing growth disorder therapeutics?

GHRHR antibodies serve several critical functions in therapeutic research:

• Antagonist development: Specific GHRHR antibody antagonists have been developed to inhibit GH secretion, offering potential therapies for acromegaly or gigantism .

• Target validation: HRP-conjugated GHRHR antibodies help validate target engagement in drug discovery programs.

• Biomarker identification: Detection of GHRHR expression levels can help stratify patients for clinical trials targeting growth disorders.

• Receptor-binding studies: Competition assays between therapeutic candidates and GHRHR antibodies can characterize binding sites and affinities.

Research has shown that antibody antagonists like AN98 can effectively inhibit GH-induced signaling in pituitary cells by competing with natural ligands for receptor binding . This approach provides important insights for developing non-antibody small molecule therapeutics targeting the same pathway.

What are the best practices for quantitative measurement of GHRHR using antibody-based assays?

Accurate quantification of GHRHR requires rigorous standardization:

• Antibody titration: Establish optimal antibody concentration through serial dilution experiments. For Western blot, a range of 1:500-1:1000 is typically effective .

• Standard curves: Include recombinant GHRHR standards when possible to establish quantitative relationships.

• Image analysis: For IHC or IF, use standardized image acquisition parameters and analysis software that allows for consistent thresholding and quantification.

• Normalization controls: Include housekeeping proteins (for Western blot) or reference tissues (for IHC) to normalize expression data.

Studies have shown that changes in GHRHR expression can be reliably quantified using immunoblotting. For example, a 3-week antithyroid treatment decreased GHRHR protein (47-kDa form) by 3.5-fold and the 65-kDa form by 1.25-fold, which correlated with changes in binding site concentration .

What emerging technologies are enhancing GHRHR antibody applications in research?

Several technological advances are expanding the utility of GHRHR antibodies:

• Super-resolution microscopy: Allows precise subcellular localization of GHRHR beyond traditional fluorescence techniques.

• Proximity ligation assays: Enable detection of GHRHR interactions with other proteins in the signaling complex.

• Automated multiplex IHC: Facilitates simultaneous detection of GHRHR with multiple markers in the same tissue section.

• CRISPR-engineered reporter cell lines: Provide clean systems for antibody validation and functional studies.