HRH4 Antibody, Biotin conjugated

What is HRH4 and why is it an important research target?

HRH4 (Histamine H4 receptor) is a G-protein coupled receptor (GPCR105) that plays crucial roles in inflammatory and immune responses. This receptor, discovered relatively recently, is considered a promising drug target for allergy, inflammation, autoimmune disorders, and cancer . HRH4 is expressed on various immune cells, particularly macrophages, where it mediates inflammatory reactions including chemotaxis, migration to inflammation sites, phagocytosis, and M1 differentiation .

Recent research has identified HRH4 as having significant pathological importance in conditions like diabetic retinopathy, where HRH4-expressing macrophages contribute to inflammation and vascular leakage . Additionally, functional studies have demonstrated HRH4's involvement in regulating steroidogenesis and proliferation in Leydig cells, suggesting broader physiological roles beyond inflammation . The growing body of evidence positions HRH4 as an important target for both fundamental research and therapeutic development.

What are the key specifications of commercially available HRH4 antibody, biotin conjugated?

Commercial HRH4 antibody, biotin conjugated preparations typically have the following specifications:

These antibodies are typically developed against specific epitopes of the human HRH4 protein and purified to ensure high specificity and minimal cross-reactivity with other histamine receptors .

How should HRH4 antibody, biotin conjugated be stored and handled?

Proper storage and handling are critical for maintaining antibody activity:

• Upon receipt, store the antibody at -20°C or -80°C as recommended by the manufacturer .

• Avoid repeated freeze-thaw cycles as these can compromise antibody integrity and binding capacity. Consider aliquoting the antibody into single-use volumes.

• The presence of 50% glycerol in the buffer helps stabilize the antibody during freeze-thaw transitions, but proper temperature management remains essential .

• When working with the antibody, keep it on ice or at 4°C. Return to frozen storage promptly after use.

• Prior to opening, briefly centrifuge the vial to ensure contents are collected at the bottom.

• For diluted working solutions, prepare fresh on the day of use whenever possible.

• Monitor the expiration date, as biotin conjugation may degrade over time, potentially affecting detection sensitivity.

Following these guidelines will help maintain optimal antibody performance throughout your experimental timeline.

How can HRH4 antibody, biotin conjugated be utilized in ELISA applications?

HRH4 antibody, biotin conjugated is particularly valuable in ELISA applications due to the high-affinity biotin-streptavidin interaction system. Here's a methodological approach for implementing sandwich ELISA:

Protocol Overview:

• Plate Preparation:

  • Coat a high-binding 96-well plate with capture antibody against HRH4 (1-10 μg/ml in carbonate/bicarbonate buffer, pH 9.6)

  • Incubate overnight at 4°C

  • Wash 3 times with washing buffer (PBS-T: PBS with 0.05% Tween-20)

• Blocking:

  • Add blocking buffer (PBS containing 1-5% BSA or non-fat dry milk)

  • Incubate for 1-2 hours at room temperature

  • Wash 3 times with washing buffer

• Sample Application:

  • Add 100 μl of standards or samples to appropriate wells

  • Incubate for 2 hours at 37°C

  • Wash 3 times with washing buffer

• Detection Antibody Application:

  • Add 100 μl of working biotin-conjugated HRH4 antibody

  • Prepare this solution 15 minutes before use by diluting concentrated antibody (typically 100×) in antibody dilution buffer

  • Incubate for 1 hour at 37°C

  • Wash 3 times with washing buffer

• Streptavidin-HRP Addition:

  • Add 100 μl of working streptavidin-HRP solution

  • Incubate for 1 hour at 37°C

  • Wash 3 times with washing buffer

• Substrate Reaction:

  • Add 100 μl of TMB substrate solution

  • Incubate for 15-20 minutes at 37°C under dark conditions

  • Monitor color development

• Reaction Termination and Measurement:

  • Add 50 μl of stop solution (typically 2N H₂SO₄)

  • Measure optical density at 450 nm within 5 minutes

  • Use 570 nm or 630 nm as correction wavelengths

For optimal results, perform antibody titration experiments to determine the ideal concentrations of both capture and detection antibodies. The biotin-conjugated antibody offers enhanced sensitivity through signal amplification when coupled with streptavidin-HRP detection systems.

What are the optimal conditions for using HRH4 antibody in immunoprecipitation studies?

For successful immunoprecipitation studies with biotinylated HRH4 antibody, the following methodological approach is recommended:

Immunoprecipitation Protocol:

• Preparation of Streptavidin-Coated Beads:

  • Add 50 μl of streptavidin-coated magnetic or agarose beads to a microcentrifuge tube

  • Wash beads 3 times with wash buffer (PBS or TBS with 0.1% Tween-20)

  • Resuspend beads in 500 μl buffer

• Antibody Binding to Beads:

  • Add 10 μl of biotinylated HRH4 antibody at optimal dilution to the beads

  • Incubate for 60 minutes at room temperature with gentle shaking

  • Centrifuge at 3,000 × g for 2 minutes at 4°C and discard supernatant

  • Wash beads 3 times with 1 ml buffer to remove unbound antibody

• Sample Preparation:

  • For cell lysates: harvest cells and lyse in a non-denaturing lysis buffer (e.g., RIPA buffer with protease inhibitors)

  • Clarify lysate by centrifugation at 14,000 × g for 10 minutes at 4°C

  • Pre-clear lysate with plain streptavidin beads to reduce non-specific binding

• Antigen Capture:

  • Add 0.1-1.0 ml of pre-cleared cell lysate to the antibody-bound beads

  • Incubate for 90 minutes to overnight at 4°C with gentle mixing

  • Collect immunoprecipitated complexes by centrifugation at 3,000 × g for 2 minutes

  • Wash the pellet thoroughly (at least 4 times) with 1 ml buffer

• Elution and Analysis:

  • For Western blot analysis: add SDS-PAGE sample buffer and heat at 95°C for 5 minutes

  • For functional assays: use appropriate non-denaturing elution methods

This protocol leverages the strong biotin-streptavidin interaction to efficiently capture HRH4 and its binding partners. The biotinylated format eliminates the need for protein A/G in the workflow, potentially reducing background from antibody heavy chains in subsequent Western blot analysis.

How can I optimize immunohistochemistry protocols using HRH4 antibody, biotin conjugated?

Optimizing immunohistochemistry protocols with biotinylated HRH4 antibody requires careful attention to tissue preparation, antigen retrieval, and detection parameters:

Optimized IHC Protocol:

• Tissue Preparation:

  • Fix tissues in 10% neutral buffered formalin for 24-48 hours

  • Process and embed in paraffin following standard protocols

  • Section at 4-6 μm thickness onto adhesive slides

  • For frozen sections, fix briefly in cold acetone or 4% paraformaldehyde

• Deparaffinization and Rehydration (for FFPE sections):

  • Heat slides at 60°C for 1 hour

  • Deparaffinize in xylene (3 × 5 minutes)

  • Rehydrate through graded ethanol series to water

• Antigen Retrieval Optimization:

  • Test multiple methods to determine optimal retrieval:

    • Heat-induced epitope retrieval (HIER): Citrate buffer (pH 6.0), EDTA buffer (pH 8.0), or Tris-EDTA (pH 9.0)

    • Enzymatic retrieval: Proteinase K or trypsin

  • For HIER, use pressure cooker or microwave heating for consistent results

  • Allow slides to cool to room temperature (approximately 20 minutes)

• Endogenous Peroxidase and Biotin Blocking:

  • Block endogenous peroxidase with 3% H₂O₂ in methanol for 10 minutes

  • If using streptavidin-HRP detection, block endogenous biotin using a commercial biotin blocking system

  • This step is critical when using biotinylated antibodies to prevent high background

• Protein Blocking:

  • Block non-specific binding with 5% normal serum (from the same species as secondary antibody) in PBS for 30 minutes

  • For tissues with high background, include 0.1% Triton X-100 and 1% BSA in blocking solution

• Primary Antibody Incubation:

  • Dilute biotinylated HRH4 antibody in blocking buffer (typical range: 1:50-1:500)

  • Perform titration experiments to determine optimal concentration

  • Incubate overnight at 4°C in a humidified chamber

  • For membrane proteins like HRH4, longer incubation periods often yield better results

• Detection Method:

  • Since the antibody is already biotinylated, directly apply streptavidin-HRP (1:100-1:500) for 30 minutes at room temperature

  • Wash thoroughly with PBS-T (3 × 5 minutes)

• Visualization and Counterstaining:

  • Develop with DAB substrate for 2-10 minutes, monitoring microscopically

  • Counterstain with hematoxylin for 30-60 seconds

  • Dehydrate through graded alcohols, clear in xylene, and mount with permanent mounting medium

Critical Optimization Parameters:

• Antibody Dilution: Test a range of dilutions (1:50, 1:100, 1:200, 1:500) to identify optimal signal-to-noise ratio

• Antigen Retrieval: This is often the most critical variable; systematic testing of different methods is essential

• Incubation Time: Adjust primary antibody incubation time (1 hour at RT vs. overnight at 4°C)

• Controls: Include appropriate positive controls (tissues with known HRH4 expression) and negative controls (omission of primary antibody, isotype controls)

Based on the search results, HRH4 has been successfully detected in tissues including retina and testis, making these potentially useful positive controls for protocol optimization .

How can HRH4 antibody be used to investigate its role in diabetic retinopathy?

Recent research has identified HRH4 as playing a significant role in diabetic retinopathy (DR) pathogenesis. The biotinylated HRH4 antibody can be strategically employed to investigate this role through several methodological approaches:

1. Tissue Expression Profiling:

• Perform immunohistochemistry on retinal sections from diabetic models and controls using biotinylated HRH4 antibody with streptavidin-HRP detection

• Quantify changes in HRH4 expression patterns associated with disease progression

• Research has shown that infiltrating macrophages in DR retinas express HRH4, and this expression increases in diabetic conditions

2. Cellular Characterization Studies:

• Use double immunofluorescence with HRH4 antibody and cell-specific markers to identify the precise cell populations expressing HRH4 in diabetic retina

• As found in the research, macrophages are a key cell type expressing HRH4 in this context

3. Mechanistic In Vitro Investigations:

• Isolate bone marrow-derived macrophages (BMDMs) and expose them to high glucose conditions (25 mM) for 48 hours to model diabetic conditions

• Assess HRH4 expression using antibody-based techniques like flow cytometry or immunocytochemistry

• Studies have demonstrated that high glucose upregulates HRH4 mRNA in macrophages, which can be correlated with protein expression

4. Functional Migration Assays:

• Use HRH4 antibody to identify and characterize cells responding to histamine in chemotaxis assays

• Research has shown that macrophages exhibit increased migration in response to histamine via HRH4, and this can be blocked by HRH4 antagonists like JNJ7777120

5. Therapeutic Intervention Assessment:

• Compare HRH4 expression patterns before and after treatment with HRH4 antagonists

• Correlate changes in HRH4+ cell infiltration with improvements in vascular permeability

• Research demonstrates that targeting HRH4 with antagonists reduces inflammation and vascular permeability in DR mouse models

6. Correlation with Clinical Parameters:

• Design studies to correlate HRH4 expression (detected via antibody-based methods) with clinical markers of DR severity

• Investigate potential of HRH4 as a biomarker for disease progression or treatment response

This multifaceted approach using HRH4 antibody allows for comprehensive characterization of receptor expression, cellular localization, and functional significance in diabetic retinopathy pathogenesis, supporting the development of HRH4-targeted therapeutic strategies.

What validation strategies ensure reliable results with HRH4 antibody?

Rigorous validation is essential to ensure reliable results with HRH4 antibody. A comprehensive validation strategy includes:

1. Genetic Validation Approaches:

• Knockout Controls: Use HRH4 knockout samples as negative controls. Search result mentions a KO-validated HRH4 antibody, indicating the feasibility of this approach.

• siRNA/shRNA Validation: Knockdown HRH4 expression in cell culture models and demonstrate corresponding reduction in antibody signal.

• Overexpression Systems: Transfect cells with HRH4-expressing vectors and confirm increased antibody binding.

2. Biochemical Validation Methods:

• Peptide Competition: Pre-incubate HRH4 antibody with excess immunizing peptide (e.g., the 204-292AA fragment mentioned in ) before application to samples. Specific binding should be significantly reduced.

• Western Blot Analysis: Confirm detection of appropriately sized bands matching the predicted molecular weight of HRH4 (~44 kDa for full-length protein).

• Mass Spectrometry Confirmation: Perform immunoprecipitation using biotinylated HRH4 antibody followed by protein identification via mass spectrometry.

3. Comparative Analysis:

• Multi-antibody Approach: Compare staining patterns using different antibodies targeting distinct HRH4 epitopes.

• Correlation with mRNA Expression: Align antibody-based protein detection with HRH4 mRNA levels measured by RT-PCR or RNA-seq.

• Cross-Species Validation: If claimed to be cross-reactive, systematically validate in each species separately.

4. Functional Correlation:

• Pharmacological Approach: Correlate antibody detection with functional responses to HRH4-specific agonists or antagonists like JNJ7777120 mentioned in the research .

• Signaling Pathway Analysis: Verify that cells positive for HRH4 by antibody staining respond appropriately in downstream signaling assays.

5. Technical Controls and Optimization:

• Titration Series: Determine optimal antibody concentration where specific signal is maximized and background is minimized.

• Isotype Controls: Use matched isotype antibodies to establish background signal levels.

• Application-Specific Controls: For each experimental technique (IHC, WB, ELISA, etc.), include appropriate positive and negative controls.

Example Validation Workflow for Biotinylated HRH4 Antibody:

• Perform Western blot analysis on lysates from cells with verified HRH4 expression (e.g., BMDMs cultured in high glucose )

• Compare with knockout or knockdown samples to confirm specificity

• Conduct peptide competition assays to verify epitope-specific binding

• Optimize staining conditions across multiple applications (IHC, ICC, ELISA)

• Correlate antibody staining with functional assays (e.g., histamine-induced migration )

This systematic validation ensures that experimental findings accurately reflect HRH4 biology rather than artifacts of non-specific antibody binding.

How can I use HRH4 antibody to study its role in inflammatory macrophage responses?

HRH4 plays a critical role in macrophage-mediated inflammatory responses. The following methodological approaches using biotinylated HRH4 antibody can elucidate these functions:

1. Expression Analysis Under Inflammatory Conditions:

• Stimulus-Dependent Regulation: Treat macrophages with different inflammatory stimuli and assess HRH4 expression:

  • High glucose (25 mM): Research shows this upregulates HRH4 mRNA in bone marrow-derived macrophages (BMDMs)

  • LPS: Studies indicate this may not affect HRH4 expression

  • Pro-inflammatory cytokines (TNF-α, IL-1β, IL-6)

  • Histamine exposure

• Detection Methods:

  • Flow cytometry using biotinylated HRH4 antibody with streptavidin-fluorophore conjugates

  • Immunocytochemistry to visualize receptor localization

  • Western blotting for quantitative expression analysis

  • ELISA for high-throughput screening

2. Functional Migration and Chemotaxis Assays:

• Transwell Migration Assay:

  • Seed macrophages in upper chambers of transwell plates

  • Add histamine to lower chambers as chemoattractant

  • Pre-treat cells with HRH4 antagonist JNJ7777120 as control

  • After migration, fix and stain migrated cells with crystal violet

  • Quantify by cell counting or colorimetric measurement

  • Research demonstrates that macrophages show increased migration in response to histamine, which is blocked by HRH4 antagonism

• Real-time Cell Migration Analysis:

  • Use live-cell imaging systems to track macrophage movement toward histamine gradients

  • Correlate migration behavior with HRH4 expression detected via antibody staining

3. Macrophage Polarization Studies:

• M1/M2 Polarization Assessment:

  • Induce M1 (classical) or M2 (alternative) activation in macrophages

  • Measure HRH4 expression in each polarization state using biotinylated antibody

  • Research suggests HRH4 may play a role in M1 differentiation

• Gene Expression Correlation:

  • Perform antibody-based cell sorting to isolate HRH4-high and HRH4-low macrophage populations

  • Compare inflammatory gene expression profiles via qPCR or RNA-seq

4. Phagocytosis Functional Assays:

• Fluorescent Particle Uptake:

  • Treat macrophages with histamine or HRH4-specific agonists/antagonists

  • Incubate with fluorescent particles (e.g., latex beads, zymosan)

  • Quantify phagocytosis via flow cytometry or microscopy

  • Correlate phagocytic activity with HRH4 expression detected by antibody staining

5. Tissue Infiltration Models:

• In Vivo Models:

  • Induce inflammatory conditions in animal models (e.g., diabetic retinopathy model using streptozotocin )

  • Use biotinylated HRH4 antibody for immunohistochemistry to identify infiltrating HRH4+ macrophages

  • Correlate macrophage infiltration with tissue pathology

• Co-localization Analysis:

  • Perform dual immunostaining for HRH4 and macrophage markers (F4/80, CD68)

  • Assess co-localization with inflammatory mediators or tissue damage markers

These methodological approaches using biotinylated HRH4 antibody can provide comprehensive insights into the receptor's role in regulating macrophage inflammatory responses, potentially identifying new therapeutic targets for inflammatory disorders.

What are common troubleshooting strategies for biotin-conjugated antibody applications?

When working with biotinylated HRH4 antibody, several technical challenges may arise. Here are methodological solutions to common problems:

1. High Background in Streptavidin-Based Detection Systems:

Problem: Non-specific binding or high background signal compromising result interpretation.

Solutions:

• Endogenous Biotin Blocking: Tissues often contain endogenous biotin, particularly liver, kidney, and adipose tissue. Use commercial biotin blocking kits before applying biotinylated antibodies.

• Streptavidin Optimization: Titrate streptavidin-HRP or streptavidin-fluorophore conjugates to identify minimal effective concentration.

• Buffer Optimization: Include 0.1-0.3% Triton X-100 in washing buffers to reduce hydrophobic interactions .

• Alternative Blocking Agents: Test different blockers (BSA, casein, commercial blockers) to identify optimal formulation for your specific tissue or cell type.

• Avidin/Biotin Pre-absorption: For particularly problematic samples, pre-absorb with avidin followed by biotin before applying biotinylated antibodies.

2. Reduced Signal Intensity:

Problem: Weak or undetectable signal despite confirmed target presence.

Solutions:

• Storage Assessment: Biotin conjugation may degrade over time. Check antibody age and storage conditions; biotinylated antibodies should be stored at -20°C or -80°C .

• Antigen Retrieval Optimization: For FFPE tissues, systematically test different antigen retrieval methods (heat-induced with varying buffers, enzymatic).

• Signal Amplification: Implement tyramide signal amplification (TSA) or other amplification systems compatible with biotin-streptavidin detection.

• Concentration Adjustment: Increase biotinylated antibody concentration incrementally while monitoring background.

• Incubation Modifications: Extend primary antibody incubation time or adjust temperature (4°C overnight instead of room temperature for 1-2 hours).

3. Inconsistent Results Across Experiments:

Problem: Variable staining intensity or patterns between experimental replicates.

Solutions:

• Standardized Protocols: Develop detailed protocols with precisely defined incubation times, temperatures, and reagent concentrations.

• Positive Controls: Include consistent positive control samples in each experiment to validate staining efficiency.

• Single-Batch Reagents: When possible, use the same lot of streptavidin conjugates and substrate reagents across experiments.

• Environmental Control: Maintain consistent temperature and humidity during incubation steps, particularly for temperature-sensitive detection systems.

• Automated Systems: Consider using automated staining platforms for improved reproducibility.

4. Cross-Reactivity Issues:

Problem: Antibody binds to proteins other than HRH4, generating misleading results.

Solutions:

• Validation in Multiple Systems: Confirm specificity using complementary techniques (Western blot, IP-MS) before proceeding with IHC or ICC applications.

• Absorption Controls: Pre-absorb antibody with recombinant HRH4 protein to confirm specificity.

• Genetic Controls: Include HRH4 knockout samples as negative controls when available .

• Peptide Competition: Perform side-by-side staining with antibody pre-incubated with immunizing peptide (204-292AA region) .

5. Technical Considerations for Special Applications:

Problem: Special applications require tailored optimization.

Solutions:

• Flow Cytometry: Use indirect methods with biotinylated primary antibody followed by streptavidin-fluorophore rather than direct conjugates for better sensitivity.

• Multiplex Immunostaining: When combining with other antibodies, use non-biotin detection systems for second primary antibody to avoid cross-reaction.

• Super-Resolution Microscopy: Consider specialized streptavidin-fluorophore conjugates optimized for nanoscopy applications.

These troubleshooting strategies represent a methodological framework for optimizing biotinylated HRH4 antibody applications across various experimental platforms.

How can I optimize Western blotting protocols for HRH4 detection?

Western blotting for HRH4 detection requires specific optimization due to the nature of this multi-pass transmembrane protein. Here's a comprehensive methodological approach:

Sample Preparation Optimization:

• Effective Membrane Protein Extraction:

  • Use specialized membrane protein extraction buffers containing 1-2% detergents (RIPA buffer with 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS)

  • Include protease inhibitor cocktail to prevent degradation

  • For difficult samples, consider using commercial membrane protein extraction kits

• Sample Denaturation Conditions:

  • Avoid boiling samples (which can cause membrane protein aggregation)

  • Heat at 37°C for 30 minutes or 65°C for 5-10 minutes in Laemmli buffer

  • Include reducing agents (β-mercaptoethanol or DTT) to disrupt disulfide bonds

Gel Electrophoresis Parameters:

• Optimal Gel Percentage:

  • Use 10-12% polyacrylamide gels for best resolution of HRH4 (predicted MW ~44 kDa)

  • Consider gradient gels (4-15%) if analyzing multiple proteins of different sizes

• Loading Controls:

  • Include membrane protein-specific loading controls (e.g., Na⁺/K⁺-ATPase, cadherin)

  • Standard housekeeping proteins (β-actin, GAPDH) may not accurately reflect membrane protein loading

Transfer Optimization:

• Transfer Conditions:

  • Use wet transfer for membrane proteins

  • Transfer at lower voltage (30V) for longer time (overnight at 4°C)

  • Add 0.05% SDS to transfer buffer to facilitate movement of hydrophobic proteins

  • Use PVDF membrane (0.2 μm pore size) instead of nitrocellulose for better protein retention

Detection Protocol:

• Blocking Optimization:

  • Test different blocking solutions (5% non-fat milk vs. 5% BSA in TBS-T)

  • BSA is often superior for phospho-specific antibodies or when using biotin-streptavidin systems

• Primary Antibody Incubation:

  • Dilute biotinylated HRH4 antibody in blocking buffer (recommended range: 1:500-1:2000)

  • Incubate overnight at 4°C with gentle rocking

  • Perform extended washing steps (5 × 5 minutes with TBS-T)

• Detection System:

  • Apply streptavidin-HRP (1:2000-1:5000) for 1 hour at room temperature

  • For enhanced sensitivity, use high-sensitivity ECL substrates

  • Consider advanced detection systems like Clarity™ Western ECL Substrate for low abundance proteins

Special Considerations for Biotinylated Antibodies:

• Endogenous Biotin Management:

  • Be aware that some tissues (especially liver, kidney) contain high levels of endogenous biotin

  • Consider pre-blocking with streptavidin followed by free biotin before applying biotinylated antibody

• Enhanced Chemiluminescence Exposure:

  • Start with short exposure times (10-30 seconds) and gradually increase if needed

  • Use multiple exposure times to capture optimal signal without saturation

Controls and Validation:

• Positive Control:

  • Include lysates from cells known to express HRH4 (e.g., macrophages cultured in high glucose conditions)

  • Consider using recombinant HRH4 protein as a reference standard

• Specificity Validation:

  • Perform peptide competition assays with the immunogen peptide (204-292AA)

  • If available, include samples from HRH4 knockout models as negative controls

• Band Identification:

  • HRH4 may show multiple bands due to post-translational modifications, glycosylation, or splice variants

  • The expected molecular weight is approximately 44 kDa, but additional bands at 35-50 kDa may represent modified forms

This optimized Western blotting protocol should enable reliable detection of HRH4 protein while mitigating common challenges associated with membrane protein analysis and biotinylated antibody detection systems.