ORM1 Antibody, FITC conjugated

What is ORM1 and why is it a target for antibody development?

ORM1 (Alpha-1-acid glycoprotein 1/Orosomucoid-1) is an acute phase protein that plays significant roles in immune regulation and disease processes. It functions as a reactant to acute inflammation and has been identified as a key protein in fatigue response mechanisms . The development of antibodies targeting ORM1 has become crucial because recent research has demonstrated that ORM1 binds to CCR5 receptors on muscle cells, enhancing muscle glycogen content and endurance . Additionally, ORM1 has been implicated in cancer progression, particularly in kidney renal clear cell carcinoma (KIRC), where higher expression levels correlate with worse survival probability . These diverse physiological and pathological roles make ORM1 a valuable target for antibody development to further investigate its mechanisms and potential therapeutic applications.

What are the key specifications of commercially available ORM1 antibody, FITC conjugated?

The commercially available ORM1 antibody, FITC conjugated, is a polyclonal antibody raised in rabbits using recombinant Human Alpha-1-acid glycoprotein 1 protein (19-201AA) as the immunogen . This antibody specifically targets human ORM1 (UniprotID: P02763) and is purified using Protein G purification methods with a purity exceeding 95% . The antibody is of IgG isotype and is provided in liquid form with a storage buffer containing 0.03% Proclin 300 as preservative, 50% Glycerol, and 0.01M PBS at pH 7.4 .

The FITC conjugation enables fluorescence visualization in applications such as ELISA, flow cytometry, and cellular binding studies . For optimal results, the antibody should be stored at -20°C or -80°C upon receipt, and repeated freeze-thaw cycles should be avoided to maintain antibody integrity and functionality .

How does ORM1 antibody, FITC conjugated differ from other ORM1 detection methods?

ORM1 antibody, FITC conjugated provides distinct advantages over other ORM1 detection methods primarily through its ability to enable direct visualization of ORM1 binding in live cells and tissues. Unlike unconjugated antibodies that require secondary detection systems, the FITC conjugation allows for one-step detection, reducing experimental complexity and potential cross-reactivity issues .

The fluorescent properties of FITC permit real-time monitoring of ORM1 interactions with cellular components, as demonstrated in studies where FITC-ORM1 binding to skeletal muscle cell membranes was visualized and quantified using flow cytometry . This approach has proven valuable in elucidating how ORM1 binds to CCR5 receptors on muscle cells, with the binding being inhibited by both excessive unlabeled ORM1 and CCR5 antagonists like Maraviroc .

Compared to mRNA detection methods or protein quantification through ELISA or Western blotting, FITC-conjugated antibodies provide spatial information about protein localization and direct evidence of receptor-ligand interactions. This capability has been instrumental in advancing our understanding of ORM1's functional mechanisms in muscle endurance and disease processes .

What are the optimal conditions for FITC conjugation to ORM1 antibodies?

The optimal conditions for FITC conjugation to antibodies, including ORM1 antibodies, require careful control of several key parameters to achieve maximal labeling efficiency without compromising antibody functionality. Based on extensive research on FITC conjugation methods, the following conditions yield optimal results:

• pH Conditions: A reaction pH of 9.5 provides the most favorable environment for FITC conjugation, as it enhances the reactivity of amino groups on the antibody with the isothiocyanate group of FITC .

• Temperature: Room temperature (approximately 20-25°C) is optimal for the conjugation reaction .

• Protein Concentration: An initial protein concentration of 25 mg/ml has been determined to achieve maximal labeling efficiency .

• Reaction Time: Under the above conditions, maximal labeling is typically obtained within 30-60 minutes .

• Starting Material Quality: Using a relatively pure IgG preparation, preferably obtained through DEAE Sephadex chromatography, and high-quality FITC significantly improves conjugation outcomes .

After conjugation, separating optimally labeled antibodies from under- and over-labeled proteins is critical, which can be achieved through gradient DEAE Sephadex chromatography . This process ensures that the final preparation contains antibodies with appropriate fluorescein/protein (F/P) ratios, maintaining both fluorescent properties and antigen-binding capacity.

How can researchers verify the specificity of ORM1 antibody, FITC conjugated in experimental settings?

Verifying the specificity of ORM1 antibody, FITC conjugated requires a multi-faceted approach to ensure reliable experimental results. Researchers should implement the following validation strategies:

• Competitive Binding Assays: Perform competition experiments using excess unlabeled ORM1 antibody or recombinant ORM1 protein. In specific binding, the FITC-labeled antibody signal should be significantly reduced or eliminated when competed with excess unlabeled counterparts . Studies have demonstrated that FITC-ORM1 binding to skeletal muscle cell membranes can be inhibited completely by excessive unlabeled ORM1, confirming specificity .

• Negative Control Cells: Utilize cell lines known not to express ORM1 or with ORM1 knockdown. Flow cytometry analysis comparing binding patterns between positive and negative control cells can demonstrate antibody specificity .

• Receptor Blocking Studies: When investigating receptor-mediated interactions, employ specific receptor antagonists or blocking antibodies. For instance, ORM1 binding to muscle cells has been shown to be inhibited by CCR5 antagonist Maraviroc and anti-CCR5 antibodies, validating the specificity of the ORM1-CCR5 interaction .

• Western Blot Correlation: Compare immunofluorescence patterns with Western blot results using the same antibody to ensure consistent target recognition across different techniques .

• siRNA Knockdown Validation: Generate ORM1 knockdown cells using siRNA and verify reduced binding of the FITC-conjugated antibody, as demonstrated in studies where ORM1 was knocked down in KIRC cell lines .

By implementing these validation approaches, researchers can confidently establish the specificity of their ORM1 antibody, FITC conjugated, enhancing the reliability and interpretability of their experimental findings.

What flow cytometry protocols are recommended for ORM1 antibody, FITC conjugated?

When utilizing ORM1 antibody, FITC conjugated for flow cytometry, the following comprehensive protocol is recommended based on successful experimental approaches in published research:

Sample Preparation:

• Harvest cells of interest (e.g., primary skeletal muscle cells, U937 monocytes, or KIRC cell lines) through gentle trypsinization for adherent cells or centrifugation for suspension cells .

• Wash cells twice with ice-cold PBS to remove media components.

• Adjust cell concentration to 1 × 10^6 cells/ml in flow cytometry buffer (PBS containing 2% FBS and 0.1% sodium azide).

Staining Procedure:

• Aliquot 100 μl of cell suspension (1 × 10^5 cells) into flow cytometry tubes.

• Add FITC-conjugated ORM1 antibody at the manufacturer's recommended concentration (typically 1-5 μg/ml) and incubate for 30 minutes at 4°C in the dark .

• For competition assays, pre-incubate cells with unlabeled ORM1 (10-fold excess) for 15 minutes before adding FITC-ORM1 antibody .

• For receptor blocking experiments, pre-incubate cells with receptor antagonists (e.g., Maraviroc for CCR5) or blocking antibodies for 30 minutes before adding FITC-ORM1 antibody .

• Wash cells twice with flow cytometry buffer by centrifugation at 400g for 5 minutes.

• Resuspend cell pellet in 500 μl of flow cytometry buffer.

• Add a viability dye (e.g., propidium iodide at 1 μg/ml) immediately before analysis to exclude dead cells.

Instrument Settings and Analysis:

• Set up flow cytometer by using unstained cells to adjust forward and side scatter parameters.

• Use appropriate single-color controls to set compensation if multiple fluorochromes are used.

• Analyze FITC signal in the FL1 channel (typically 530/30 nm bandpass filter).

• Collect at least 10,000 events per sample within the viable cell gate.

• Compare median fluorescence intensity (MFI) between experimental and control samples.

• For binding affinity studies, perform titration experiments with increasing concentrations of FITC-ORM1 antibody to generate saturation curves .

This protocol has successfully demonstrated specific binding of FITC-ORM1 to skeletal muscle cells and U937 cells, with binding inhibition observed in competition assays with unlabeled ORM1 and CCR5 antagonists .

How has ORM1 antibody, FITC conjugated contributed to our understanding of fatigue mechanisms?

ORM1 antibody, FITC conjugated has been instrumental in elucidating the molecular mechanisms underlying fatigue, particularly through its ability to visualize and quantify ORM1-receptor interactions in skeletal muscle cells. Research using this tool has led to several groundbreaking discoveries:

Firstly, studies utilizing FITC-conjugated ORM1 demonstrated that ORM1 specifically binds to skeletal muscle cell membranes, with this binding being completely inhibitable by excessive unlabeled ORM1 . This finding was critical in establishing a direct link between circulating ORM1 and muscle tissue, suggesting a potential mechanism for systemic-to-local signaling in fatigue response.

Flow cytometry analysis with FITC-ORM1 further revealed that all skeletal muscle cells bind specifically with ORM1, indicating a widespread sensitivity of muscle tissue to this protein . This discovery helped explain how elevated ORM1 levels during fatigue could broadly impact muscle function throughout the body.

Perhaps most significantly, FITC-labeled ORM1 binding experiments with receptor blocking identified CCR5 as the receptor through which ORM1 exerts its effects on muscle cells . Flow cytometry analysis showed that FITC-ORM1 binding to U937 cells (which express functional CCR5) was significantly inhibited by an anti-CCR5 antibody and by the CCR5 antagonist Maraviroc . Similar inhibition was observed with primary skeletal muscle cells, confirming that ORM1 acts through CCR5 on muscle cells .

These findings collectively established a novel pathway: fatigue induces increased ORM1 levels, which then bind to CCR5 on muscle cells to enhance muscle glycogen content and endurance—representing a positive feedback mechanism to resist fatigue and maintain homeostasis . This understanding has opened potential new avenues for therapeutic intervention in fatigue management through modulation of the ORM1/CCR5 system.

What is the relationship between ORM1, CCR5, and muscle endurance as revealed through FITC-conjugated studies?

The relationship between ORM1, CCR5, and muscle endurance has been elegantly delineated through studies employing FITC-conjugated techniques, revealing a functional pathway with significant implications for fatigue management. Research has established a comprehensive mechanistic framework:

FITC-ORM1 binding experiments demonstrated that ORM1 directly interacts with the skeletal muscle cell membrane . Flow cytometry analysis confirmed this binding was specific and occurred across all skeletal muscle cells, indicating a ubiquitous muscle response system to ORM1 . The critical discovery came when researchers found that this binding could be partially inhibited by both the CCR5 antagonist Maraviroc and anti-CCR5 antibodies, definitively identifying CCR5—previously known primarily for its role in inflammation and immune response—as the receptor through which ORM1 exerts its effects on muscle cells .

Functionally, this ORM1-CCR5 interaction has been shown to increase muscle glycogen content, a factor directly related to muscle endurance . The molecular pathway appears to involve enhanced glucose uptake, as ORM1 has been reported to increase glucose uptake in adipocytes, and CCR5 activation enhances glucose uptake in activated T cells . This suggests that ORM1 may activate CCR5 on muscle cells to increase glucose uptake, thereby elevating glycogen content and enhancing endurance .

Supporting this mechanism, administration of exogenous ORM1 increased muscle glycogen and enhanced muscle endurance, while ORM1 deficiency resulted in significantly decreased muscle endurance both in vivo and in vitro . Notably, the endurance deficits in ORM1-deficient models could largely be restored by exogenous ORM1, confirming the causal relationship .

The physiological relevance of this pathway is further underscored by the observation that one of the major side effects of CCR5 antagonists used in HIV treatment is fatigue . This clinical finding provides additional evidence that the ORM1-CCR5 axis plays a significant role in regulating muscle endurance and fatigue resistance in humans.

How can researchers design experiments to investigate ORM1's role in fatigue using FITC-conjugated antibodies?

Researchers investigating ORM1's role in fatigue using FITC-conjugated antibodies should design comprehensive experiments that address both molecular interactions and physiological outcomes. The following experimental approach is recommended:

1. Fatigue Model Characterization and ORM1 Expression Analysis:

• Establish appropriate fatigue models (e.g., exercise-induced, sleep deprivation, or disease-related fatigue) .

• Quantify ORM1 expression in multiple tissues (blood, muscle, liver) using standard immunoassays.

• Correlate ORM1 levels with objective fatigue markers and subjective fatigue assessments.

2. ORM1-Receptor Interaction Studies:

• Isolate primary skeletal muscle cells from relevant models (human biopsies or animal models) .

• Incubate cells with FITC-conjugated ORM1 and visualize binding using confocal microscopy to determine subcellular localization.

• Perform flow cytometry analysis with FITC-ORM1 to quantify binding parameters (Kd, Bmax) .

• Include competitive binding experiments with unlabeled ORM1 to confirm specificity .

• Conduct receptor blocking studies using anti-CCR5 antibodies and CCR5 antagonists (e.g., Maraviroc) to confirm receptor engagement .

3. Downstream Signaling Investigation:

• Examine changes in glucose uptake, glycogen synthesis, and energy metabolism pathways following ORM1 treatment.

• Use fluorescent glucose analogs in combination with FITC-ORM1 to simultaneously track receptor binding and glucose uptake.

• Analyze CCR5 pathway activation through phosphorylation of downstream targets.

4. Functional Outcome Assessment:

• Design in vitro muscle endurance assays using electrical stimulation of muscle cells treated with or without ORM1.

• Measure glycogen content in muscle cells after various treatments (ORM1, ORM1 + CCR5 antagonists) .

• Perform in vivo studies with FITC-ORM1 to track biodistribution in fatigued vs. non-fatigued animals.

• Utilize ORM1 knockout or knockdown models with subsequent rescue experiments using exogenous ORM1 .

5. Translational Correlation Studies:

• Collect human samples from subjects with various fatigue conditions.

• Perform ex vivo binding studies with FITC-ORM1 on muscle biopsies from fatigued vs. non-fatigued individuals.

• Correlate CCR5 expression levels with ORM1 binding capacity and clinical fatigue parameters.

6. Therapeutic Intervention Design:

• Test ORM1 mimetics or CCR5 pathway modulators in fatigue models.

• Use FITC-conjugated compounds to verify target engagement before proceeding to functional studies.

This comprehensive experimental approach leverages FITC-conjugated antibodies to establish not only molecular interactions but also their functional relevance to fatigue mechanisms, potentially identifying novel therapeutic targets for fatigue management.

What is the significance of ORM1 in kidney renal clear cell carcinoma progression?

ORM1 has emerged as a significant factor in kidney renal clear cell carcinoma (KIRC) progression, with research utilizing tools such as FITC-conjugated antibodies revealing its multifaceted roles in cancer development and potential therapeutic implications.

Analysis of TCGA database has demonstrated that ORM1 protein expression is significantly elevated in primary KIRC tumor tissues compared to normal tissues . This overexpression appears to be regulated at the epigenetic level, as the methylation level of the ORM1 promoter is substantially lower in tumor tissues than in normal tissues, suggesting that hypomethylation contributes to increased ORM1 expression in KIRC .

The clinical significance of ORM1 overexpression is substantial—patients with high ORM1 expression exhibit significantly worse survival probability compared to those with low/medium expression . This correlation establishes ORM1 as a potential prognostic biomarker for KIRC patient outcomes.

At the cellular level, functional studies involving ORM1 knockdown in KIRC cell lines (786-O and Caki-2) have revealed that ORM1 plays crucial roles in multiple cancer-promoting processes . When ORM1 was knocked down, cell proliferation was potently inhibited compared to controls—at 72 hours, proliferation in ORM1-knockdown cells was reduced to approximately 53.7% (786-O) and 51.9% (Caki-2) of the control levels . Additionally, cell migration and invasion capabilities were dramatically suppressed following ORM1 knockdown .

Mechanistically, ORM1 appears to regulate apoptotic pathways in KIRC cells. ORM1 knockdown resulted in upregulation of pro-apoptotic proteins (active caspase-3 and Bax) and downregulation of anti-apoptotic Bcl-2 . Further investigation revealed that CALR (calreticulin) acts as a downstream effector of ORM1, as CALR expression decreased following ORM1 knockdown, and rescued expression of CALR reversed the pro-apoptotic effects .

Perhaps most importantly for clinical applications, ORM1 affects the efficacy of sorafenib, a standard treatment for advanced KIRC. The inhibition rate of sorafenib was enhanced in ORM1 knockdown cells but reduced after ORM1 overexpression, suggesting that ORM1 levels may influence treatment response .

How can ORM1 antibody, FITC conjugated be utilized in cancer research protocols?

ORM1 antibody, FITC conjugated offers valuable applications in cancer research protocols, enabling researchers to investigate ORM1's role in tumor biology through fluorescence-based detection systems. The following protocol outlines optimal utilization strategies:

1. Tumor Cell Expression and Localization Studies:

• Prepare single-cell suspensions from tumor tissues or cultured cancer cell lines (e.g., KIRC lines like 786-O, A498, and Caki-2) .

• Fix cells with 4% paraformaldehyde (10 minutes at room temperature) followed by permeabilization with 0.1% Triton X-100 if intracellular staining is required.

• Block with 3% BSA in PBS for 30 minutes to reduce non-specific binding.

• Incubate with FITC-conjugated ORM1 antibody (1:100-1:500 dilution) for 1 hour at room temperature in the dark .

• For specificity controls, include samples with pre-incubation using unlabeled ORM1 antibody.

• Analyze by flow cytometry or confocal microscopy to determine expression levels and subcellular localization.

2. Tumor Microenvironment Infiltration Analysis:

• Prepare thin sections (5-8 μm) from fresh-frozen or formalin-fixed paraffin-embedded tumor tissues.

• For FFPE sections, perform antigen retrieval using citrate buffer (pH 6.0) followed by blocking with 10% normal serum.

• Apply FITC-conjugated ORM1 antibody (optimized concentration) to tissue sections and incubate overnight at 4°C in a humidified chamber .

• Co-stain with antibodies against tumor markers or immune cell markers (using different fluorophores) to identify cell-specific ORM1 expression patterns.

• Counterstain nuclei with DAPI and mount with anti-fade medium.

• Image using confocal microscopy and perform quantitative analysis of expression in different regions and cell types.

3. Receptor-Ligand Interaction Studies:

• Isolate membrane fractions from cancer cells or prepare whole-cell lysates.

• Perform binding assays using FITC-ORM1 with increasing concentrations to generate saturation curves.

• Include competition assays with unlabeled ORM1 or receptor antagonists to confirm binding specificity.

• For receptor identification, use candidate receptor antibodies (e.g., anti-CCR5) to block FITC-ORM1 binding and identify potential interaction partners in cancer cells .

• Quantify binding parameters (Kd, Bmax) through fluorescence intensity measurements.

4. Functional Response Monitoring:

• Treat cancer cells with FITC-ORM1 and track real-time cellular responses using live-cell imaging.

• Monitor changes in calcium signaling, receptor internalization, or other rapid responses.

• Perform time-course analysis of FITC-ORM1 binding and subsequent changes in cell morphology, migration, or proliferation.

• Combine with genetically-encoded fluorescent reporters for relevant signaling pathways to correlate ORM1 binding with pathway activation.

5. Therapeutic Response Assessment:

• Treat cancer cells with anti-cancer drugs (e.g., sorafenib for KIRC) in the presence or absence of ORM1 .

• Use FITC-ORM1 binding as a biomarker to predict or monitor treatment response.

• Analyze changes in ORM1 binding patterns before and after treatment to identify resistant vs. sensitive populations.

This protocol framework enables comprehensive investigation of ORM1's role in cancer progression through visualization and quantification of its expression, interactions, and functional impacts in tumor biology.

What molecular mechanisms link ORM1 to cancer cell proliferation, migration, and apoptosis?

Research employing tools like FITC-conjugated antibodies has revealed complex molecular mechanisms through which ORM1 influences cancer cell proliferation, migration, and apoptosis, particularly in kidney renal clear cell carcinoma (KIRC). These interconnected pathways provide insight into ORM1's role as a tumor promoter:

1. Cell Proliferation Regulation:
ORM1 significantly enhances cancer cell proliferation, as demonstrated by the dramatic reduction in KIRC cell growth following ORM1 knockdown . The proliferative effects likely stem from ORM1's influence on multiple pathways:

• Apoptotic Resistance: By maintaining high Bcl-2 levels and suppressing pro-apoptotic proteins, ORM1 creates an environment conducive to continued proliferation rather than programmed cell death .

• Metabolic Support: Given ORM1's known role in increasing glucose uptake in other cell types, it may similarly enhance cancer cell metabolism, providing energy resources needed for rapid proliferation .

• Cell Cycle Regulation: While not explicitly detailed in the search results, ORM1's impact on proliferation suggests potential interactions with cell cycle regulatory proteins.

2. Migration and Invasion Mechanisms:
ORM1 knockdown dramatically suppresses cancer cell migration and invasion capabilities , indicating its critical role in these processes. Potential mechanisms include:

• Extracellular Matrix Interaction: As a glycoprotein, ORM1 may influence cell-matrix interactions crucial for migration.

• Cytoskeletal Reorganization: ORM1 may impact pathways controlling cytoskeletal dynamics necessary for cell movement.

• Inflammatory Signaling: Given ORM1's role in inflammation and its interaction with CCR5 (a chemokine receptor), it may activate chemotactic pathways that facilitate directional migration .

3. Apoptosis Regulation Pathways:
ORM1 exerts significant anti-apoptotic effects through a well-defined molecular cascade:

• CALR (Calreticulin) Regulation: ORM1 maintains expression of CALR, which appears to be a crucial downstream effector. When ORM1 is knocked down, CALR decreases; conversely, rescued expression of CALR reverses pro-apoptotic effects .

• Bcl-2/Bax Balance: ORM1 promotes anti-apoptotic Bcl-2 expression while suppressing pro-apoptotic Bax. This tilts the balance toward cell survival .

• Caspase-3 Inhibition: ORM1 suppresses the activation of caspase-3, a key executioner of apoptosis. ORM1 knockdown increases both the expression and activity of caspase-3 .

4. Treatment Resistance Mechanisms:
ORM1 contributes to treatment resistance, particularly to sorafenib in KIRC:

• Drug Efficacy Modulation: The inhibitory effect of sorafenib is enhanced when ORM1 is knocked down but reduced when ORM1 is overexpressed .

• Survival Pathway Maintenance: By sustaining anti-apoptotic signaling even during drug treatment, ORM1 may allow cancer cells to evade therapy-induced cell death .

Understanding these molecular mechanisms not only clarifies ORM1's role in cancer progression but also identifies potential points for therapeutic intervention, particularly through targeting the ORM1-CALR-apoptotic pathway axis.

What are common challenges in using ORM1 antibody, FITC conjugated and how can they be addressed?

Researchers working with ORM1 antibody, FITC conjugated may encounter several technical challenges that can affect experimental outcomes. Here are the most common issues and evidence-based solutions:

1. Background Fluorescence Issues:

Problem: High background fluorescence can mask specific ORM1 signals, particularly in tissues with autofluorescence or when using suboptimal antibody concentrations.

Solutions:

• Optimize antibody concentration through titration experiments (typically 1-5 μg/ml provides optimal signal-to-noise ratio) .

• Include rigorous negative controls including isotype controls and competitive inhibition with unlabeled ORM1 .

• For tissue sections, use Sudan Black B (0.1-0.3% in 70% ethanol) to quench autofluorescence.

• Employ spectral unmixing during confocal microscopy to distinguish FITC signal from autofluorescence.

• Consider time-resolved fluorescence to overcome autofluorescence issues.

2. Signal Degradation and Photobleaching:

Problem: FITC is susceptible to photobleaching, leading to signal loss during extended imaging or analysis.

Solutions:

• Add anti-fade reagents to mounting media for fixed samples.

• Minimize exposure to light during sample preparation and storage.

• During flow cytometry, analyze FITC-stained samples first or within 4 hours of staining.

• For long-term storage, keep FITC-conjugated antibodies at -20°C or -80°C in single-use aliquots to avoid repeated freeze-thaw cycles .

• When possible, use confocal microscopy with reduced laser power and increased detector sensitivity.

3. pH Sensitivity Affecting Signal Intensity:

Problem: FITC fluorescence is pH-sensitive, decreasing significantly at lower pH values.

Solutions:

• Maintain all buffers at pH 7.4-8.0 for optimal FITC fluorescence .

• For endosomal or lysosomal studies where pH is naturally lower, consider using alternative fluorophores.

• Include pH buffers in fixation and permeabilization solutions.

• For flow cytometry, ensure consistent buffer composition across all samples.

4. Non-specific Binding:

Problem: FITC-conjugated antibodies may show non-specific binding, especially in tissues with high Fc receptor expression.

Solutions:

• Pre-block samples with 5-10% serum from the same species as the secondary antibody.

• Add 1% BSA to staining buffers to reduce non-specific interactions.

• Include Fc receptor blocking reagents when working with immune cells or tissues.

• Validate specificity through competition experiments with unlabeled ORM1 .

• Use flow cytometry to quantitatively assess binding parameters in the presence of specific inhibitors .

5. Suboptimal FITC-to-Protein Ratio:

Problem: Commercial antibodies may have variable FITC-to-protein ratios, affecting sensitivity and specificity.

Solutions:

• Request fluorescein/protein (F/P) ratio information from manufacturers.

• The optimal F/P ratio for antibodies is typically between 3:1 and 8:1 .

• For critical applications, consider purifying optimally labeled fractions using gradient DEAE Sephadex chromatography .

• Compare multiple commercial sources using standardized samples to identify optimal preparations.

6. Receptor Saturation in Binding Studies:

Problem: Excessive antibody concentration can lead to receptor saturation and non-specific binding.

Solutions:

• Perform binding saturation curves to determine appropriate concentrations.

• Include displacement assays with unlabeled ORM1 to confirm specific binding sites .

• For receptor studies, use incremental concentrations of FITC-ORM1 to calculate binding kinetics.

By addressing these common challenges through methodical troubleshooting, researchers can optimize the use of ORM1 antibody, FITC conjugated for more reliable and reproducible results across diverse experimental applications.

How do experimental conditions affect ORM1 antibody, FITC conjugated performance in different assays?

The performance of ORM1 antibody, FITC conjugated varies considerably across different experimental conditions and assay types. Understanding these variations is crucial for optimizing experimental design and interpreting results accurately:

1. Temperature Effects:

Temperature significantly impacts both antibody binding kinetics and FITC fluorescence properties:

• 4°C Conditions: Lower temperatures reduce internalization of antibody-receptor complexes, making this optimal for surface binding studies and flow cytometry applications . Flow cytometry analysis of FITC-ORM1 binding to skeletal muscle cells and U937 cells has been successfully performed at 4°C to maintain surface binding .

• Room Temperature (20-25°C): This represents an optimal compromise between binding efficiency and fluorescence stability for most applications . The conjugation reaction between FITC and antibodies achieves maximal labeling at room temperature within 30-60 minutes .

• 37°C Conditions: While physiologically relevant, higher temperatures accelerate photobleaching of FITC and may promote internalization of antibody-antigen complexes, potentially reducing surface detection but enhancing studies of receptor trafficking.

2. pH Influence:

FITC fluorescence is highly pH-dependent:

• Acidic Conditions (pH <6.0): FITC fluorescence is significantly quenched, making detection less sensitive. This is particularly problematic when tracking ORM1 to endosomal/lysosomal compartments where pH is naturally acidic.

• Neutral to Slightly Basic Conditions (pH 7.4-9.0): FITC fluorescence is optimal, with maximal brightness at pH 8.0-9.0 . The recommended buffer for ORM1 antibody storage and use maintains pH 7.4 .

• Conjugation Reaction: A pH of 9.5 provides optimal conditions for FITC conjugation to antibodies, enhancing the labeling efficiency .

3. Buffer Composition:

Different buffer components can significantly affect antibody performance:

• Preservatives: Proclin 300 (0.03%) in storage buffers helps maintain antibody integrity without interfering with fluorescence .

• Glycerol Content: The 50% glycerol in storage formulations prevents freeze-thaw damage and maintains antibody stability .

• Blocking Agents: For specific binding studies, the inclusion of BSA or serum in experimental buffers reduces non-specific interactions without compromising specific ORM1 detection.

4. Fixation Effects:

Cell preparation methods influence epitope accessibility and fluorescence properties:

• Live Cell Analysis: Preferred for receptor binding studies as seen in FITC-ORM1 binding to skeletal muscle and U937 cells , preserving native receptor conformation.

• Paraformaldehyde Fixation: Maintains cellular architecture but may alter some epitopes. Low concentration (2-4%) and short duration fixation minimizes this risk.

• Alcohol-Based Fixation: May extract membrane lipids, potentially affecting membrane-associated ORM1 or its receptors like CCR5.

5. Assay-Specific Considerations:

Different applications require tailored optimization:

• Flow Cytometry: Requires single-cell suspensions with minimal clumping. The successful quantification of FITC-ORM1 binding to skeletal muscle cells demonstrated specific binding that could be competed with unlabeled ORM1 .

• Fluorescence Microscopy: Benefits from optimized signal-to-noise ratio through careful titration of antibody concentration and appropriate mounting media to prevent photobleaching.

• ELISA Applications: While the FITC conjugate is reported suitable for ELISA , the detection method must accommodate fluorescence rather than chromogenic endpoints.

• Competitive Binding Assays: Require careful equilibration times and washing procedures to distinguish bound from unbound antibody. Studies with CCR5 antagonist Maraviroc and anti-CCR5 antibodies successfully demonstrated inhibition of FITC-ORM1 binding .

By systematically optimizing these conditions for specific experimental requirements, researchers can maximize the performance of ORM1 antibody, FITC conjugated across diverse applications, from basic binding studies to complex functional investigations of ORM1's role in fatigue mechanisms and cancer progression.

What emerging research directions might benefit from advanced applications of ORM1 antibody, FITC conjugated?

Several cutting-edge research directions could leverage ORM1 antibody, FITC conjugated to address critical questions in biomedicine. These emerging areas represent significant opportunities for innovative applications:

1. Single-Cell Analysis of ORM1 Receptor Heterogeneity:

The advent of high-dimensional cytometry and single-cell technologies opens possibilities for detailed characterization of ORM1 receptor expression patterns across diverse cell populations:

• Apply FITC-ORM1 in mass cytometry (CyTOF) or spectral flow cytometry alongside panels of lineage markers to map receptor distribution across tissues.

• Combine with single-cell RNA sequencing to correlate ORM1 binding capacity with transcriptional states of individual cells.

• Investigate heterogeneity in CCR5 expression and ORM1 binding across muscle fiber types to explain differential fatigue susceptibility .

• Examine whether cancer stem cell populations exhibit distinct ORM1 binding patterns compared to bulk tumor cells, potentially explaining treatment resistance mechanisms .

2. Intravital Microscopy for Real-Time ORM1 Dynamics:

Advances in intravital microscopy enable visualization of molecular interactions in living organisms:

• Track FITC-ORM1 biodistribution in real-time during exercise-induced fatigue to map temporal and spatial patterns of ORM1-target engagement .

• Visualize ORM1-receptor interactions within the tumor microenvironment, potentially revealing how ORM1 influences cancer cell-stromal interactions .

• Monitor dynamic changes in ORM1 binding during therapeutic interventions, providing mechanistic insights into treatment responses.

• Combine with genetically encoded calcium indicators to correlate ORM1 binding with immediate signaling responses in target tissues.

3. Organoid and Tissue-Chip Applications:

Three-dimensional culture systems provide physiologically relevant contexts for investigating ORM1 function:

• Apply FITC-ORM1 to muscle organoids to study fatigue mechanisms in a controlled yet tissue-like environment .

• Utilize kidney organoids derived from patient samples to investigate how ORM1 influences KIRC progression in personalized models .

• Incorporate FITC-ORM1 binding assays in microfluidic tissue-chips that recreate muscle-immune interfaces during exercise or systemic inflammation.

• Test how persistent vs. pulsatile ORM1 exposure affects receptor dynamics and downstream responses in these complex models.

4. Nanoscale Imaging of ORM1-Receptor Interactions:

Super-resolution microscopy and proximity labeling approaches can reveal molecular details beyond the diffraction limit:

• Employ FITC-ORM1 with techniques like STORM or PALM to visualize nanoscale receptor clustering and internalization dynamics.

• Combine with proximity labeling methods to identify novel molecular partners that associate with ORM1-receptor complexes.

• Investigate the molecular architecture of ORM1-CCR5 complexes on muscle cell membranes, potentially revealing how this interaction enhances glucose uptake and glycogen synthesis .

• Examine whether ORM1 induces different receptor clustering patterns in cancer cells versus normal cells, possibly explaining differential responses .

5. Therapeutic Targeting and Companion Diagnostics:

The potential therapeutic relevance of the ORM1 pathway creates opportunities for translational applications:

• Develop FITC-ORM1 as a companion diagnostic to predict response to CCR5 modulators in fatigue management .

• Utilize ORM1 binding patterns as biomarkers for cancer progression and treatment response prediction in KIRC .

• Screen for small molecule mimetics or antagonists of ORM1 using competition assays with FITC-ORM1.

• Investigate whether ORM1 antibodies could sensitize resistant tumors to treatments like sorafenib by blocking its protective effects .

6. Multi-Omics Integration with ORM1 Binding Profiles:

Combining ORM1 binding data with multi-omics approaches could reveal comprehensive biological networks:

• Correlate FITC-ORM1 binding capacity with proteomic profiles to identify molecular signatures associated with high ORM1 sensitivity.

• Integrate metabolomic data with ORM1 binding patterns to understand how this protein influences cellular metabolism, particularly in the context of muscle glycogen regulation .

• Apply FITC-ORM1 binding assays to patient-derived samples and correlate with genomic alterations to identify genetic determinants of ORM1 pathway activation.

These emerging research directions represent the frontier of ORM1 biology investigation, where FITC-conjugated antibodies can serve as critical tools for visualization, quantification, and mechanistic understanding of ORM1's diverse physiological and pathological roles.