NOMO1 Antibody, FITC conjugated

What is NOMO1 and what are its primary biological functions?

NOMO1 (Nodal Modulator 1) is a 1,222 amino acid highly conserved single-pass type I membrane protein expressed in various tissues including colon tumor tissue and normal colonic mucosa . It functions as a critical determinant of heart formation and is known to be involved in the pathological process of congenital heart disease (CHD) . Recent structural studies have revealed that NOMO comprises twelve Ig-like domains that reside in the ER lumen, followed by a single transmembrane domain and a short cytosolic tail . NOMO proteins (including NOMO1, NOMO2, and NOMO3) act as antagonists of Nodal signaling and interact with Nicalin to form a Nicalin-NOMO complex, where they are either rapidly degraded or stabilized by Nicalin .

What are the recommended applications for NOMO1 antibody, FITC conjugated?

The NOMO1 antibody with FITC conjugation is primarily recommended for flow cytometry (at dilutions of 1:20-100) and immunofluorescence on paraffin-embedded tissues (IF/IHC-P at dilutions of 1:50-200) . These applications allow researchers to investigate NOMO1 expression patterns in various cell types and tissues. The antibody demonstrates reactivity with human, mouse, and rat samples, providing versatility across different experimental models . Beyond basic detection, this antibody can be applied in studies examining NOMO1's role in cardiomyocyte differentiation, ER morphology maintenance, and nodal signaling pathways .

How can NOMO1 antibody, FITC conjugated be used to investigate its role in cardiac development and congenital heart disease?

NOMO1 antibody with FITC conjugation provides a valuable tool for investigating NOMO1's critical role in heart formation and congenital heart disease (CHD). Research has shown that upregulation of miR-33a-5p inhibits human cardiomyocyte progenitor cells (hCMPCs) proliferation, cell cycle G0/S transition, and differentiation into cardiomyocytes while promoting apoptosis by targeting NOMO1 . To study this relationship, researchers can use FITC-conjugated NOMO1 antibodies to:

• Track NOMO1 expression levels in cardiomyocyte progenitor cells during differentiation

• Assess co-localization with cardiac differentiation markers like GATA4, cTnT, and MEF2C

• Evaluate changes in NOMO1 expression following miR-33a-5p manipulation

Methodology should include flow cytometric analysis of NOMO1 expression in conjunction with cell cycle markers and apoptotic markers to correlate NOMO1 levels with cardiomyocyte proliferation and apoptosis rates in normal versus CHD models .

What approaches can be used to study NOMO1's role as a force-bearing transmembrane protein using FITC-conjugated antibodies?

Recent structural studies have identified NOMO as a force-bearing transmembrane protein with critical interfaces between distal Ig domains that enable it to maintain ER morphology and buffer mechanical forces . To investigate this function:

• Combine FITC-NOMO1 antibody staining with live cell imaging to track NOMO1 dynamics in response to mechanical stimuli

• Perform Fluorescence Recovery After Photobleaching (FRAP) experiments using the FITC signal to measure NOMO1 mobility within the ER membrane

• Compare wild-type NOMO1 mobility with mutant variants (particularly those affecting the Ig 1/10/11 interface)

FRAP experiments have revealed that NOMO exhibits markedly slow and restricted lateral mobility compared to other ER proteins like Sec61β, and even slower than LBR, which is limited by attachment to the nuclear lamina . The specific experimental approach would involve transfecting cells with NOMO variants and measuring FITC fluorescence recovery over time following photobleaching of a defined region of interest (ROI) .

How can researchers integrate NOMO1 antibody, FITC conjugated into studies of Nodal signaling pathways?

NOMO proteins function as antagonists of Nodal signaling through interaction with Nicalin . To study this pathway:

• Use FITC-conjugated NOMO1 antibodies in co-immunoprecipitation experiments followed by flow cytometry or fluorescence microscopy to identify binding partners in the Nodal signaling pathway

• Perform dual-labeling experiments with antibodies against Nodal pathway components to assess co-localization

• Design time-course experiments measuring NOMO1 expression (via FITC signal intensity) during developmental processes regulated by Nodal signaling

For Nodal pathway activation studies, researchers can treat cells with recombinant Nodal protein and track changes in NOMO1 expression and localization using flow cytometry and confocal microscopy with the FITC-conjugated antibody .

What are the optimal sample preparation protocols for NOMO1 antibody, FITC conjugated in flow cytometry?

For optimal flow cytometry results with FITC-conjugated NOMO1 antibody:

• Begin with single-cell suspensions at a concentration of 10^5 to 10^8 cells per test in 100 μL final volume

• Carefully titrate the antibody for optimal performance (recommended starting dilution: 1:20-100)

• Use ≤0.125 μg per test to avoid potential quenching of FITC fluorescence that can occur at higher concentrations

• Include appropriate controls:

  • Unstained cells

  • Isotype control (Rabbit IgG-FITC)

  • Single-color controls if performing multicolor flow cytometry

For confirming specificity in complex samples (e.g., differentiated cardiomyocytes), researchers should include additional markers such as CD45 FITC and CD33 PE-Cy7 antibodies as used in published protocols . When analyzing data, compensate for spectral overlap if using multiple fluorophores, and be aware that FITC signals can be quenched by anti-FITC antibodies in some applications .

What are the critical parameters for optimizing immunofluorescence staining with NOMO1-FITC antibodies?

For optimal immunofluorescence results with FITC-conjugated NOMO1 antibody on paraffin-embedded tissues (IHC-P):

Table 1: Optimization Parameters for NOMO1-FITC Immunofluorescence

For co-localization studies with ER markers, confocal microscopy should be employed with appropriate excitation (488 nm) and emission (515-530 nm) filters for FITC detection . Given NOMO1's role as a transmembrane protein in the ER, counterstaining with ER markers can provide valuable context for interpreting NOMO1 localization patterns .

How can researchers quantitatively assess NOMO1 expression levels using FITC-conjugated antibodies?

Quantitative assessment of NOMO1 expression using FITC-conjugated antibodies can be accomplished through:

• Flow Cytometry Quantification:

  • Use standardized FITC beads to establish a fluorescence calibration curve

  • Express results as Molecules of Equivalent Soluble Fluorochrome (MESF) or antibody binding capacity (ABC)

  • Apply the calibration to convert mean fluorescence intensity (MFI) values to absolute protein numbers

• Fluorescence Microscopy Quantification:

  • Employ software-based image analysis (ImageJ/FIJI with appropriate plugins)

  • Establish consistent exposure settings across all experimental conditions

  • Perform background subtraction and normalize to cell number or area

• Quantitative RT-PCR Correlation:

  • Combine FITC-based protein quantification with qRT-PCR analysis of NOMO1 mRNA

  • This approach has been validated in studies examining miR-33a-5p targeting of NOMO1 in cardiomyocytes

For comparative studies between different cell types or experimental conditions, researchers should standardize their protocols and include appropriate positive and negative controls to account for autofluorescence and non-specific binding.

How can researchers address potential signal quenching issues with FITC-conjugated NOMO1 antibodies?

FITC signal quenching is a documented concern with FITC-conjugated antibodies and can impact experimental outcomes . To address this issue:

• Titration Optimization:

  • Carefully titrate the antibody concentration (recommended ≤0.125 μg per test for flow cytometry)

  • Test multiple concentrations to identify the optimal signal-to-noise ratio

• Alternative Detection Strategies:

  • Consider secondary detection using anti-FITC antibodies conjugated to alternative fluorophores

  • Compare direct FITC detection with secondary amplification methods

• Environmental Factors:

  • Maintain pH between 7.2-8.5 where FITC fluorescence is optimal

  • Avoid exposure to strong light sources during sample preparation

  • Use antifade reagents in mounting media for microscopy applications

• Validation Approaches:

  • Confirm NOMO1 detection patterns using alternative antibodies with different conjugates

  • Correlate FITC-based detection with orthogonal methods (Western blot, qPCR)

If signal quenching is suspected, researchers can compare the fluorescence intensity of cells stained directly with the FITC-conjugated NOMO1 antibody versus those stained with unconjugated primary followed by FITC-conjugated secondary antibody as a control .

What strategies can be employed when analyzing contradictory data from NOMO1-FITC antibody experiments?

When faced with contradictory results from NOMO1-FITC antibody experiments:

• Antibody Validation:

  • Verify antibody specificity through knockdown/knockout controls

  • Test multiple NOMO1 antibodies targeting different epitopes

  • Consider potential cross-reactivity with NOMO2 and NOMO3, as these proteins share high sequence similarity

• Experimental Design Assessment:

  • Evaluate fixation and permeabilization protocols, as these can affect epitope accessibility

  • Consider subcellular localization - NOMO1 is a transmembrane protein in the ER with specific domains in different cellular compartments

  • Assess potential interference from other experimental factors (e.g., overexpression constructs, treatment conditions)

• Data Integration:

  • Combine FITC-based flow cytometry data with western blot quantification

  • Correlate protein detection with mRNA expression data

  • Consider the impact of post-translational modifications on antibody binding

• Context-Specific Expression:

  • NOMO1 expression and function may vary by cell type and developmental stage

  • In cardiac development studies, consider the differentiation state of cardiomyocytes

  • For ER morphology studies, evaluate the cell's mechanical environment

A systematic approach to troubleshooting contradictory data includes designing controlled experiments that isolate variables and progressively eliminate potential sources of inconsistency.

How can NOMO1-FITC antibodies be incorporated into multi-parameter flow cytometry panels?

Designing effective multi-parameter flow cytometry panels including NOMO1-FITC antibodies requires careful consideration of spectral overlap and antibody compatibility:

Table 2: Multi-Parameter Panel Design with NOMO1-FITC

For compensating spectral overlap:

• Prepare single-stained controls for each fluorophore

• Include an unstained control and FMO (Fluorescence Minus One) controls

• Perform automated compensation using flow cytometry software

For analyzing intracellular NOMO1 along with surface markers:

• First stain for surface markers (e.g., CD45, CD33)

• Fix and permeabilize cells using a commercial kit appropriate for intracellular staining

• Then stain with NOMO1-FITC antibody at the optimal concentration

This approach has been validated in studies examining myeloid cell populations and has been used to confirm AML presence by flow cytometry .

What emerging applications are being developed for NOMO1 antibody, FITC conjugated in mechanobiology research?

Recent structural studies have identified NOMO as a force-bearing transmembrane protein with critical interfaces between distal Ig domains . This finding opens new research directions for FITC-conjugated NOMO1 antibodies:

• Live-Cell Force Measurements:

  • Using FRET-based biosensors combined with NOMO1-FITC antibodies to measure tension across NOMO1 in living cells

  • Correlating NOMO1 localization with cellular force generation and transmission

• ER-Cytoskeleton Interactions:

  • Investigating NOMO1's role in mediating connections between the ER membrane and cytoskeletal elements

  • Studying how mechanical forces affect NOMO1 distribution and dynamics in the ER membrane

• Differentiation Mechanics:

  • Exploring how NOMO1-mediated force sensing contributes to myogenesis and cardiomyocyte differentiation

  • Experiments have shown that NOMO-silenced myoblast lines exhibit delays in myotube formation

These research directions build on findings that NOMO exhibits markedly slow and restricted lateral mobility within the ER membrane, which depends partly on the presence of the Ig 1/10/11 interface . FITC-conjugated antibodies can enable real-time tracking of NOMO1 dynamics during mechanical stimulation experiments.

How can NOMO1-FITC antibodies contribute to understanding the relationship between NOMO1 and miRNA regulation in disease models?

Studies have established that miR-33a-5p targets NOMO1 to modulate human cardiomyocyte activity, suggesting an important regulatory relationship with implications for congenital heart disease . Future research applications include:

• miRNA Target Validation:

  • Using NOMO1-FITC antibodies to quantify protein expression changes following miRNA manipulation

  • Flow cytometric analysis can provide single-cell resolution of NOMO1 regulation by miRNAs

• Therapeutic Development:

  • Screening potential therapeutic compounds that modulate the miR-33a-5p/NOMO1 axis

  • Using FITC-conjugated antibodies to rapidly assess NOMO1 expression in drug screening platforms

• Disease Modeling:

  • Applying NOMO1-FITC antibodies in patient-derived iPSC models of congenital heart disease

  • Correlating NOMO1 expression patterns with disease phenotypes and severity

These approaches could help elucidate the complex relationship between miRNA regulation of NOMO1 and disease pathogenesis, potentially identifying new therapeutic targets for conditions like congenital heart disease .