NUBP2 Antibody, FITC conjugated

What is NUBP2 and why is it important in cellular research?

NUBP2 (Nucleotide-binding protein 2) functions as a component of the cytosolic iron-sulfur (Fe-S) protein assembly machinery. It plays a critical role in the maturation of extramitochondrial Fe-S proteins and has been implicated in regulating cilium formation and structure. NUBP2 is an MRP/MinD-type P-loop NTPase with sequence similarity to bacterial division site-determining proteins and is conserved throughout eukaryotes. Research shows it negatively regulates ciliogenesis, making it a crucial target for studying cellular development and function .

What are the storage and handling recommendations for NUBP2 Antibody, FITC conjugated?

For optimal results, NUBP2 Antibody, FITC conjugated should be stored at -20°C or -80°C upon receipt. Repeated freeze-thaw cycles should be avoided to maintain antibody integrity and fluorescence. The antibody is typically provided in liquid form with a buffer composition of 0.03% Proclin 300 as preservative, 50% glycerol, and 0.01M PBS at pH 7.4 . When working with FITC-conjugated antibodies, it's important to protect them from light exposure to prevent photobleaching of the fluorophore, as FITC is susceptible to photodegradation .

What species reactivity can be expected with NUBP2 Antibody, FITC conjugated?

The commercially available NUBP2 Antibody, FITC conjugated has confirmed reactivity with human samples . Some variants also demonstrate cross-reactivity with mouse and rat samples, allowing for comparative studies across these mammalian models . For research involving other species, validation experiments are strongly recommended prior to application in critical studies.

How does FITC conjugation affect antibody performance and detection?

FITC (Fluorescein Isothiocyanate) conjugation enables direct visualization of the antibody through fluorescence techniques. FITC is conjugated to proteins via primary amines (lysines), with typically 3-6 FITC molecules per antibody. Higher conjugation rates can lead to solubility problems and internal quenching, reducing brightness. The fluorophore is excited at 495 nm with emission at 519 nm, making it compatible with standard FITC filter sets in fluorescence microscopy and flow cytometry . When designing multi-color experiments, researchers should consider spectral overlap with other fluorophores.

What are the recommended dilutions and applications for NUBP2 Antibody, FITC conjugated?

NUBP2 Antibody, FITC conjugated has been validated for ELISA applications . For immunofluorescence and immunohistochemistry-paraffin (IHC-P) applications, recommended dilutions generally range from 1:50 to 1:200, though optimal dilutions should be determined experimentally for each specific application and protocol . When adopting methods from similar FITC-conjugated antibodies, researchers should consider that typical applications may include flow cytometry, immunocytochemistry/immunofluorescence, immunohistochemistry, and western blot, each requiring specific optimization .

How should researchers design control experiments when using NUBP2 Antibody, FITC conjugated?

Robust control experiments should include:

• Negative controls: Samples treated with isotype-matched FITC-conjugated IgG from the same host species (rabbit)

• Blocking peptide controls: Pre-incubation of the antibody with the immunizing peptide to confirm specificity

• Positive controls: Samples known to express NUBP2 (based on literature)

• siRNA knockdown controls: Comparing staining between NUBP2-silenced and normal cells

• Cross-validation: Using alternative detection methods like western blot to confirm findings

These controls help distinguish specific binding from background fluorescence, particularly important with FITC conjugates which may exhibit some non-specific binding .

What counterstaining methods are compatible with NUBP2 Antibody, FITC conjugated?

DAPI (4′,6-diamidino-2-phenylindole) is highly compatible for nuclear counterstaining with FITC-conjugated antibodies, as demonstrated in similar applications . For multi-color imaging experiments, researchers should select counterstains with minimal spectral overlap with FITC's emission (519 nm). Compatible counterstains include propidium iodide (when using appropriate filter sets), DRAQ5, or far-red nuclear dyes. For organelle visualization, markers conjugated to fluorophores such as Texas Red, Cy3, or far-red dyes would minimize bleed-through issues when imaging NUBP2 localization.

How can researchers effectively study NUBP2's role in ciliogenesis using the FITC-conjugated antibody?

To investigate NUBP2's function in ciliogenesis, researchers can implement a multi-faceted approach:

• Co-localization studies: Use the NUBP2 Antibody, FITC conjugated alongside markers for basal bodies (e.g., γ-tubulin) and ciliary structures (e.g., acetylated α-tubulin) using confocal microscopy

• Temporal dynamics: Track NUBP2 localization throughout cell cycle phases, particularly during cilium formation and resorption

• RNAi experiments: Combine NUBP2 knockdown with immunofluorescence to assess changes in cilia number and morphology

• Rescue experiments: Reintroduce NUBP2 in knockdown cells to confirm specificity of observed phenotypes

Research has shown that downregulation of NUBP2 markedly increases the number of ciliated cells, confirming its role as a negative regulator of ciliogenesis .

What approaches can be used to study the interaction between NUBP2 and the CCT/TRiC molecular chaperone complex?

To explore NUBP2's interaction with the CCT/TRiC molecular chaperone complex, researchers can employ:

• Co-immunoprecipitation: Using NUBP2 Antibody to pull down the protein complex, followed by western blot analysis for CCT/TRiC components

• Proximity ligation assay (PLA): Combining NUBP2 Antibody, FITC conjugated with antibodies against CCT/TRiC components to visualize protein-protein interactions in situ

• FRET analysis: Employing FITC-conjugated NUBP2 Antibody with acceptor fluorophore-labeled CCT/TRiC antibodies

• Domain mapping: Using truncated NUBP2 constructs to identify specific interaction domains

• Functional assays: Assessing how CCT/TRiC inhibition affects NUBP2 localization and function

Evidence indicates that NUBP2, along with NUBP1, interacts with several members of the CCT/TRiC molecular chaperone complex, which is enriched at the basal body and may play a role in ciliogenesis regulation .

How can researchers optimize dual immunofluorescence protocols involving NUBP2 Antibody, FITC conjugated?

For successful dual immunofluorescence involving NUBP2 Antibody, FITC conjugated:

• Sequential staining: Apply unconjugated primary antibodies first, followed by non-FITC secondary antibodies, then apply the NUBP2 Antibody, FITC conjugated last

• Cross-reactivity prevention: Use secondary antibodies raised against species different from the NUBP2 Antibody host (rabbit)

• Fixation optimization: Test both paraformaldehyde (4%) and methanol fixation to determine optimal epitope preservation

• Blocking optimization: Include normal serum from the species of all secondary antibodies in blocking solution

• Signal amplification: Consider tyramide signal amplification if NUBP2 expression is low

• Controls: Include single-stained samples to establish proper exposure settings and assess bleed-through

Proper optimization ensures clear distinction between NUBP2 and other proteins of interest while minimizing background and cross-reactivity .

What are common challenges when using FITC-conjugated antibodies and how can they be overcome?

Common challenges and solutions include:

These strategies can significantly improve the quality of data when working with FITC-conjugated NUBP2 antibody .

How should researchers interpret NUBP2 localization patterns in centrioles and cilia?

When interpreting NUBP2 localization in centrioles and cilia:

• Spatial distribution: NUBP2 typically localizes to centrioles throughout the cell cycle and to the basal body of primary cilia

• Co-localization analysis: Quantify overlap with established markers for specific centriolar/ciliary compartments

• Cell cycle context: Interpret localization in the context of cell cycle stage (G0/G1 for primary cilia)

• Comparison with NUBP1: Evaluate whether NUBP2 and NUBP1 show identical or distinct localization patterns

• 3D reconstruction: Use z-stack imaging to fully capture the three-dimensional distribution

Research indicates that both NUBP1 and NUBP2 are integral components of centrioles throughout the cell cycle and localize at the basal body of primary cilia in quiescent cells, suggesting their role in regulating cilium formation .

What considerations are important when quantifying immunofluorescence results from NUBP2 Antibody, FITC conjugated staining?

For accurate quantification:

• Signal-to-noise ratio: Calculate and report the ratio between specific signal and background

• Dynamic range optimized imaging: Avoid pixel saturation by optimizing exposure times

• Standardized acquisition: Maintain identical microscope settings across all experimental conditions

• Thresholding methods: Document and consistently apply thresholding methods for signal detection

• Normalization: Consider normalizing NUBP2 signal to a reference protein or structure

• Technical replicates: Include multiple fields and biological replicates

• Blinded analysis: Perform quantification blinded to experimental conditions to prevent bias

• Statistical validation: Apply appropriate statistical tests based on data distribution

These practices ensure robust and reproducible quantification of NUBP2 expression and localization patterns .

How does NUBP2 function in the cytosolic iron-sulfur (Fe-S) protein assembly machinery?

NUBP2 functions as a key component of the cytosolic iron-sulfur (Fe-S) protein assembly (CIA) machinery. It forms a heterotetramer with NUBP1, creating a Fe-S scaffold complex that mediates the de novo assembly of Fe-S clusters and their transfer to target apoproteins. This process is essential for the maturation of extramitochondrial Fe-S proteins . Research approaches to study this function include:

• Biochemical reconstitution: In vitro reconstitution of Fe-S cluster assembly with purified components

• Spectroscopic analysis: UV-visible and EPR spectroscopy to monitor Fe-S cluster formation

• Mutagenesis: Strategic mutations in NUBP2's nucleotide-binding motifs to assess functional requirements

• Metabolic labeling: Using radioactive iron (⁵⁵Fe) to track the flow of iron through the Fe-S assembly pathway

• Proteomic profiling: Identifying NUBP2-dependent changes in Fe-S proteome

Understanding NUBP2's role in Fe-S protein biogenesis provides insights into fundamental cellular processes dependent on these critical cofactors .

What is the relationship between NUBP2, NUBP1, and KIFC5A in regulating ciliogenesis?

The relationship between these proteins in ciliogenesis involves complex regulatory mechanisms:

• Antagonistic functions: NUBP1/NUBP2 and KIFC5A appear to have opposing effects on ciliogenesis - knockdown of NUBP1/NUBP2 increases ciliated cells, while KIFC5A knockdown reduces ciliogenesis

• Compensatory mechanisms: Double silencing of NUBP1 and KIFC5A restores the percentage of ciliated cells to control levels

• Independent recruitment: NUBP1 and NUBP2 are recruited to centrioles independently of KIFC5A

• Molecular interaction: KIFC5A is a minus-end directed motor protein that interacts with NUBP1/NUBP2

• Temporal regulation: These proteins likely coordinate their activities during specific phases of the cell cycle to regulate ciliogenesis

This complex interplay suggests these proteins form a regulatory module controlling the balance of ciliogenesis, potentially through effects on basal body maturation or initiation of axoneme extension .

How can NUBP2 Antibody, FITC conjugated be used in live-cell imaging applications?

While challenging due to the need for intracellular delivery, live-cell applications could include:

• Cell permeabilization techniques: Mild detergents, pore-forming toxins, or cell-penetrating peptides to introduce the antibody

• Microinjection: Direct introduction of the antibody into individual cells

• Electroporation: Temporary membrane disruption to allow antibody entry

• Photoswitchable FITC derivatives: Using photoactivatable or photoswitchable FITC variants for pulse-chase experiments

• Correlative microscopy: Live imaging followed by fixation and antibody staining to correlate dynamic events with NUBP2 localization

• Proximity labeling: Combining with APEX2 or BioID approaches for temporal protein interaction studies

These approaches could provide valuable insights into the dynamic localization and function of NUBP2 during cellular processes like ciliogenesis or cell division .