UBAC2 Antibody, FITC conjugated

What is UBAC2 and what is its role in cellular processes?

UBAC2 (Ubiquitin-Associated Domain-Containing Protein 2) is a 344-amino acid protein with a calculated molecular weight of approximately 39 kDa, though it often appears at around 35 kDa in Western blots . This protein contains UBA (Ubiquitin-Associated) domains and is involved in protein trafficking pathways. Specifically, UBAC2 restricts the trafficking of FAF2 (FAS-associated factor 2) from the endoplasmic reticulum to lipid droplets .

UBAC2 is encoded by gene ID 337867 and has a UniProt ID of Q8NBM4 . Expression analysis shows that UBAC2 is detectable in various human cell types, including HEK-293 and MCF-7 cells, as well as in mouse kidney tissue . The protein's involvement in ubiquitin-associated pathways makes it relevant to research on protein degradation and cellular stress responses.

What are the key characteristics of FITC-conjugated antibodies in research applications?

Fluorescein Isothiocyanate (FITC) is a widely-used fluorophore in immunological research with distinct spectral properties. FITC absorbs blue light with an excitation maximum around 498 nm and emits green fluorescence with an emission maximum around 519 nm . When conjugated to antibodies, FITC enables direct visualization of target proteins without requiring secondary detection systems.

The FITC-conjugated UBAC2 antibody offers several advantages in research applications:

The UBAC2 Antibody-FITC conjugate is particularly valuable for direct immunofluorescence applications, reducing protocol complexity and potential cross-reactivity issues that might arise with secondary antibody systems .

What optimal dilution ranges should be used for different experimental applications?

The effectiveness of UBAC2 antibody detection is highly dependent on using appropriate dilutions for specific applications. Based on experimental validation, the following dilution ranges are recommended:

It is critical to note that these ranges serve as starting points, and researchers should perform titration experiments to determine optimal concentrations for their specific experimental systems . Sample-dependent variations may necessitate adjustments to achieve optimal signal-to-noise ratios.

How should UBAC2 Antibody-FITC conjugates be stored to maintain optimal activity?

Proper storage of FITC-conjugated antibodies is essential to maintaining their fluorescence intensity and binding specificity. The UBAC2 Antibody-FITC conjugate should be:

• Stored as aliquots at -20°C to minimize freeze-thaw cycles

• Protected from light exposure during storage and handling to prevent photobleaching

• Maintained in appropriate buffer conditions (typically 0.01 M PBS, pH 7.4, with 0.03% Proclin-300 and 50% glycerol) to ensure stability

Repeated freeze-thaw cycles significantly reduce antibody activity and fluorescence intensity. Therefore, preparing smaller working aliquots during initial receipt is strongly recommended. When properly stored, the antibody maintains activity for approximately 12 months .

What are the optimal protocols for multiplexing UBAC2 Antibody-FITC with other fluorophore-conjugated antibodies?

Multiplexing with FITC-conjugated UBAC2 antibodies requires careful selection of compatible fluorophores to avoid spectral overlap. Recommended fluorophores for multiplexing include:

When designing multiplex experiments, consider these methodological recommendations:

• Stagger your primary antibody hosts (e.g., use rabbit anti-UBAC2-FITC with mouse antibodies against other targets)

• Implement proper compensation controls when using flow cytometry

• For microscopy applications, acquire single-channel images sequentially rather than simultaneously to minimize bleed-through

• Include appropriate single-stain controls to verify specificity of signal in each channel

For longer imaging sessions where photobleaching is a concern, consider using Cyanine 5.5 labeled antibodies in combination with FITC-conjugated UBAC2 antibody, as Cyanine 5.5 exhibits superior photostability for extended imaging .

How can researchers validate the specificity of UBAC2 Antibody-FITC conjugates?

Rigorous validation is essential for ensuring the reliability of UBAC2 antibody-based experiments. Recommended validation approaches include:

• Knockout/Knockdown Controls: Several publications have utilized UBAC2 knockdown/knockout systems for antibody validation . Researchers should compare staining between wild-type and UBAC2-deficient samples.

• Multiple Detection Methods: Confirm UBAC2 detection across different techniques:

  • Western blot (expected band at ~35 kDa)

  • Immunofluorescence (with subcellular localization pattern consistent with ER distribution)

  • Immunohistochemistry (with tissue-specific expression patterns)

• Peptide Competition Assay: Pre-incubate the antibody with excess immunizing peptide to demonstrate signal specificity.

• Cross-Species Reactivity Assessment: Verify consistent detection patterns in both human and mouse samples, as the antibody has been validated for both species .

• Orthogonal Antibody Comparison: Compare staining patterns with alternative UBAC2 antibodies from different clones or manufacturers.

What are effective troubleshooting strategies for non-specific binding in UBAC2-FITC immunofluorescence?

Non-specific binding can significantly impact the interpretability of UBAC2 immunofluorescence experiments. To minimize background and ensure specific staining:

• Optimize Blocking Conditions:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Extend blocking time from 30 minutes to 1-2 hours at room temperature

  • Consider using species-specific Fc receptor blocking reagents for samples containing immune cells

• Antibody Optimization:

  • Begin with higher dilutions (1:500) and titrate to find optimal concentration

  • Reduce primary antibody incubation time or temperature if background persists

• Sample Processing Modifications:

  • For IHC applications, optimize antigen retrieval methods (TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 may also be tested)

  • Increase wash duration and volume between steps

  • Test different fixation methods (paraformaldehyde vs. methanol) for cell culture samples

• Signal Enhancement Techniques:

  • If signal is weak, consider signal amplification systems compatible with FITC

  • Adjust image acquisition settings (exposure time, gain) to optimize signal-to-noise ratio without introducing artifacts

How does UBAC2 expression vary across cell and tissue types, and what are the implications for detection methods?

UBAC2 expression exhibits notable variation across different tissues and cell types, which has important implications for detection strategies:

For tissues with variable expression, consider these methodological approaches:

• Include positive control tissues known to express UBAC2 in each experiment

• For IHC applications, optimize antigen retrieval methods specifically for each tissue type

• In tissues with lower expression, consider using more concentrated antibody dilutions or signal amplification systems

• For single-cell analysis techniques like flow cytometry, establish appropriate gating strategies based on known expression patterns

What are the optimal methods for reducing photobleaching of FITC during extended imaging sessions?

FITC is susceptible to photobleaching during extended imaging sessions, which can limit its utility in time-lapse experiments. To minimize photobleaching while maintaining high-quality data:

• Anti-Fade Reagents:

  • Incorporate commercial anti-fade mounting media specifically formulated for FITC

  • Create fresh mounting media immediately before use for optimal performance

• Microscopy Adjustments:

  • Reduce excitation light intensity to the minimum required for adequate signal

  • Implement neutral density filters in the excitation path

  • Use shorter exposure times with more sensitive cameras

  • Consider confocal systems with acousto-optic tunable filters (AOTFs) to precisely control excitation intensity

• Alternative Approaches:

  • For experiments requiring extensive time-lapse imaging, consider antibodies conjugated to more photostable fluorophores like Cyanine 5.5

  • Implement deconvolution algorithms to extract maximum information from lower-intensity images

  • Use computational methods to correct for photobleaching in time series data

How do buffer conditions affect the performance of UBAC2 Antibody-FITC conjugates?

• pH Considerations:

  • FITC fluorescence is optimal at slightly alkaline pH (7.5-8.5)

  • Acidic environments significantly reduce FITC quantum yield

  • For applications in acidic cellular compartments, consider pH-insensitive alternatives

• Stabilizing Agents:

  • BSA (0.1-1%) can be added to diluted antibody solutions to prevent adsorption to tubes

  • Avoid sodium azide in working solutions when using peroxidase-based detection systems

• Experimental Buffers:

  • For flow cytometry, PBS with 1-2% FBS is typically suitable

  • For immunofluorescence washes, PBS with 0.05-0.1% Tween-20 reduces background

  • For antigen retrieval in IHC, TE buffer at pH 9.0 provides optimal results, though citrate buffer at pH 6.0 can serve as an alternative

What are the considerations for quantitative analysis of UBAC2 expression using FITC-conjugated antibodies?

Quantitative analysis of UBAC2 expression using FITC-conjugated antibodies requires careful experimental design and appropriate controls:

• Standardization Approaches:

  • Include calibration standards with known fluorophore concentrations

  • Normalize FITC signal to cell number or total protein content

  • Use internal reference proteins with stable expression across experimental conditions

• Imaging Considerations:

  • Maintain consistent exposure settings across all experimental groups

  • Account for potential flat-field correction in microscopy

  • Implement background subtraction methods appropriate to the imaging modality

• Flow Cytometry Optimization:

  • Use quantitative beads to convert mean fluorescence intensity to molecules of equivalent soluble fluorochrome (MESF)

  • Implement compensation to correct for spectral overlap in multiplex experiments

  • Consider the impact of cell size/autofluorescence on signal interpretation

• Analytical Approaches:

  • For Western blot quantification, use standard curves with recombinant UBAC2 protein

  • For image-based quantification, employ automated analysis workflows to reduce subjective bias

  • Consider machine learning approaches for complex pattern recognition in tissue samples

How can UBAC2 Antibody-FITC conjugates be utilized in studying protein trafficking mechanisms?

Given UBAC2's role in restricting trafficking of FAF2 from the endoplasmic reticulum to lipid droplets , FITC-conjugated UBAC2 antibodies offer valuable tools for investigating protein trafficking mechanisms:

• Co-localization Studies:

  • Combine UBAC2-FITC with markers for different cellular compartments (ER, Golgi, lipid droplets)

  • Implement super-resolution microscopy techniques to precisely map UBAC2's subcellular distribution

  • Quantify co-localization coefficients under different cellular stress conditions

• Live Cell Imaging Applications:

  • For cell-permeable applications, consider modified delivery systems for FITC-conjugated antibodies

  • Correlate UBAC2 localization with lipid droplet formation in real-time

  • Monitor dynamic changes in response to lipid loading or ER stress

• Proximity Ligation Approaches:

  • Combine UBAC2-FITC antibodies with proximity ligation assays to identify interaction partners

  • Map the protein interaction network of UBAC2 under different physiological conditions

What are the implications of UBAC2 mutations or altered expression in disease states?

Research into UBAC2's role in disease states is an emerging field where FITC-conjugated antibodies can provide valuable insights:

• Expression Analysis in Disease Models:

  • Compare UBAC2 expression levels between normal and pathological tissue samples

  • Correlate expression patterns with disease progression or therapeutic response

  • Develop tissue microarray approaches for high-throughput screening

• Mutation Impact Studies:

  • Use UBAC2-FITC antibodies to assess localization changes of mutant variants

  • Implement flow cytometry to quantify expression differences across patient-derived samples

  • Combine with functional assays to correlate expression with cellular phenotypes

• Therapeutic Targeting Potential:

  • Evaluate UBAC2 as a potential biomarker for diseases involving ER stress or lipid metabolism

  • Assess the impact of therapeutic compounds on UBAC2 expression and localization

  • Develop screening assays for compounds that modulate UBAC2 function