AASS Antibody, FITC conjugated

What is AASS and why is it studied as a research target?

AASS (Alpha-aminoadipic semialdehyde synthase, mitochondrial) is a bifunctional enzyme involved in lysine degradation pathways. It contains two enzymatic activities: Lysine ketoglutarate reductase (LKR/SDH) and Saccharopine dehydrogenase (SDH), as indicated by its alternative name LKR/SDH . This mitochondrial protein plays important roles in signal transduction and metabolic processes. Researchers typically study AASS to understand lysine metabolism disorders, mitochondrial function, and related pathological conditions. The human AASS protein consists of distinct domains, with commercially available antibodies often targeting specific regions, such as amino acids 224-364 of the human protein .

How does FITC conjugation work and what advantages does it offer in antibody applications?

FITC (Fluorescein isothiocyanate) conjugation involves chemical attachment of the fluorophore to antibodies through reaction with primary amine groups, forming stable thiourea bonds. The conjugation chemistry is relatively straightforward and typically preserves the biological activity of the labeled protein . FITC offers several advantages as an antibody label:

• High fluorescence quantum yield with excitation/emission peaks at approximately 495nm/525nm (yellow-green fluorescence)

• Direct visualization capability without requiring secondary detection steps

• Compatibility with standard fluorescence microscope filter sets

• Well-established protocols with extensive literature support

• Simplified experimental procedures by eliminating secondary antibody incubation steps

What is the molecular structure of FITC conjugation to antibodies?

FITC conjugation typically occurs through reaction between the isothiocyanate group of FITC and primary amines (mainly lysine residues) on the antibody. The isothiocyanate group forms a stable thiourea bond with these amines. The conjugation process follows established protocols, such as those described by Harlow and Lane (1988) . Most commercially available FITC-conjugated antibodies are prepared through controlled reactions to achieve optimal fluorophore-to-protein ratios, ensuring sufficient labeling while maintaining antibody functionality .

How are FITC-conjugated antibodies stored and what precautions should be taken?

FITC-conjugated antibodies require specific storage conditions to maintain their activity and fluorescence properties:

• Temperature: Store at -20°C to -80°C for long-term preservation

• Buffer composition: Typically supplied in Phosphate-Buffered Saline (PBS) with 0.01% sodium azide as a preservative

• Light protection: Critical to prevent photobleaching; continuous exposure to light causes gradual loss of fluorescence

• Physical state: Often provided with stabilizers such as 50% glycerol

• Aliquoting: Create single-use aliquots to avoid repeated freeze-thaw cycles

When handling these antibodies, minimize exposure to light during all experimental procedures and work efficiently to reduce the time samples are exposed to environmental conditions that could affect antibody performance .

What is the recommended protocol for immunofluorescence using AASS Antibody, FITC conjugated?

The following protocol is recommended for immunofluorescence detection using AASS Antibody, FITC conjugated:

• Sample preparation:

  • Culture cells on coverslips or appropriate imaging chambers to 70-80% confluence

  • Fix cells with 4% paraformaldehyde for 15-20 minutes at room temperature

  • Permeabilize with 0.1-0.5% Triton X-100 in PBS for 5-10 minutes

  • Block with PBS containing 10% fetal bovine serum (FBS) for 20-30 minutes

• Antibody staining:

  • Dilute AASS Antibody, FITC conjugated to appropriate concentration (typically 1:100-1:500) in blocking solution

  • Apply diluted antibody to samples and incubate for 1 hour at room temperature in the dark

  • Wash cells 2-3 times with PBS, 5 minutes each wash

  • Mount slides with anti-fade mounting medium containing DAPI for nuclear counterstaining

• Imaging:

  • Observe cells using a fluorescence microscope equipped with appropriate FITC filter set (excitation ~495nm, emission ~525nm)

  • Acquire images promptly to minimize photobleaching

This protocol may require optimization based on specific sample types, fixation methods, and expression levels of AASS in your experimental system.

What controls should be included when using AASS Antibody, FITC conjugated?

Proper controls are critical for validating results with FITC-conjugated AASS antibodies:

• Negative controls:

  • Isotype control: FITC-conjugated rabbit IgG at the same concentration as the AASS antibody

  • Unstained samples: To assess autofluorescence levels

  • Blocking peptide control: Pre-incubate antibody with immunizing peptide to confirm specificity

  • AASS knockdown/knockout cells: To verify antibody specificity

• Positive controls:

  • Known AASS-expressing tissue or cell line

  • Recombinant AASS protein for Western blot validation

• Technical controls:

  • Single-color controls: When performing multiplex staining to establish bleed-through parameters

  • FITC quenching control: Pre-incubation with anti-FITC antibody to confirm signal specificity

  • Mitochondrial co-localization: Since AASS is a mitochondrial protein, co-staining with mitochondrial markers can confirm proper localization

For flow cytometry applications, additional controls should include unstained cells and fluorescence-minus-one (FMO) controls to properly set gating parameters .

How can AASS Antibody, FITC conjugated be used in flow cytometry?

For flow cytometric analysis using AASS Antibody, FITC conjugated:

• Cell preparation:

  • Harvest cells (1-5×10^6 cells per sample)

  • Fix with 2-4% paraformaldehyde for 10-15 minutes at room temperature

  • Permeabilize with 0.1-0.5% saponin or commercial permeabilization buffer (critical for accessing the mitochondrial AASS protein)

  • Block with 2-5% BSA in PBS for 10-15 minutes

• Antibody staining:

  • Prepare antibody dilution in staining buffer (PBS with 1-2% BSA)

  • Incubate cells with diluted antibody (typically 1:50-1:200) for 30-60 minutes at room temperature in the dark

  • Wash twice with staining buffer

  • Resuspend in appropriate volume for flow cytometry analysis

• Instrument setup:

  • Use 488nm laser excitation and appropriate emission filters (~525/40nm)

  • Include unstained cells and single-stained controls for compensation when performing multicolor analysis

  • Collect sufficient events (minimum 10,000-20,000) for statistical validity

Flow cytometry can provide quantitative data on AASS expression levels across different cell populations and can be combined with other markers to correlate AASS expression with specific cell states or functions .

What are the optimal antibody dilutions for different applications?

Optimal dilutions for AASS Antibody, FITC conjugated vary by application:

For optimal results, it's recommended to perform titration experiments starting with the manufacturer's suggested dilution. For AASS Antibody, FITC conjugated products like CSB-PA883373LC01HU, the antibody is typically supplied at 1 mg/mL concentration in a purified form , allowing for accurate dilution calculations.

What are common causes of high background when using FITC-conjugated antibodies?

High background is a common challenge with FITC-conjugated antibodies. Major causes and solutions include:

• Antibody concentration issues:

  • Problem: Excessive antibody concentration increases non-specific binding

  • Solution: Titrate antibody to optimal concentration; typically start at 1:500 dilution in PBS/10% FBS

• Inadequate blocking:

  • Problem: Insufficient blocking allows non-specific antibody binding

  • Solution: Extend blocking time to 20-30 minutes with PBS/10% FBS or increase blocking agent concentration

• Insufficient washing:

  • Problem: Residual unbound antibody creates diffuse background

  • Solution: Increase number or duration of wash steps; use gentle agitation during washes

• Fixation artifacts:

  • Problem: Over-fixation can increase autofluorescence or non-specific binding sites

  • Solution: Optimize fixation protocol; consider different fixatives or shorter fixation times

• Sample-specific autofluorescence:

  • Problem: Natural cellular autofluorescence in the FITC channel

  • Solution: Include unstained controls; consider autofluorescence quenching techniques

• Light exposure:

  • Problem: FITC photobleaching creating artifacts

  • Solution: Minimize light exposure during all experimental steps

How can researchers validate the specificity of AASS Antibody, FITC conjugated?

Validating antibody specificity is crucial for reliable results. For AASS Antibody, FITC conjugated:

• Genetic validation approaches:

  • CRISPR knockout or siRNA knockdown of AASS

  • Overexpression systems with tagged AASS constructs

  • Comparison of tissues/cells with known differential AASS expression

• Biochemical validation:

  • Western blot analysis to confirm binding to protein of expected molecular weight

  • Immunoprecipitation followed by mass spectrometry

  • Peptide competition assays using the immunogen (e.g., amino acids 224-364 of human AASS protein)

• Localization-based validation:

  • Co-localization with mitochondrial markers (AASS is a mitochondrial protein)

  • Comparison with other validated AASS antibodies targeting different epitopes

  • Subcellular fractionation to confirm enrichment in mitochondrial fraction

• Technical validation:

  • Comparison across multiple applications (IF, flow cytometry, Western blot)

  • Testing reactivity against recombinant AASS protein

  • Evaluating cross-reactivity with related proteins

Proper validation should demonstrate that the antibody specifically recognizes the intended target protein in its native environment and experimental conditions .

How does photobleaching affect FITC-conjugated antibodies and what strategies minimize this effect?

FITC is particularly susceptible to photobleaching compared to newer fluorophores. Understanding and mitigating this issue is important:

• Photobleaching mechanism and effects:

  • FITC molecules undergo irreversible photochemical destruction upon repeated excitation

  • Results in progressive signal loss during imaging sessions

  • Creates challenges for quantitative analysis and time-lapse imaging

  • Can lead to inconsistent results between samples imaged at different times

• Preventive strategies:

  • Store antibody preparations protected from light

  • Minimize exposure time during microscopic examination

  • Use anti-fade mounting media containing anti-oxidants

  • Reduce excitation light intensity with neutral density filters

  • Optimize instrument settings to use minimum required illumination

• Experimental approaches:

  • Image control samples and experimental samples in alternating order

  • Standardize exposure times across samples

  • Consider alternative fluorophores like Alexa Fluor 488 for extended imaging

  • For quantitative work, correct for photobleaching mathematically

Understanding FITC's photobleaching characteristics is particularly important when studying AASS distribution in mitochondria, where high-resolution imaging might require longer exposure times or repeated imaging of the same field .

What factors affect the signal-to-noise ratio when using AASS Antibody, FITC conjugated?

Multiple factors influence the signal-to-noise ratio in experiments using FITC-conjugated antibodies:

• Antibody-related factors:

  • Quality of antibody preparation (purity, specificity)

  • Fluorophore-to-protein ratio (optimal is typically 3-6 FITC molecules per antibody)

  • Antibody concentration and incubation conditions

  • Storage conditions affecting antibody integrity

• Sample-related factors:

  • Expression level of AASS in the sample

  • Sample thickness affecting antibody penetration

  • Fixation and permeabilization efficiency for accessing mitochondrial AASS

  • Autofluorescence in the FITC emission range

• Technical factors:

  • Blocking effectiveness (PBS/10% FBS recommended)

  • Washing stringency to remove unbound antibody

  • Mounting medium quality and anti-fade properties

  • Microscope optical quality and filter set specifications

• Imaging parameters:

  • Camera sensitivity and dynamic range

  • Exposure settings and signal digitization

  • Background subtraction methods

  • Deconvolution or other image processing techniques

Optimizing each of these factors through systematic testing can significantly improve detection of specific AASS signals while minimizing background interference.

How can AASS Antibody, FITC conjugated be used in multiplex immunofluorescence studies?

Multiplex immunofluorescence with AASS Antibody, FITC conjugated requires careful planning:

• Compatible fluorophore combinations:

  • FITC (excitation/emission: ~495/525nm) can be combined with:

    • DAPI for nuclear counterstaining (excitation/emission: ~358/461nm)

    • TRITC or Cy3 for a second target (excitation/emission: ~550/570nm)

    • Far-red fluorophores like Cy5 (excitation/emission: ~650/670nm)

• Experimental design strategies:

  • Sequential staining approach for antibodies from the same species

  • Simultaneous staining with antibodies from different species

  • Careful optimization of each antibody individually before combining

  • Include all necessary single-stain controls for spectral unmixing

• Biological applications:

  • Co-localization of AASS with other mitochondrial proteins

  • Correlation of AASS expression with metabolic state markers

  • Investigation of AASS distribution during mitochondrial dynamics

• Technical considerations:

  • Use of spectral unmixing for closely overlapping fluorophores

  • Confocal microscopy to reduce out-of-focus fluorescence

  • Sequential scanning to minimize bleed-through

  • Standardized acquisition settings for quantitative comparisons

Multiplex approaches are particularly valuable for studying mitochondrial proteins like AASS in their native context, allowing simultaneous visualization of organelle morphology, functional state, and protein distribution .

What approaches enable high-resolution imaging of AASS distribution using FITC-conjugated antibodies?

High-resolution imaging of AASS requires specialized techniques:

• Confocal microscopy approaches:

  • Standard confocal microscopy (resolution ~250nm laterally)

  • Airyscan or HyVolution processing for resolution enhancement

  • 3D reconstruction of z-stacks for volumetric distribution analysis

  • Spectral confocal for distinguishing FITC from autofluorescence

• Super-resolution techniques:

  • Structured Illumination Microscopy (SIM): Compatible with standard FITC preparation

  • Stimulated Emission Depletion (STED): Requires optimization for FITC

  • Single-molecule localization microscopy: May require specialized buffers

  • Expansion microscopy: Physical expansion of samples for enhanced resolution

• Sample preparation optimization:

  • Thinner sections for improved resolution

  • Optimal fixation to preserve mitochondrial ultrastructure

  • Enhanced permeabilization for antibody access to mitochondrial proteins

  • Careful blocking to maximize signal-to-noise ratio

• Analysis approaches:

  • Deconvolution algorithms to enhance image clarity

  • Quantitative co-localization with mitochondrial markers

  • Machine learning segmentation of subcellular structures

  • Correlative light and electron microscopy for ultrastructural context

These advanced imaging approaches can reveal AASS distribution patterns within mitochondria at unprecedented resolution, potentially uncovering functional microdomains or dynamic reorganization during cellular processes .

How can site-specific conjugation improve FITC-labeled antibody performance?

Site-specific conjugation represents an advanced approach to improving antibody performance:

• Limitations of conventional conjugation:

  • Random attachment to lysine residues throughout the antibody

  • Potential interference with antigen-binding regions

  • Heterogeneous products with variable performance

  • Possible multivalent interactions leading to aggregation

• Site-specific conjugation advantages:

  • Controlled attachment at specific locations away from binding sites

  • Homogeneous products with consistent performance

  • Preserved antibody affinity and specificity

  • Reduced aggregation and improved stability

• Enzymatic methods for site-specific modification:

  • Transglutaminase (MTGase) approach: Creates isopeptide bonds at specific glutamine residues

  • Prior deglycosylation using PNGase F to expose attachment sites in Fc region

  • Introduction of azide-functional handles for subsequent click chemistry

  • Strain-promoted click chemistry for fluorophore attachment

• Performance improvements:

  • More consistent fluorophore-to-protein ratio

  • Better lot-to-lot reproducibility

  • Enhanced signal-to-noise ratio in challenging applications

  • Improved stability during storage and experimental procedures

Site-specific conjugation represents the cutting edge of antibody technology, potentially offering superior FITC-conjugated AASS antibodies with enhanced performance characteristics compared to conventionally prepared conjugates .

What quantitative approaches can measure AASS expression using FITC-conjugated antibodies?

Quantitative measurement of AASS expression requires rigorous approaches:

• Flow cytometry quantification:

  • Median fluorescence intensity (MFI) measurements

  • Calibration with standardized FITC beads

  • Conversion to molecules of equivalent soluble fluorochrome (MESF)

  • Comparative analysis across multiple samples or conditions

• Quantitative microscopy approaches:

  • Standardized image acquisition parameters

  • Integrated intensity measurements within defined regions

  • Background subtraction methodologies

  • Internal reference standards for normalization

• Calibration strategies:

  • Standard curves using cells with known AASS expression levels

  • Correlation with orthogonal measures (qPCR, Western blot)

  • Inclusion of calibrated fluorescent beads in microscopy samples

  • Standardized units of measurement for cross-study comparison

• Advanced analysis methods:

  • Machine learning algorithms for automated segmentation

  • 3D quantification in confocal z-stacks

  • Correlation of AASS levels with mitochondrial morphology

  • Multi-parametric analysis combining multiple markers

Proper quantification requires careful consideration of FITC's photophysical properties, including photobleaching rates, pH sensitivity, and potential quenching in cellular environments .

How is AASS Antibody, FITC conjugated being used in current mitochondrial research?

Current mitochondrial research applications for AASS Antibody, FITC conjugated include:

• Metabolic pathway investigations:

  • Lysine degradation pathway dynamics in different metabolic states

  • Relationship between AASS levels and mitochondrial respiration

  • Correlation of AASS expression with other metabolic enzymes

  • Changes in AASS distribution during metabolic reprogramming

• Mitochondrial dynamics studies:

  • AASS localization during mitochondrial fusion and fission events

  • Distribution patterns in relation to inner membrane organization

  • Potential role in mitochondrial quality control mechanisms

  • Colocalization with mitochondrial functional domains

• Disease-related research:

  • Altered AASS expression or localization in metabolic disorders

  • Potential biomarker applications in mitochondrial dysfunction

  • Correlation with oxidative stress responses

  • Implications in neurological disorders with mitochondrial components

• Technical applications:

  • Mitochondrial isolation quality control marker

  • Evaluation of mitochondrial purification techniques

  • Benchmark for mitochondrial protein detection methods

  • Reference marker for mitochondrial subcompartment studies

These applications leverage the specificity of AASS antibodies and the direct detection capabilities of FITC conjugation to advance understanding of fundamental mitochondrial biology .

What improvements in FITC technology are enhancing antibody performance?

Recent advancements in FITC technology are improving antibody performance:

• Photobleaching resistance:

  • Modified FITC derivatives with improved photostability

  • Specialized anti-fade mounting media formulations

  • Photoprotective additives during imaging

  • Computational correction of photobleaching effects

• Conjugation chemistry improvements:

  • Site-specific conjugation methods as described earlier

  • Controlled fluorophore-to-protein ratios for optimal brightness

  • Spacer molecules to reduce fluorophore-antibody interactions

  • Purification techniques to remove unconjugated fluorophore

• Detection technology enhancements:

  • More sensitive cameras and photomultipliers

  • Advanced filter sets with improved signal separation

  • Computational approaches for signal extraction

  • Machine learning algorithms for image analysis

• Alternative approaches:

  • Brighter and more stable green fluorophores

  • pH-insensitive fluorescein derivatives

  • Quantum dot conjugation for extreme photostability

  • Enzymatic amplification of FITC signals

These improvements are enabling more sensitive and reliable detection of AASS and other targets in challenging experimental contexts, pushing the boundaries of what's possible in both basic research and clinical applications .

How can AASS Antibody, FITC conjugated contribute to understanding mitochondrial disease mechanisms?

AASS Antibody, FITC conjugated offers valuable insights into mitochondrial disease mechanisms:

• Diagnostic applications:

  • Evaluation of AASS expression patterns in patient samples

  • Correlation of AASS levels with disease severity

  • Potential biomarker for specific mitochondrial disorders

  • Monitoring therapeutic responses in mitochondrial diseases

• Mechanistic investigations:

  • AASS redistribution during mitochondrial stress

  • Role in metabolic adaptation to disease states

  • Interaction with other mitochondrial proteins in pathological conditions

  • Contribution to mitochondrial quality control mechanisms

• Therapeutic development support:

  • Target validation for drugs affecting lysine metabolism

  • Screening assays for compounds affecting AASS function

  • Monitoring mitochondrial responses to experimental therapies

  • Evaluation of mitochondrial-targeted drug delivery systems

• Model system validation:

  • Verification of disease models (cell culture, animal models)

  • Comparative analysis between model systems and human samples

  • Evaluation of genetic manipulation effects on mitochondrial function

  • Assessment of environmental factors affecting mitochondrial proteins

The ability to directly visualize AASS in cellular and tissue contexts provides unique opportunities to understand its role in health and disease, potentially leading to new diagnostic approaches or therapeutic targets .

What emerging applications are being developed for site-specifically conjugated antibodies?

Emerging applications for site-specifically conjugated antibodies like AASS Antibody, FITC conjugated include:

• Virus-based nanoparticle (VNP) functionalization:

  • Site-specific antibody attachment to VNPs for targeted delivery

  • Enhanced stability compared to conventional conjugation methods

  • Reduced aggregation through controlled orientation

  • Applications in cancer targeting and imaging

• Multimodal imaging approaches:

  • Dual-labeled antibodies with precisely positioned fluorophores and MRI contrast agents

  • Combined optical and PET imaging agents

  • Correlative light and electron microscopy probes

  • Integrated therapeutic and diagnostic (theranostic) agents

• Advanced therapeutic applications:

  • Antibody-drug conjugates with defined drug-to-antibody ratios

  • Enhanced pharmacokinetics through controlled conjugation

  • Reduced immunogenicity of conjugated products

  • Improved tissue penetration and target engagement

• Technological integration:

  • Combination with microfluidic systems for high-throughput analysis

  • Integration with in vivo imaging technologies

  • Application in protein-protein interaction screens

  • Development of advanced biosensors with precise FRET characteristics

These emerging applications represent the cutting edge of antibody technology, with site-specific FITC conjugation serving as a platform for numerous innovative research and clinical tools .