ATAT1 Antibody, FITC conjugated

What is ATAT1 and what cellular functions does it regulate?

ATAT1 (Alpha-tubulin N-acetyltransferase 1) specifically acetylates 'Lys-40' in alpha-tubulin on the lumenal side of microtubules. This enzyme plays several critical roles in cellular function:

• Promotes microtubule destabilization and accelerates microtubule dynamics, which may be independent of its acetylation activity

• Enters microtubules through each end and diffuses throughout the lumen

• Selectively acetylates long/old microtubules due to its slow enzymatic rate

• Required for normal sperm flagellar function

• Promotes directional cell locomotion and chemotaxis through AP2A2-dependent acetylation of alpha-tubulin at clathrin-coated pits concentrated at the leading edge of migrating cells

• May facilitate primary cilium assembly

Research has demonstrated that ATAT1 is indispensable for tubulin hyperacetylation in response to cellular stressors including high salt, high glucose, and hydrogen peroxide-induced oxidative stress .

What are the key applications for ATAT1 antibody, FITC conjugated?

ATAT1 antibody, FITC conjugated is primarily used in the following applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): For quantitative detection of ATAT1 in samples

• Flow cytometry: For analysis of ATAT1 expression at the single-cell level

• Immunofluorescence microscopy: For visualization of ATAT1 localization in cells and tissues

While ELISA is the most commonly validated application, researchers have adapted these antibodies for various specialized techniques depending on their specific experimental requirements.

What are the typical reactivity and specificity parameters for ATAT1 antibodies?

The ATAT1 antibody, FITC conjugated typically shows the following characteristics:

• Species reactivity: Primarily human, though some cross-reactivity with mouse and rat has been reported

• Isotype: IgG (typically from rabbit sources)

• Molecular weight detection: Approximately 42-47 kDa (observed range)

• Immunogen: Usually a recombinant fragment of human ATAT1 protein (amino acids 194-238)

Researchers should verify the specific reactivity parameters for their chosen antibody, as cross-reactivity with other proteins in the acetyltransferase family may occur depending on the epitope recognized.

How can ATAT1 antibody, FITC conjugated be optimized for studying microtubule acetylation dynamics?

For researchers investigating microtubule acetylation dynamics using ATAT1 antibody, FITC conjugated:

• Dual staining approach: Combine ATAT1 antibody with acetylated tubulin antibodies to correlate enzyme localization with substrate modification. This allows tracking of both the enzyme and its activity product.

• Live cell imaging optimization:

  • Use lower antibody concentrations (1:500-1:1000) to minimize potential interference with enzyme function

  • Employ microinjection techniques rather than cell permeabilization when possible

  • Utilize low-intensity illumination to prevent photobleaching of the FITC conjugate

• Vesicular transport analysis: Since ATAT1 is enriched in vesicles that transport along axons, researchers should design time-lapse imaging protocols to capture the dynamic movement of ATAT1-positive vesicles along microtubules .

• Stress response studies: When examining stress-induced tubulin hyperacetylation, include appropriate positive controls (cells exposed to high salt, high glucose, or oxidative stress) and negative controls (Atat1 knockout cells) to validate antibody specificity under these conditions .

What methodological considerations should be addressed when using ATAT1 antibody, FITC conjugated in neuronal studies?

When employing ATAT1 antibody, FITC conjugated in neuronal research:

• Fixation protocol optimization:

  • For cultured neurons: 4% paraformaldehyde for 15 minutes at room temperature preserves both microtubule structure and ATAT1 localization

  • For tissue sections: 2% paraformaldehyde with 0.1% glutaraldehyde maintains both enzyme localization and neuronal architecture

• Subcellular localization considerations:

  • ATAT1 exists in both cytosolic and vesicle-associated pools

  • The cytosolic ATAT1 is predominantly located at the external surface of vesicles, not encapsulated within the vesicular lumen

  • Mild proteinase K digestion can be employed to distinguish between internal and external vesicle proteins, with ATAT1 showing sensitivity to this treatment

• Axonal transport experimental design:

  • ATAT1 is crucial for proper axonal transport, and knockout/knockdown of Atat1 impairs both anterograde and retrograde transport

  • Researchers should consider the bidirectional nature of ATAT1's role in axonal transport when designing experiments

  • The acetyl mimic α-tubulin K40Q can rescue transport deficits in ATAT1-deficient neurons, providing a valuable control for specificity

How does the binding domain of ATAT1 impact antibody selection and experimental design?

The binding domain of ATAT1 has significant implications for antibody selection and experiment design:

$**Table 1**: ATAT1 Isoform Characteristics and Associated Antibody Considerations$

What is the recommended approach for validating ATAT1 antibody, FITC conjugated specificity?

A comprehensive validation strategy for ATAT1 antibody, FITC conjugated should include:

• Genetic validation:

  • Test antibody in Atat1 knockout or knockdown models to confirm specificity

  • Include wild-type controls for comparative analysis

• Peptide competition assays:

  • Pre-incubate antibody with excess immunizing peptide before application

  • Loss of signal confirms epitope specificity

• Western blot confirmation:

  • Verify detection of a single band at the expected molecular weight (42-47 kDa)

  • Compare with multiple ATAT1 antibodies recognizing different epitopes

• Immunofluorescence validation:

  • Compare staining patterns with multiple ATAT1 antibodies

  • Confirm colocalization with known ATAT1 interactors (e.g., vesicular markers)

  • Validate subcellular distribution patterns against published literature

• Functional validation:

  • Confirm that detected protein correlates with known ATAT1 functions (e.g., tubulin acetylation)

What controls are essential when performing flow cytometry with ATAT1 antibody, FITC conjugated?

When performing flow cytometry with ATAT1 antibody, FITC conjugated, the following controls are critical:

• Isotype control:

  • Use FITC-conjugated rabbit IgG (for rabbit-derived antibodies) at the same concentration

  • Essential for distinguishing non-specific binding from true signal

• Unstained control:

  • Include cells without any antibody to establish autofluorescence baseline

  • Particularly important for cells with high autofluorescence (e.g., neurons)

• Single-color controls:

  • When performing multicolor cytometry, include single-stained samples for compensation

  • Critical for accurate signal separation in FITC channel

• Biological controls:

  • Positive control: Cells known to express high levels of ATAT1

  • Negative control: Atat1 knockout/knockdown cells

  • Treatment controls: Cells with induced ATAT1 expression or activity

• Titration controls:

  • Perform antibody titration to determine optimal signal-to-noise ratio

  • For ATAT1 antibody, FITC conjugated, the recommended dilution range is typically 1:50-1:500 depending on application

How should researchers distinguish between specific binding and background when using ATAT1 antibody, FITC conjugated?

To differentiate specific binding from background when using ATAT1 antibody, FITC conjugated:

• Optimize blocking conditions:

  • Use 5-10% normal serum from the species in which the secondary antibody was raised

  • Include 0.1-0.3% Triton X-100 for intracellular targets

  • Consider adding 1% BSA to reduce non-specific binding

• Implement appropriate negative controls:

  • Include samples stained with isotype control antibodies

  • Use Atat1 knockout or knockdown samples when available

  • Include secondary-only controls to assess background

• Signal quantification strategies:

  • Calculate signal-to-noise ratio by comparing mean fluorescence intensity (MFI) of test samples to isotype controls

  • MFI ratio >3 typically indicates specific binding

  • For flow cytometry, define positive populations using fluorescence minus one (FMO) controls

• Spectral considerations:

  • Account for potential spectral overlap when using multiple fluorophores

  • FITC excitation/emission (494/518 nm) may overlap with other green fluorophores

  • Proper compensation is essential for multicolor experiments

What are the recommended storage and handling conditions for ATAT1 antibody, FITC conjugated?

For optimal performance of ATAT1 antibody, FITC conjugated:

• Storage recommendations:

  • Store at -20°C for long-term preservation

  • For short-term storage (1 month), 4°C is acceptable

  • Avoid repeated freeze-thaw cycles by preparing single-use aliquots

• Buffer composition:

  • Typically stored in PBS with 0.02-0.03% sodium azide and 50% glycerol, pH 7.3-7.4

  • Some formulations may include 0.1% BSA as a stabilizer

• Light sensitivity precautions:

  • Store FITC-conjugated antibodies in amber vials or wrapped in aluminum foil

  • Minimize exposure to light during all handling steps to prevent photobleaching

  • Work under reduced ambient lighting when possible

• Reconstitution of lyophilized antibodies:

  • Use sterile ddH₂O or buffer recommended by the manufacturer

  • Allow complete dissolution before use (typically 5-10 minutes at room temperature)

  • Centrifuge briefly to collect all liquid at the bottom of the vial

What strategies can researchers employ to optimize antibody dilution for ATAT1 antibody, FITC conjugated?

To determine the optimal dilution for ATAT1 antibody, FITC conjugated:

• Application-specific titration approach:

  • ELISA: Start with a dilution series from 1:100 to 1:5000

  • Flow cytometry: Begin with 1:50 to 1:500 dilution range

  • Immunofluorescence: Test dilutions from 1:50 to 1:500

• Titration methodology:

  • Prepare serial dilutions of antibody (e.g., 1:50, 1:100, 1:250, 1:500, 1:1000)

  • Apply to identical samples under standardized conditions

  • Evaluate signal-to-noise ratio at each dilution

  • Select the highest dilution that maintains robust specific signal

• Sample-specific considerations:

  • Higher antibody concentrations may be needed for fixed tissues compared to cell lines

  • Primary cells often require more antibody than established cell lines

  • Expression level of target protein impacts optimal dilution

• Signal intensity assessment:

  • For flow cytometry: Calculate staining index (SI) = (MFI positive - MFI negative)/2 × SD of negative

  • Higher SI values indicate better separation of positive and negative populations

How can researchers troubleshoot common issues with ATAT1 antibody, FITC conjugated?

For troubleshooting problems with ATAT1 antibody, FITC conjugated:

• Weak or no signal:

  • Verify target protein expression in your sample

  • Increase antibody concentration or incubation time

  • Check fluorescence microscope/flow cytometer settings (FITC channel)

  • Ensure proper permeabilization for intracellular targets

  • Consider alternative fixation methods that better preserve the epitope

• High background:

  • Increase blocking time or concentration

  • Reduce primary antibody concentration

  • Include additional wash steps

  • Use centrifugation to remove cell debris before analysis

  • Consider autofluorescence quenching reagents

• Non-specific binding:

  • Verify antibody specificity using knockout/knockdown controls

  • Increase blocking agent concentration

  • Add 0.1-0.5% Tween-20 to wash buffers

  • Filter antibody solutions to remove aggregates

• FITC-specific issues:

  • FITC is pH-sensitive (optimal at pH 8.0), check buffer pH

  • Photobleaching can occur; minimize light exposure

  • FITC has relatively low photostability compared to Alexa Fluors; consider alternative conjugates for prolonged imaging

How can ATAT1 antibody, FITC conjugated be used to study vesicular transport mechanisms?

ATAT1 antibody, FITC conjugated is particularly valuable for investigating vesicular transport mechanisms:

• Live cell imaging applications:

  • ATAT1 is transported on the cytosolic side of motile vesicles

  • Time-lapse microscopy can track ATAT1-positive vesicles moving bidirectionally along axons

  • Combine with lysosomal markers (e.g., LAMP1) to study co-transport dynamics

• Quantitative vesicular transport analysis:

  • Measure parameters including average velocity, instantaneous velocity, run length, and pausing time

  • Compare wild-type to ATAT1-deficient neurons to assess functional importance

  • Analyze both anterograde and retrograde transport independently

• Vesicular proteomics integration:

  • ATAT1 has been detected in vesicular proteomes alongside motor proteins

  • LC-MS/MS analysis can identify ATAT1-interacting proteins in vesicular fractions

  • Subcellular fractionation followed by Western blot can confirm ATAT1 enrichment in vesicular fractions

• Methodological approach:

  • Transfect neurons with ATAT1-GFP and vesicular markers (e.g., BDNF-mCherry)

  • Culture neurons in microfluidic chambers to isolate axons

  • Perform time-lapse imaging using low-intensity illumination

  • Use kymograph analysis to quantify transport parameters

What considerations apply when using ATAT1 antibody, FITC conjugated for studying neurodevelopmental processes?

When using ATAT1 antibody, FITC conjugated to investigate neurodevelopmental processes:

• Developmental timing:

  • ATAT1 expression varies across developmental stages

  • Ventricular dilation in ATAT1-deficient mice begins at postnatal day 5

  • Early embryonic development may be less affected than postnatal processes

• Region-specific considerations:

  • ATAT1 is highly expressed throughout the brain, including septum, striatum, and cerebral cortex

  • Different brain regions show varying sensitivity to ATAT1 deficiency

  • Septum and striatum show hypoplasia in ATAT1-deficient mice

• Neuronal migration analysis:

  • ATAT1 deficiency impairs neuronal migration to septum and striatum

  • Birth-dating experiments with BrdU can quantify migration defects

  • Consider using in utero electroporation to label and track specific neuronal populations

• Experimental approaches:

  • For cortical development studies, combine ATAT1 antibody, FITC conjugated with markers for neural stem/progenitor cells (Sox2, Tbr2) and lamination (Cux1, Cux2, Tbr1, Ctip2)

  • For migration studies, pulse-chase experiments with BrdU or EdU labeling

  • For cellular architecture analysis, combine with Golgi staining or neuronal tracing methods

How can ATAT1 antibody, FITC conjugated be used to investigate stress-induced tubulin modifications?

For studying stress-induced tubulin modifications using ATAT1 antibody, FITC conjugated:

• Experimental stress induction protocols:

  • High salt stress: 100-150 mM NaCl added to culture medium

  • High glucose stress: 25-30 mM glucose in culture medium

  • Oxidative stress: 50-200 μM hydrogen peroxide treatment

  • Control conditions: standard culture medium

• Time-course analysis considerations:

  • Tubulin hyperacetylation occurs rapidly (within 15-30 minutes)

  • Include multiple time points (baseline, 15 min, 30 min, 1 hour, 3 hours)

  • ATAT1 is required for stress-induced tubulin hyperacetylation

• Multi-parameter analysis:

  • Combine ATAT1 antibody, FITC conjugated with antibodies against acetylated tubulin

  • Include markers for other tubulin PTMs (polyglutamylation, tyrosination)

  • Assess ATAT1 localization changes in response to stress

• Controls and validation:

  • Include ATAT1 knockout/knockdown cells as negative controls

  • Use HDAC6 inhibitors (e.g., tubastatin A) as positive controls for tubulin hyperacetylation

  • Verify specificity by showing that other tubulin PTMs remain unchanged

How does ATAT1 antibody, FITC conjugated compare to other STAT protein antibodies in flow cytometry applications?

When comparing ATAT1 antibody, FITC conjugated to STAT protein antibodies (STAT1, STAT6) in flow cytometry:

• Protocol considerations:

  • STAT protein detection typically requires methanol fixation (80%) for 5 minutes

  • ATAT1 detection may be more effective with paraformaldehyde fixation

  • Both require permeabilization (0.1% PBS-Triton X-100 for 15 minutes)

• Signal intensity and resolution:

  • STAT1 phospho-specific antibodies may require stronger signals due to lower abundance

  • ATAT1 antibodies detect total protein rather than specific phosphorylation states

  • Both benefit from similar blocking conditions (10% normal goat serum)

• Dilution optimization:

  • STAT6-FITC antibodies show optimal performance at approximately 1:50 dilution

  • ATAT1-FITC antibodies may require similar dilutions (1:50-1:100)

  • Both require acquisition of >5,000 events for reliable analysis

• Control considerations:

  • Similar isotype controls are used (FITC-conjugated rabbit IgG)

  • Both require unstained samples as additional controls

  • STAT proteins may benefit from stimulation controls (e.g., IFN treatment for STAT1)

$**Table 2**: Comparative Flow Cytometry Parameters for FITC-Conjugated Antibodies$

What are the comparative advantages of using FITC conjugation versus other fluorophores for ATAT1 antibodies?

When comparing FITC conjugation to other fluorophores for ATAT1 antibodies:

• Spectral characteristics:

  • FITC: Excitation 494 nm, Emission 518 nm (green)

  • Alternatives: PE (yellow-orange), APC-Cy7 (far red), Alexa Fluor 647 (far red)

  • FITC is compatible with standard FITC/GFP filter sets found in most laboratories

• Sensitivity and brightness:

  • FITC has moderate brightness (lower than PE, Alexa Fluor 488)

  • FITC shows higher background autofluorescence in some tissues

  • PE offers 5-10× higher brightness but is more susceptible to photobleaching

  • Alexa Fluor dyes provide greater photostability and brightness

• Application-specific considerations:

  • For flow cytometry: PE or APC-Cy7 may provide better separation of populations

  • For imaging: Alexa Fluor 488 offers better photostability than FITC

  • For multiplexing: Far-red fluorophores (Alexa 647) reduce spectral overlap

• Technical limitations:

  • FITC is pH-sensitive (optimal at pH 8.0)

  • FITC photobleaches more rapidly than newer fluorophores

  • Biological samples often have higher autofluorescence in the FITC/GFP channel

  • Alexa Fluor conjugates are generally more expensive but offer superior performance

What methodological differences exist when comparing antibody validation approaches across different research applications?

When comparing antibody validation approaches across different research applications:

• Application-specific validation hierarchy:

  • Western blot: Focus on molecular weight and band specificity

  • Immunohistochemistry: Emphasize tissue distribution patterns and cellular localization

  • Flow cytometry: Prioritize population separation and signal-to-noise ratio

  • ELISA: Concentrate on sensitivity and dynamic range

• Validation stringency requirements:

  • Basic research: Knockout/knockdown controls strongly recommended

  • Diagnostic applications: Requires FDA/regulatory validation protocols

  • Reproducibility across multiple antibody lots becomes crucial for longitudinal studies

• Technical validation approaches:

  • Western blot validation is common for flow cytometry antibodies

  • Immunoprecipitation followed by mass spectrometry provides rigorous validation

  • For ATAT1, validation should include assessment of tubulin acetylation levels

• Cross-platform consideration:

  • FITC conjugates validated for flow cytometry may require additional optimization for microscopy

  • Antibodies performing well in fixed tissues may not work optimally in live-cell applications

  • Different fixation methods may be required for preserving epitopes across applications