ACSL6 Antibody, FITC conjugated

What is ACSL6 antibody and what cellular functions does it target?

ACSL6 antibody targets Long-chain acyl-CoA synthetase 6 (also known as Long-chain-fatty-acid--CoA ligase 6), a 77 kDa protein involved in fatty acid metabolism . This protein plays a critical role in lipid metabolism pathways and has been acknowledged for its substantial effect on fatty acid metabolism in the brain and its involvement in facilitating spermatogenesis . Recent research has expanded our understanding of ACSL6's role in pathological conditions, particularly its involvement in cancer progression and radioresistance mechanisms .

When using ACSL6 antibody in your research, remember that you're investigating a protein that participates in critical metabolic processes, particularly in the incorporation of long-chain fatty acids into cellular metabolism. Methodologically, this antibody can be used to identify, localize, and quantify ACSL6 protein expression across various experimental models.

What are the optimal storage conditions for FITC-conjugated ACSL6 antibody?

FITC-conjugated ACSL6 antibodies require special storage considerations due to their light sensitivity. For optimal performance:

• Store the antibody in light-protected vials or wrap containers with aluminum foil to protect from light exposure

• Conjugated antibodies are stable for at least 12 months when stored at 4°C

• For longer storage (up to 24 months), dilute with up to 50% glycerol and store at -20°C to -80°C

• Avoid repeated freeze-thaw cycles as they will compromise both enzyme activity and antibody binding

The methodological approach to properly handling these conjugated antibodies is essential for maintaining their performance. When retrieving the antibody for experiments, allow it to equilibrate to room temperature in dark conditions before opening to prevent condensation, which could affect antibody concentration and stability.

What techniques can ACSL6 antibody, FITC conjugated be used for in research applications?

The FITC-conjugated ACSL6 antibody can be utilized in multiple research techniques:

When implementing these techniques, it's important to include appropriate controls. For immunofluorescence or flow cytometry applications, include an isotype control to account for non-specific binding. The FITC conjugation eliminates the need for secondary antibody incubation steps, simplifying protocols and reducing background in multi-color experiments.

What species cross-reactivity can be expected with commercially available ACSL6 antibodies?

Based on immunogen sequence analysis, ACSL6 antibodies show varying degrees of cross-reactivity:

When planning cross-species experiments, validate the antibody in your specific species of interest even if homology is predicted to be high. The methodological approach should include a positive control sample known to express ACSL6 from your species of interest, alongside negative controls where the protein is not expressed or is knocked down.

How can ACSL6 antibody be used to investigate the ACSL6-FLI1-COL-autophagy pathway in cancer research?

Recent studies have uncovered that ACSL6 interacts with Friend leukemia integration 1 transcription factor (FLI1) to regulate collagens (COLs) and ultimately affect autophagy pathways, particularly in lung cancer radiotherapy response . To investigate this pathway:

• Design coimmunoprecipitation (Co-IP) experiments using ACSL6 antibody to capture protein complexes involving ACSL6 and FLI1

  • Successful Co-IP has demonstrated direct protein-protein interaction between ACSL6 and FLI1 in H460 and H1299 lung cancer cell lines

• Implement dual immunofluorescence using FITC-conjugated ACSL6 antibody alongside a differently labeled FLI1 antibody

  • Confocal imaging has shown that colocalization between ACSL6 and FLI1 increases immediately after irradiation and remains elevated for 8 hours

• Complement immunostaining with molecular techniques to assess expression levels

  • RT-qPCR and Western blot analyses can confirm changes in expression levels of ACSL6, FLI1, and COLs (particularly COL2A1, COL4A3, and COL9A3)

• Assess autophagy markers in relation to ACSL6 expression

  • Monitor LC3, P62, and BCL2 levels as indicators of autophagy flux in response to ACSL6 manipulation

Methodologically, control experiments should include ACSL6 knockout or knockdown models to verify pathway specificity. Time-course experiments are crucial when studying radiation response, as the research indicates temporal dynamics in the interaction between ACSL6 and FLI1, with colocalization fading after 24 hours post-irradiation .

What controls should be implemented when using FITC-conjugated ACSL6 antibody for quantitative fluorescence imaging?

For rigorous quantitative analysis with FITC-conjugated ACSL6 antibody:

• Autofluorescence control

  • Include unstained samples to measure natural tissue/cell autofluorescence in the FITC channel

  • This is particularly important in tissues with high endogenous fluorescence (e.g., lung tissue)

• Isotype control

  • Use a FITC-conjugated isotype-matched control antibody (same host species, same immunoglobulin class)

  • Apply at the same concentration as the ACSL6 antibody

• Negative control

  • If possible, include ACSL6 knockout or knockdown samples

  • Alternatively, use samples known not to express ACSL6

• Positive control

  • Include samples with confirmed high ACSL6 expression

  • Consider using lung cancer cell lines such as H460 or H1299, which have been documented to express ACSL6

• Fluorescence calibration

  • Use calibration beads with known FITC fluorescence intensities

  • Create a standard curve to enable quantitative comparison between experiments

The methodological approach should include standardized image acquisition parameters (exposure time, gain, offset) across all samples. When analyzing colocalization, as seen in studies examining ACSL6-FLI1 interaction , proper channel alignment and correction for chromatic aberration are essential for accurate results.

How can FITC-conjugated ACSL6 antibody be used to investigate the role of ACSL6 in radiotherapy resistance?

Studies have indicated that ACSL6 may influence radiotherapy resistance in lung cancer through the ACSL6-FLI1-COL-autophagy axis . To investigate this:

• Design radiation dose-response experiments

  • Culture cells with varying ACSL6 expression levels (normal, overexpressed, knocked down)

  • Treat with increasing radiation doses (e.g., 0, 2, 4, 8 Gy)

  • Use FITC-conjugated ACSL6 antibody to monitor expression and localization changes

• Perform time-course experiments post-irradiation

  • Monitor ACSL6 expression at multiple timepoints (0h, 8h, 24h, 48h)

  • Research has shown that ACSL6-FLI1 colocalization changes over time after irradiation

• Assess autophagy flux in relation to ACSL6 expression

  • Use FITC-conjugated ACSL6 antibody in combination with autophagy markers

  • Compare autophagy levels between ACSL6-normal and ACSL6-deficient cells after radiation

• Evaluate clonogenic survival

  • Conduct colony formation assays with irradiated cells

  • Compare survival between ACSL6-manipulated and control cells

  • Research has shown that ACSL6 knockout combined with autophagy inhibition produces the greatest radiosensitizing effect

Methodologically, it is crucial to include proper controls and to standardize radiation delivery. The research indicates that IR-elevated ACSL6 expression modulates lung cancer radiosensitivity by tuning the FLI1-COL-autophagy pathway , suggesting that analyzing these components in tandem would provide the most comprehensive understanding.

What approaches can resolve potential discrepancies in ACSL6 antibody staining patterns between different experimental models?

When facing inconsistent staining patterns across different models:

• Validate antibody specificity in each model

  • Perform Western blots to confirm the antibody detects a protein of the expected size (~77 kDa)

  • Include ACSL6 knockout or knockdown controls specific to each model system

• Compare fixation and permeabilization methods

  • Different tissue/cell types may require optimized protocols

  • Test multiple fixation methods (paraformaldehyde, methanol, acetone)

  • Adjust permeabilization conditions (detergent type, concentration, duration)

• Analyze potential post-translational modifications

  • ACSL6 function may be regulated by modifications that affect antibody binding

  • Consider using phospho-specific antibodies if phosphorylation is suspected

• Examine expression levels quantitatively

  • Use RT-qPCR to measure mRNA levels

  • Compare protein levels via Western blot

  • Correlate with fluorescence intensity from immunostaining

Methodologically, a systematic approach to troubleshooting is essential. Begin with antibody validation in each model, then optimize sample preparation, and finally compare results across multiple detection methods. When studying ACSL6 in relation to cancer radiotherapy resistance , remember that radiation exposure itself alters ACSL6 expression patterns, which must be accounted for in experimental design.

What analytical techniques can differentiate between specific and non-specific binding of FITC-conjugated ACSL6 antibody in complex tissue samples?

For distinguishing specific from non-specific binding:

• Competitive inhibition analysis

  • Pre-incubate the antibody with excess blocking peptide (e.g., catalog #AAP41858 for ARP41858_P050-FITC antibody)

  • Compare staining with and without peptide competition

  • Specific staining should be significantly reduced or eliminated

• Fluorescence spectral unmixing

  • Acquire full emission spectra at each pixel

  • Computationally separate FITC signal from autofluorescence

  • Particularly useful in tissues with high autofluorescence (e.g., lung tissue in ACSL6 cancer studies)

• Multi-antibody validation

  • Use two different ACSL6 antibodies targeting different epitopes

  • True positive signals should show colocalization

• Correlation with functional assays

  • Compare FITC-ACSL6 staining with enzymatic activity measurements

  • ACSL6 has acyl-CoA synthetase activity that can be measured biochemically

• Single-cell analysis techniques

  • Use flow cytometry to correlate FITC-ACSL6 signal with cell type-specific markers

  • Apply machine learning algorithms to identify staining patterns associated with specific cell populations

The methodological approach should include quantitative image analysis, with signal-to-noise ratio calculations for each sample. When studying ACSL6 in the context of the FLI1-COL-autophagy axis , combining immunofluorescence with functional assays of autophagy will provide more conclusive evidence of specific binding related to the biological pathway of interest.

How can FITC-conjugated ACSL6 antibody be optimized for multi-color immunofluorescence studies examining the ACSL6-FLI1-COL pathway?

For effective multi-color immunofluorescence examining the ACSL6-FLI1-COL pathway:

• Strategic fluorophore selection

  • Pair FITC-conjugated ACSL6 antibody (green fluorescence) with spectrally distinct fluorophores

  • For FLI1: Consider red fluorophores (e.g., Cy3, TRITC) as successfully used in previous colocalization studies

  • For COLs: Far-red fluorophores (e.g., Cy5, AF647) work well to complete the panel

• Sequential staining protocol

  • Begin with the weakest signal (often ACSL6) and end with the strongest

  • Block between sequential antibody applications to prevent cross-reactivity

  • Consider tyramide signal amplification for weak signals

• Spectral overlap correction

  • Perform single-color controls for each fluorophore

  • Use these to establish compensation settings in confocal microscopy or flow cytometry

  • Apply spectral unmixing algorithms for closely overlapping signals

• Validation controls

  • Use cells with known expression patterns of ACSL6, FLI1, and COLs

  • Include radiation-treated samples to capture the dynamic relationship between these proteins

Research has shown that the interaction between ACSL6 and FLI1 changes temporally after irradiation, with colocalization visible immediately after irradiation and remaining increased for 8 hours before fading at 24 hours . This temporal dynamic should inform your experimental design, with multiple timepoints collected to capture the changing interactions.

What methodological approaches can address inconsistent results when using ACSL6 antibody for detecting radiation-induced changes in protein expression?

To address variability in radiation response studies:

• Standardize radiation delivery

  • Use calibrated radiation sources with verified dose rates

  • Ensure uniform exposure across all samples

  • Document radiation parameters (dose, dose rate, energy)

• Establish time-course protocols

  • Studies show that ACSL6-FLI1 interactions change over time post-irradiation

  • Collect samples at multiple timepoints (0h, 8h, 24h, 48h)

  • Analyze both immediate and delayed responses

• Control for cell cycle effects

  • Radiation sensitivity varies with cell cycle phase

  • Consider synchronizing cells before irradiation

  • Use cell cycle markers alongside ACSL6 staining

• Quantify expression changes systematically

  • Use digital image analysis for immunofluorescence

  • Employ Western blot with densitometry for protein quantification

  • Apply RT-qPCR for transcriptional changes

• Address heterogeneity in cell populations

  • Consider single-cell analysis techniques

  • Use flow cytometry to correlate ACSL6 expression with cell type markers

  • Apply spatial analysis in tissue samples to identify microenvironmental effects

Research has demonstrated that IR exposure significantly increases ACSL6 expression in human lung tumor samples , but this response may vary across different tumor types or genetic backgrounds. The methodological approach should account for this biological variability by including appropriate controls and sufficiently large sample sizes.

How can researchers validate that observed fluorescence signals from FITC-conjugated ACSL6 antibody accurately represent functional protein in the ACSL6-FLI1-COL-autophagy pathway?

To validate that fluorescence signals correlate with functional protein:

• Combine imaging with functional assays

  • Correlate FITC-ACSL6 signal intensity with acyl-CoA synthetase activity measurements

  • Use cell fractionation to confirm subcellular localization suggested by imaging

• Apply genetic manipulation controls

  • Use CRISPR/Cas9 to introduce tagged ACSL6 (e.g., with a different fluorophore)

  • Compare endogenous (antibody-detected) vs. tagged protein localization

  • Employ ACSL6 knockdown or knockout controls

• Verify pathway functionality

  • Measure downstream effects on FLI1 nuclear translocation

  • Assess COL expression levels (particularly COL2A1, COL4A3, and COL9A3)

  • Quantify autophagy markers (LC3, P62, BCL2) in relation to ACSL6 levels

• Confirm protein-protein interactions

  • Supplement imaging with biochemical techniques like Co-IP

  • Use proximity ligation assay (PLA) to verify close associations between ACSL6 and FLI1

  • Apply FRET (Fluorescence Resonance Energy Transfer) to confirm direct interactions

• Correlate with physiological outcomes

  • Link ACSL6 expression to radiation sensitivity in clonogenic assays

  • Connect ACSL6-FLI1-COL-autophagy pathway activity to tumor growth in animal models

Research has shown that the ACSL6-FLI1-COL-autophagy axis affects radiotherapy outcomes in lung cancer models . A methodological approach that combines fluorescence imaging with functional readouts will provide the strongest evidence that the observed signals represent biologically relevant protein activity.

What emerging applications of FITC-conjugated ACSL6 antibody might advance our understanding of ACSL6 in disease processes beyond current research focus?

While current research has focused on ACSL6's role in lung cancer radiotherapy , several emerging applications may expand our understanding:

• Neurodegenerative disease research

  • ACSL6 has known effects on fatty acid metabolism in the brain

  • FITC-conjugated antibodies could help map ACSL6 distribution across different brain regions

  • May provide insights into metabolic aspects of neurodegeneration

• Metabolic disorder investigations

  • As a key enzyme in fatty acid metabolism, ACSL6 may play roles in metabolic syndromes

  • Flow cytometry with FITC-ACSL6 antibody could identify cellular subpopulations with altered expression

• Developmental biology

  • ACSL6's involvement in spermatogenesis suggests roles in developmental processes

  • Time-lapse imaging with FITC-conjugated antibodies in cellular models could reveal dynamic expression patterns

• Single-cell analysis technologies

  • Combining FITC-ACSL6 antibody with single-cell RNA-seq or mass cytometry

  • Could reveal previously unrecognized heterogeneity in ACSL6 expression across cell populations

The methodological approach for these emerging applications should build on established protocols while adapting to specific requirements of each research area. As new connections between ACSL6 and disease processes are discovered, researchers should consider how the ACSL6-FLI1-COL-autophagy axis might function in different cellular contexts.

How might technological advances in imaging and antibody development enhance the utility of FITC-conjugated ACSL6 antibodies in future research?

Future technological advances will likely expand research capabilities:

• Super-resolution microscopy applications

  • Techniques like STORM, PALM, or STED could reveal nano-scale organization of ACSL6

  • May identify previously undetectable protein complexes involving ACSL6 and FLI1

• Multiplex imaging systems

  • Advanced spectral imaging allowing simultaneous detection of 10+ targets

  • Could enable comprehensive mapping of the entire ACSL6-FLI1-COL-autophagy pathway in a single sample

• Live-cell imaging adaptations

  • Development of cell-permeable antibody fragments for real-time visualization

  • Would allow tracking of ACSL6 dynamics during radiation response without fixation

• Antibody engineering improvements

  • Site-specific conjugation techniques for more consistent FITC labeling

  • Potentially smaller antibody formats (nanobodies, affibodies) for better tissue penetration

• Artificial intelligence-assisted analysis

  • Machine learning algorithms for automated detection of ACSL6 expression patterns

  • Could identify subtle changes in subcellular localization not apparent to human observers

The methodological implications of these advances include the need for standardized calibration procedures, careful validation against existing techniques, and development of new quantitative analysis frameworks. As the field moves toward higher dimensional data, computational approaches will become increasingly important for extracting meaningful biological insights about ACSL6 function in normal physiology and disease states like cancer .