IST1 Antibody, FITC conjugated

What is IST1 protein and what are its primary cellular functions?

IST1 is an ESCRT-III-like protein involved in specific functions of the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery. This approximately 40 kDa protein plays several critical roles in cellular processes, including efficient abscission during cytokinesis and recruitment of VPS4A/VPS4B to the midbody of dividing cells . During late anaphase, IST1 participates in nuclear envelope reassembly and mitotic spindle disassembly through interaction with the ESCRT-III complex, mediating the recruitment of SPAST to the nuclear membrane and facilitating microtubule severing . Additionally, IST1 regulates early endosomal tubulation together with the ESCRT-III complex by mediating recruitment of SPAST .

IST1 (also known as hIST1, Putative MAPK-activating protein PM28, and KIAA0174) is encoded by the KIAA0174 gene with UniProt ID P53990 . Its involvement in the multivesicular body (MVB) pathway is not fully established, though it plays documented roles in endosomal sorting and recycling pathways .

What applications can FITC-conjugated IST1 antibodies be used for in research?

FITC-conjugated IST1 antibodies are versatile research tools that can be employed in multiple applications:

The FITC-conjugated antibody is particularly advantageous for direct detection applications, eliminating the need for secondary antibodies in fluorescence-based techniques like immunofluorescence microscopy .

What is the significance of FITC conjugation for IST1 antibodies?

FITC (Fluorescein isothiocyanate) conjugation provides several advantages in IST1 antibody applications:

• Enables direct visualization of IST1 without secondary antibodies, simplifying experimental protocols and reducing background

• FITC emits green fluorescence (emission maximum ~519 nm) when excited with appropriate wavelength light (excitation maximum ~495 nm)

• Facilitates multicolor imaging when combined with antibodies labeled with spectrally distinct fluorophores

• Reduces potential cross-reactivity issues that may occur with two-step detection systems

• Streamlines protocols by eliminating secondary antibody incubation steps

When using FITC-conjugated antibodies, researchers must protect samples from light during processing to prevent photobleaching of the fluorophore, which can affect signal intensity and experimental outcomes .

How should researchers handle and store FITC-conjugated IST1 antibodies for optimal performance?

Proper handling and storage are essential for maintaining the functionality of FITC-conjugated IST1 antibodies:

• Store at -20°C in the dark to protect the FITC fluorophore from photobleaching

• Maintain antibody stability for up to one year when properly stored

• Aliquoting may be unnecessary for -20°C storage according to some manufacturers, though dividing into small aliquots is generally recommended to avoid repeated freeze-thaw cycles

• Typical storage buffer composition includes PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Some preparations may contain 0.1% BSA as a stabilizer

• Always protect from light during handling and experimental procedures to preserve fluorescence signal

• Follow manufacturer-specific recommendations, as storage conditions may vary between products

What protocols should be followed for immunofluorescence studies using FITC-conjugated IST1 antibodies?

When designing immunofluorescence experiments with FITC-conjugated IST1 antibodies, researchers should follow these methodological steps:

• Sample preparation:

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

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

• Blocking and antibody incubation:

  • Block with 1-5% BSA or 5-10% normal serum in PBS for 30-60 minutes

  • Incubate with FITC-conjugated IST1 antibody at recommended dilution (1:100-1:500 for immunofluorescence) overnight at 4°C or 1-2 hours at room temperature

  • Protect from light during all incubation steps to prevent photobleaching

• Nuclear counterstaining and mounting:

  • Counterstain nuclei with DAPI (1 μg/ml) for 5 minutes

  • Mount with anti-fade mounting medium to minimize photobleaching

Expected IST1 localization patterns include cytoplasmic distribution, punctate endosomal structures, and enrichment at cell bridges and midbodies during cytokinesis . For co-localization studies, consider markers for early endosomes (EEA1), ESCRT machinery components, or cytokinetic structures .

How can researchers validate the specificity of IST1 antibodies in their experimental systems?

Validation of IST1 antibody specificity is crucial for reliable research outcomes:

• Genetic knockdown/knockout controls:

  • Use siRNA knockdown of IST1 (>90% depletion has been achieved with specific siRNA duplexes)

  • Compare antibody signal between control and knockdown samples

• Recombinant protein controls:

  • Test antibody recognition of recombinant IST1 protein (available commercial antibodies often use recombinant human IST1 protein spanning amino acids 172-284 as immunogen)

  • Perform peptide competition assays with the immunogenic peptide

• Cross-reactivity assessment:

  • Test in multiple species if cross-reactivity is claimed (most IST1 antibodies are human-specific)

  • Test in verified IST1-expressing cell lines (A549, HeLa, PC-3 cells)

• Correlation with other detection methods:

  • Compare results with other validated antibodies targeting different epitopes of IST1

  • Correlate with overexpression studies using tagged IST1 constructs

• Expected localization patterns:

  • Verify cytoplasmic distribution and punctate structures

  • Confirm enrichment at cell bridges and midbodies during cytokinesis

  • Validate co-localization with known interaction partners like CHMP1B or SNX15

This comprehensive validation approach increases confidence in experimental results and helps troubleshoot unexpected findings.

What are the recommended protocols for using FITC-conjugated IST1 antibodies in ELISA?

For optimal use of FITC-conjugated IST1 antibodies in ELISA applications:

Recommended dilution range: 1:100-1:500 (optimize for specific antibody lot and experimental conditions)

Direct ELISA protocol:

• Coat plate with antigen (recombinant IST1 protein or cell lysate) in carbonate buffer (pH 9.6) overnight at 4°C

• Wash 3 times with PBS-T (PBS + 0.05% Tween-20)

• Block with 1-5% BSA in PBS for 1-2 hours at room temperature

• Wash 3 times with PBS-T

• Add FITC-conjugated IST1 antibody at optimized dilution in antibody dilution buffer

• Incubate 1-2 hours at room temperature or overnight at 4°C (protect from light)

• Wash 5 times with PBS-T

• Detect fluorescence signal directly using a fluorescence plate reader (excitation ~495 nm, emission ~520 nm)

Optimization considerations:

• Perform antibody titration to determine optimal concentration

• Include standard curve using recombinant IST1 protein (amino acids 172-284 as used in many commercial antibodies)

• Protect from light throughout the procedure to prevent photobleaching of FITC

• Consider plate background fluorescence in data analysis

How can IST1 antibodies be used to study endosomal tubulation and recycling pathways?

IST1 plays a critical role in endosomal trafficking, particularly in regulating endosomal tubulation and recycling pathways. Researchers can use IST1 antibodies to investigate these processes through several approaches:

• Visualization of endosomal tubulation dynamics:

  • Deplete IST1 using validated siRNA (>90% knockdown efficiency)

  • Use GFP-Rab5b to mark early endosomes

  • Perform live-cell spinning disc confocal microscopy to measure tubule dynamics

  • IST1 depletion increases both tubule frequency and lifetime

• Co-localization with endosomal markers:

  • Use FITC-conjugated IST1 antibodies with markers for:

    • Early endosomes (EEA1) - increased after IST1 depletion

    • Sorting nexins (SNX1, SNX4, SNX15) - IST1 co-assembles with SNX15 on endosomes

    • Recycling endosomes (Rab4, Rab11)

• Structure-function analysis:

  • The C-terminal fragment (CTD) of IST1 can be recruited to endosomes by overexpressed SNX15

  • Deletion of the C-terminal MIM1 motif abolishes this recruitment

  • SNX15 overexpression redistributes endogenous IST1 onto endosomes while depleting it from the cytoplasm

These approaches allow researchers to characterize IST1's role in endosomal dynamics and recycling pathways, particularly its interactions with the SNX15-positive subdomain of early/sorting endosomes .

What approaches can be used to investigate IST1's role during cell division using FITC-conjugated antibodies?

IST1 plays crucial roles during cell division, particularly in abscission during cytokinesis and nuclear envelope reassembly. To investigate these functions:

• Synchronization and time-course analysis:

  • Synchronize cells using cell cycle synchronization methods

  • Collect cells at defined time points during mitosis and cytokinesis

  • Use FITC-conjugated IST1 antibodies to track localization changes

  • Co-stain with markers for mitotic phases

• High-resolution imaging of cytokinetic structures:

  • Focus on midbody and cytokinetic bridge structures where IST1 localizes

  • Co-stain with VPS4A/B to observe recruitment mediated by IST1

  • IST1 functions in efficient abscission during cytokinesis

• Nuclear envelope reassembly analysis:

  • Monitor IST1 during late anaphase when nuclear envelope reassembly occurs

  • IST1 mediates recruitment of SPAST to the nuclear membrane for microtubule severing

  • Investigate interaction with ESCRT-III complex components

• Functional perturbation approaches:

  • siRNA knockdown of IST1 (>90% depletion)

  • Quantify cytokinesis defects (multinucleation, delayed abscission)

  • Measure nuclear envelope reassembly kinetics

These approaches provide a comprehensive framework for investigating IST1's role during cell division using FITC-conjugated antibodies to track its dynamic localization and interactions.

How do the IST1 MIM motifs affect experimental approaches when using antibodies targeting different regions?

IST1 contains MIT-interacting motifs (MIMs) that mediate interactions with MIT domain-containing proteins like VPS4A/B and SNX15. These motifs have important implications for antibody selection:

IST1 domain structure considerations:

• N-terminal ESCRT-III core: Structural, membrane association

• C-terminal MIM motifs: Mediate protein-protein interactions with MIT domains

• C-terminal fragment (CTD) can be recruited to endosomes by SNX15

• Deletion of the C-terminal MIM1 motif abolishes SNX15-mediated recruitment

Methodological implications:

• Epitope masking effects:

  • Antibodies targeting MIM regions may have reduced accessibility when IST1 is engaged with binding partners

  • This can create false negative signals when IST1 is functionally active

  • Solution: Use antibodies targeting different epitopes or regions (many commercial antibodies target amino acids 172-284)

• Structure-function investigations:

  • When studying SNX15-IST1 interactions, consider that SNX15's MIT domain interacts with IST1's MIM motifs

  • Antibodies recognizing regions outside the MIM1 motif may be preferable for certain interaction studies

  • Research shows SNX15 overexpression redistributes endogenous IST1 onto endosomes

• Distinguishing IST1 pools:

  • Different antibodies may preferentially detect cytoplasmic versus membrane-associated IST1

  • For studying endosomal IST1, confirm antibody can detect the membrane-associated pool

Understanding these domain-specific considerations is crucial when selecting antibodies for specific research questions about IST1 function.

What are the best practices for quantitative analysis of IST1 localization using FITC-conjugated antibodies?

Quantitative analysis of IST1 using FITC-conjugated antibodies requires rigorous methodology:

Image acquisition best practices:

• Microscopy settings:

  • Use identical acquisition parameters across all samples

  • Set exposure times to avoid saturation while maximizing signal

  • Acquire at optimal resolution for structures of interest

  • Use confocal microscopy for improved signal-to-noise ratio

• Controls for quantification:

  • Include technical replicates (multiple fields of view)

  • Include biological replicates (multiple experiments)

  • Use positive control (known IST1-expressing cells like HeLa)

  • Include negative control (IST1 knockdown)

Quantitative analysis approaches:

• Intensity-based measurements:

  • Mean fluorescence intensity of IST1 signal in whole cells or specific compartments

  • Integrated density measurements with background subtraction

  • Normalization to cell area or volume

• Object-based analysis:

  • Identification and counting of IST1-positive puncta

  • Measurement of puncta size, intensity, and distribution

  • Colocalization analysis with markers like EEA1 or SNX15

• Expected patterns to quantify:

  • Cytoplasmic distribution throughout the cell

  • Punctate structures (endosomes)

  • Enrichment at cell bridges and midbodies during cytokinesis

  • Co-localization with SNX15 on discrete endosomal structures

These best practices ensure robust quantitative analysis of IST1 localization and expression using FITC-conjugated antibodies, particularly important when studying the dynamic distribution of IST1 between cytoplasmic and endosomal pools.

How can researchers troubleshoot issues with FITC-conjugated IST1 antibody experiments?

When encountering problems with FITC-conjugated IST1 antibody experiments, systematic troubleshooting approaches can help identify and resolve issues:

Low or no signal issues:

• Photobleaching: Protect from light; use anti-fade mounting media; minimize exposure time

• Low expression level: Use validated positive control cells (A549, HeLa, PC-3)

• Epitope masking: Try different fixation methods; consider antibody recognizing different epitope

• Improper dilution: Titrate antibody; try concentrations from 1:50-1:500 for various applications

• Degraded fluorophore: Use fresh aliquots; check fluorescence directly

High background issues:

• Non-specific binding: Optimize blocking (5% BSA or normal serum); increase blocking time

• Autofluorescence: Use alternative fixation; include quenching step

• Over-concentration: Titrate antibody; optimize dilution

• Insufficient washing: Increase wash steps and duration

Methodological optimization strategies:

• Test different fixation methods (PFA, methanol, acetone)

• Optimize permeabilization (Triton X-100 concentration and timing)

• Try antigen retrieval for fixed tissues (citrate buffer pH 6.0 or TE buffer pH 9.0)

• Use confocal microscopy to improve signal-to-noise ratio

Systematically testing these parameters can help resolve issues with FITC-conjugated IST1 antibody performance across various applications.

How can IST1 antibodies be used to investigate the relationship between IST1 and the ESCRT machinery?

IST1 functions as an ESCRT-III-like protein with specific roles in the ESCRT machinery. Here are approaches to study this relationship:

• Co-localization and interaction studies:

  • Immunofluorescence co-localization of IST1 with core ESCRT components

  • FITC-conjugated IST1 antibody can be combined with antibodies against other ESCRT components

  • Co-immunoprecipitation to detect physical interactions

  • IST1 is involved in recruiting VPS4A and/or VPS4B to the midbody of dividing cells

• Functional perturbation approaches:

  • Compare phenotypes of IST1 vs. other ESCRT component knockdowns

  • IST1 is required for efficient abscission during cytokinesis, but not for HIV-1 budding

  • During late anaphase, IST1 is involved in nuclear envelope reassembly and mitotic spindle disassembly together with the ESCRT-III complex

  • IST1 mediates the recruitment of SPAST to the nuclear membrane

• Structure-function relationship analysis:

  • Map domains required for interaction with ESCRT components

  • Test the importance of the C-terminal MIM motifs in ESCRT functions

  • The involvement of IST1 in the MVB pathway is not fully established

This multi-faceted approach provides comprehensive insights into IST1's roles within the ESCRT machinery, helping to distinguish its specific functions from those of other ESCRT-III proteins.

What approaches can combine IST1 antibodies with other molecular tools for comprehensive pathway analysis?

To gain comprehensive insights into IST1's functions, researchers should employ complementary approaches that integrate antibody-based detection with other molecular tools:

• Combined imaging approaches:

  • FITC-conjugated IST1 antibodies for fixed cell analysis

  • Live-cell imaging with fluorescently tagged proteins (GFP-Rab5b for early endosomes, mApple-IST1)

  • Super-resolution microscopy to resolve endosomal subdomains where IST1 localizes

• Functional perturbation combined with antibody detection:

  • siRNA knockdown of IST1 (>90% depletion efficiency)

  • Expression of dominant-negative constructs (IST1 with MIM1 motif deletion)

  • Track effects on EEA1-positive endosome size and number

  • Analyze SNX15 localization and function in IST1-depleted cells

  • Monitor endosomal tubulation frequency and persistence

• Cargo trafficking assays:

  • Transferrin uptake and recycling studies (IST1 co-localizes with internalized transferrin on endosomes)

  • Quantitative endocytosis and recycling assays in IST1-depleted versus control cells

Research findings provide a foundation for these approaches, showing that:

• IST1 depletion increases levels of endosomal EEA1

• Loss of IST1 increases both Rab5b tubule frequency and lifetime

• IST1 co-assembles with SNX15 on endosomes

• The C-terminal fragment of IST1 can be recruited to endosomes by SNX15, dependent on the MIM1 motif

This integrated approach provides mechanistic insights into IST1's roles in endosomal recycling pathways beyond what any single technique could reveal.

What are the current technical limitations of IST1 antibodies and how might researchers address them?

Current IST1 antibodies, including FITC-conjugated versions, have several technical limitations that researchers should consider:

Current limitations and potential solutions:

• Epitope accessibility issues:

  • Conformational changes in IST1 may mask epitopes

  • Solution: Use multiple antibodies targeting different regions (common commercial antibodies target amino acids 172-284)

  • Consider antibodies against different regions when studying protein interactions

• Species cross-reactivity constraints:

  • Many IST1 antibodies are human-specific with limited cross-reactivity

  • Solution: Carefully validate in non-human systems; use species-specific antibodies

  • Some antibodies show reactivity with mouse and rat samples

• FITC conjugation limitations:

  • Photobleaching; relatively low quantum yield compared to newer fluorophores

  • Solution: Use anti-fade reagents; minimize exposure; consider signal amplification

  • Protect samples from light during all procedures

• Application-specific performance:

  • Antibodies may work well in some applications but not others

  • Solution: Validate for each specific application (ELISA, WB, IHC, IF)

  • Optimize protocols for each application with proper controls

Alternative approaches:

• CRISPR knock-in of tags for endogenous IST1 labeling

• Fluorescent protein fusions for live-cell imaging

• Proximity labeling approaches for interaction studies

• Advanced microscopy techniques for improved resolution of IST1-containing structures

Addressing these limitations and considering alternative approaches will advance our ability to study IST1 biology with greater precision and reliability.