TWIST1 Antibody, FITC conjugated

What is TWIST1 and why is it significant in research?

TWIST1 functions as a critical transcriptional regulator involved in multiple cellular processes. It inhibits myogenesis by sequestering E proteins, inhibiting MEF2 trans-activation, and preventing MYOD1 DNA-binding through physical interaction. Additionally, TWIST1 represses proinflammatory cytokines including TNFA and IL1B, while regulating cranial suture patterning and fusion through differential gene expression depending on dimer composition . The significance of TWIST1 extends to its central role in promoting epithelial-mesenchymal transition (EMT) and tumor metastasis, making it a valuable target for cancer research .

What applications are TWIST1 antibodies typically used for?

TWIST1 antibodies are employed across various experimental applications, with immunofluorescence/immunocytochemistry (IF/ICC) being particularly common, typically at dilutions between 1/50 and 1/100 . When selecting TWIST1 antibodies, researchers should consider the specific experimental requirements, including:

• Detection of TWIST1-protein interactions through co-immunoprecipitation assays

• Visualization of subcellular localization via immunofluorescence microscopy

• Assessment of TWIST1 recruitment to target gene promoters through chromatin immunoprecipitation (ChIP) assays

• Quantification of TWIST1 expression levels across different experimental conditions

What is the significance of FITC conjugation in TWIST1 antibodies?

FITC (Fluorescein Isothiocyanate) conjugation provides direct fluorescent detection capability without requiring secondary antibodies, streamlining experimental workflows. The FITC fluorophore emits green fluorescence when excited with appropriate wavelengths, enabling direct visualization of TWIST1 protein in cellular contexts. This conjugation is particularly valuable for multi-color immunostaining protocols where multiple targets need to be detected simultaneously using distinguishable fluorophores .

What cross-reactivity concerns exist for TWIST1 antibodies?

Cross-reactivity with related proteins represents a significant concern when using TWIST1 antibodies. For instance, certain TWIST1 polyclonal antibodies demonstrate less than 16% cross-reactivity with recombinant human Twist-2 in direct ELISAs . This limited cross-reactivity is important when studying TWIST1-specific functions, particularly in experimental systems where both TWIST1 and TWIST2 may be expressed simultaneously.

How can TWIST1 antibodies be optimized for detecting protein complexes in EMT research?

TWIST1 forms different protein complexes that determine its function in EMT regulation. Research has demonstrated that TWIST1 can interact with the NuRD complex to repress epithelial genes, while its association with the TIP60-Com complex activates mesenchymal genes . When designing experiments to study these complexes:

• Optimize immunoprecipitation conditions with protease and phosphatase inhibitors to preserve native protein interactions

• Consider lysine acetylation status, as TWIST1-KK (acetylated) preferentially associates with TIP60-Com, while non-acetylated TWIST1 interacts with the NuRD complex

• Use BRD8 antibodies to co-precipitate TWIST1 in TWIST1/TIP60-Com complexes and HDAC2 antibodies for TWIST1/NuRD complexes

• Apply targeted mass spectrometry approaches to confirm complex composition and identify novel interacting partners

What methodological considerations should be addressed when using TWIST1 antibodies for ChIP-Seq experiments?

ChIP-Seq experiments with TWIST1 antibodies require careful optimization to accurately identify TWIST1 binding sites across the genome. When designing ChIP-Seq protocols:

• Validate antibody specificity and efficiency through preliminary ChIP-qPCR at known TWIST1 binding sites (e.g., SNAI2, ESR1, CDH1, and MYC promoters)

• Optimize chromatin fragmentation to generate 200-500bp fragments for high-resolution binding site identification

• Include appropriate controls such as non-immune IgG and input chromatin

• Normalize data using spike-in controls to account for technical variations

• Confirm ChIP-Seq findings through targeted ChIP-qPCR validation at selected genomic loci

How can TWIST1 antibodies be employed to study invadopodia formation in cancer metastasis models?

TWIST1 has been demonstrated to promote invadopodia formation, which contributes to tumor metastasis through localized extracellular matrix degradation . When investigating this phenomenon:

• Combine TWIST1 immunostaining with markers of invadopodia such as cortactin and Tks5

• Implement FITC-gelatin degradation assays to assess functional invadopodia

• Correlate TWIST1 expression levels with matrix degradation capabilities

• Employ time-lapse microscopy to track the dynamic formation and function of TWIST1-induced invadopodia

• Utilize TWIST1 knockdown or overexpression models to demonstrate causality in invadopodia formation

What are the critical factors for interpreting TWIST1 antibody data in glioblastoma invasion studies?

TWIST1 plays a significant role in promoting glioblastoma multiforme (GBM) invasion through mechanisms distinct from the classic EMT observed in carcinomas . When analyzing TWIST1 antibody data in GBM research:

• Recognize that TWIST1 does not generate a classic E- to N-cadherin "switch" in GBM cell lines, unlike in carcinoma EMT

• Correlate TWIST1 expression with changes in cell adhesion, extracellular matrix proteins, cell motility, and actin cytoskeleton organization

• Examine TWIST1-regulated genes involved in GBM invasion, including SNAI2, MMP2, HGF, FAP, and FN1

• Compare in vitro findings with orthotopic xenotransplant models to validate invasion patterns

What is the recommended protocol for immunofluorescence using FITC-conjugated TWIST1 antibodies?

For optimal immunofluorescence results with FITC-conjugated TWIST1 antibodies:

• Fix cells using 4% paraformaldehyde for 15 minutes at room temperature

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

• Block with 3% BSA in PBS for 1 hour

• Apply FITC-conjugated TWIST1 antibody at 1:50-1:100 dilution in blocking buffer

• Incubate overnight at 4°C in a humidified chamber protected from light

• Wash 3x with PBS for 5 minutes each

• Counterstain nuclei with DAPI and mount with anti-fade mounting medium

• Store slides at 4°C protected from light until imaging

What approaches can resolve inconsistent TWIST1 antibody staining patterns?

Inconsistent staining patterns may result from several factors. To troubleshoot:

• Optimize fixation conditions - overfixation can mask epitopes while underfixation may compromise cell morphology

• Test different permeabilization methods - methanol, acetone, or varying concentrations of detergents

• Implement antigen retrieval techniques if appropriate for your sample type

• Titrate antibody concentration to determine optimal signal-to-noise ratio

• Include positive and negative control samples in each experiment

• Consider epitope accessibility issues in different cell types or tissue contexts

How can researchers quantitatively analyze TWIST1 expression in relation to EMT markers?

Quantitative analysis of TWIST1 expression in relation to EMT markers requires:

• Implement multi-color immunofluorescence to simultaneously visualize TWIST1 and EMT markers

• Utilize high-content imaging systems for automated, unbiased quantification

• Apply appropriate image analysis software to:

  • Segment individual cells

  • Quantify nuclear vs. cytoplasmic TWIST1 localization

  • Correlate TWIST1 expression with EMT markers at the single-cell level

• Employ flow cytometry with FITC-conjugated TWIST1 antibodies for high-throughput quantification across cell populations

• Validate imaging findings with complementary techniques such as western blotting and qRT-PCR

What storage and handling practices maximize FITC-conjugated TWIST1 antibody performance?

To maintain optimal antibody performance:

• Aliquot antibodies upon receipt to minimize freeze-thaw cycles

• Store at -20°C in the dark to prevent photobleaching of the FITC fluorophore

• Avoid repeated freeze/thaw cycles that can compromise antibody activity

• Include preservatives such as sodium azide (0.02%) in storage buffer

• Maintain antibodies in 50% glycerol for enhanced stability

• When working with the antibody, keep on ice and protect from light

• Follow manufacturer's expiration guidelines and quality control recommendations

How should researchers interpret TWIST1 nuclear vs. cytoplasmic localization?

TWIST1 functions primarily as a nuclear transcription factor, but its cellular distribution can provide important functional insights:

• Predominant nuclear localization generally indicates active transcriptional function

• Cytoplasmic retention may suggest regulatory mechanisms that prevent TWIST1-mediated transcription

• Dynamic shuttling between compartments may occur in response to signaling events

• Post-translational modifications, particularly lysine acetylation at positions 73/76, can influence subcellular localization and protein interactions

• Co-localization with specific protein partners (HDAC2 for repressive functions, BRD8 for activating functions) provides functional context

What experimental design considerations are critical when studying TWIST1 in tumor invasion models?

When investigating TWIST1's role in tumor invasion:

• Select appropriate in vitro models that recapitulate specific aspects of invasion:

  • FITC-gelatin degradation assays for invadopodia function

  • Transwell migration/invasion assays for cell motility

  • 3D spheroid invasion models for more physiologically relevant contexts

• Complement in vitro findings with in vivo orthotopic xenotransplant models

• Implement TWIST1 genetic manipulation (knockdown, overexpression, or mutation) to establish causality

• Assess cell-cell adhesion, cell-substrate interactions, migration capability, and actin cytoskeleton organization as functional readouts of TWIST1 activity

• Analyze TWIST1-regulated target genes including those involved in EMT and invasion (SNAI2, MMP2, HGF, FAP, FN1)

How can dual-color analysis with TWIST1-FITC antibodies resolve heterogeneity in tumor models?

Tumor heterogeneity presents significant challenges in understanding TWIST1 function. Dual-color analysis strategies include:

• Co-staining for TWIST1 and EMT markers (E-cadherin, N-cadherin, Vimentin) to identify cells undergoing transition

• Combining TWIST1 detection with markers of cancer stem cells to examine correlations with stemness properties

• Analyzing TWIST1 expression in relation to proliferation markers (Ki67) to assess relationships between EMT and growth rates

• Implementing single-cell analytics to resolve population heterogeneity:

  • Flow cytometry for high-throughput quantification

  • Single-cell RNA-seq to correlate TWIST1 protein levels with transcriptional profiles

  • Spatial transcriptomics to preserve tissue context when analyzing heterogeneity

What controls are essential when validating TWIST1 antibody specificity?

Proper validation of TWIST1 antibody specificity requires:

• Positive controls: Cell lines with confirmed TWIST1 expression (e.g., 4T1, 168FARN)

• Negative controls: Cell lines with minimal TWIST1 expression (e.g., 67NR)

• Genetic validation approaches:

  • TWIST1 knockdown samples to demonstrate signal reduction

  • TWIST1 overexpression models to confirm signal enhancement

• Peptide competition assays to verify epitope specificity

• Cross-validation with multiple antibodies targeting different TWIST1 epitopes

• Western blot analysis to confirm detection of a single band at the expected molecular weight

How might TWIST1 antibodies contribute to understanding post-translational regulation mechanisms?

TWIST1 function is regulated through various post-translational modifications (PTMs). Future research directions include:

• Developing modification-specific antibodies that recognize acetylated TWIST1 (acK73/76) to discriminate between TWIST1 bound to TIP60-Com versus NuRD complexes

• Investigating phosphorylation-specific antibodies to monitor TWIST1 activation status

• Combining TWIST1 detection with analysis of E3 ubiquitin-protein ligases (e.g., MIB1) to explore degradation mechanisms

• Implementing proximity ligation assays to visualize specific TWIST1 interactions in situ

• Applying mass spectrometry approaches to comprehensively map the TWIST1 "PTM code" and its functional consequences

What emerging applications exist for TWIST1 antibodies in cancer diagnostics and therapeutics?

TWIST1 antibodies may contribute to translational applications including:

• Developing diagnostic assays to detect TWIST1 expression as a biomarker for metastatic potential

• Creating imaging tools to identify invasive tumor boundaries in surgical settings

• Monitoring treatment response through quantitative assessment of TWIST1 expression and localization

• Generating therapeutic antibodies targeting TWIST1-dependent tumor cell functions

• Supporting drug discovery efforts by enabling high-throughput screening for compounds that modulate TWIST1 expression or activity