OFUT4 Antibody

What is the difference between OCT4 and FUT4 antibodies in research applications?

OCT4 antibodies detect the protein encoded by the human gene POU5F1, a transcription factor critical for embryonic stem cell pluripotency with a molecular weight of approximately 38-45 kDa . They are primarily used as pluripotent stem cell markers. In contrast, FUT4 antibodies target fucosyltransferase 4, an enzyme that catalyzes the synthesis of fucosylated glycans with α1,3-linkage, particularly the Lewis x antigen . FUT4 antibodies are often used in cancer research, especially for studying aberrant glycosylation patterns in tumors.

How do I select the appropriate OCT4 antibody to ensure specific detection of pluripotency-associated OCT4A isoform?

When selecting an OCT4 antibody for pluripotency research, verify that it specifically targets the N-terminal domain (amino acids 1-134) of OCT4A, which is absent in OCT4B . The OCT4A isoform is localized in the nucleus and associated with pluripotency, while OCT4B is cytoplasmic and found in non-pluripotent cell types . Review the antibody documentation carefully to confirm it was generated against OCT4A-specific epitopes and validated in pluripotent stem cells. Cross-reference with positive controls such as embryonic stem cells and negative controls like differentiated cells to ensure specificity .

Why do some FUT4 antibodies show variable effectiveness in different experimental contexts?

Variability in FUT4 antibody effectiveness may stem from:

• Differential recognition of glycosylated forms of FUT4

• Expression levels that fluctuate temporally in cell culture (as observed in MC38-FUT4 cells where Lewis x expression decreased 96 hours after enrichment)

• Tissue-specific glycosylation patterns affecting epitope accessibility

• Post-translational modifications altering antibody recognition sites

For consistent results, validate FUT4 antibodies in your specific experimental system and consider including fucosyltransferase inhibitors (e.g., 2F-peracetyl fucose) as controls to confirm specificity .

What are the optimal conditions for detecting OCT4 protein in western blot analysis?

For optimal OCT4 western blot detection:

• Use 8-12% SDS-PAGE gels to properly resolve the 38-45 kDa OCT4 protein

• Employ reducing conditions with fresh β-mercaptoethanol or DTT

• Transfer proteins to PVDF membranes (preferred over nitrocellulose for some antibodies)

• Block with 5% non-fat milk or BSA depending on the antibody specifications

• Apply primary antibody at 1:1000-1:5000 dilution (optimize for each antibody)

• Include positive controls (embryonic stem cells) and negative controls

• Be aware that OCT4 often appears at slightly higher molecular weight (45-52 kDa) than predicted (38 kDa) due to post-translational modifications

• Use secondary antibodies at 1:5000 dilution and develop with enhanced chemiluminescence

How can I ensure accurate immunofluorescence detection of OCT4 in pluripotent stem cells while avoiding false positives?

To ensure accurate immunofluorescence detection while minimizing false positives:

• Sample preparation:

  • Use 4% paraformaldehyde fixation for 15-20 minutes

  • Perform antigen retrieval if necessary

  • Permeabilize with 0.1-0.5% Triton X-100 for nuclear proteins

• Antibody validation:

  • Use antibodies targeting OCT4A-specific regions (N-terminal domain)

  • Include multiple OCT4 antibodies from different sources/clones

  • Always run parallel negative controls (differentiated cells) and positive controls (ES cells)

• Critical controls:

  • Perform secondary antibody-only controls to check for non-specific binding

  • Include isotype controls to detect non-specific binding

  • Validate with OCT4 knockout/knockdown cells if available

• Confirmation methods:

  • Confirm nuclear localization of OCT4 staining (OCT4A is exclusively nuclear)

  • Validate expression with RT-qPCR for OCT4A transcripts

  • Co-stain with other pluripotency markers (SOX2, NANOG) to confirm cell state

What approaches can effectively distinguish between true and false-positive signals in OCT4 antibody applications?

Effectively distinguishing true from false-positive OCT4 signals requires a multi-faceted approach:

• Multiple antibody validation:

  • Use at least two OCT4 antibodies targeting different epitopes

  • Select antibodies with validated specificity for OCT4A

• Western blot confirmation:

  • Verify single band at expected molecular weight (38-45 kDa)

  • Check for absence of non-specific bands

• mRNA expression analysis:

  • Perform RT-qPCR with isoform-specific primers

  • Use RNA-seq to confirm OCT4A transcript presence

• Knockdown/knockout validation:

  • Demonstrate signal reduction with OCT4 siRNA/shRNA

  • Use CRISPR-based approaches for complete validation

• Functional assays:

  • Correlate OCT4 detection with pluripotency phenotypes

  • Demonstrate loss of pluripotency with OCT4 reduction

This comprehensive approach is essential as some commercial OCT4 antibodies have been documented to produce misleading results in testis-derived cells that do not express OCT4 mRNA .

How can FUT4 antibodies be utilized to investigate the relationship between fucosylation patterns and cancer progression?

FUT4 antibodies can be strategically employed to investigate cancer progression through:

• Tumor tissue analysis:

  • Immunohistochemistry of patient samples to correlate FUT4 expression with clinical outcomes

  • Multivariate analysis with patient survival data to establish prognostic value

• Glycoprotein profiling:

  • Immunoprecipitation with FUT4 antibodies followed by mass spectrometry to identify FUT4-modified proteins

  • Co-immunoprecipitation to detect interactions between FUT4 and downstream signaling proteins

• Functional studies:

  • Use FUT4 antibodies to neutralize enzyme activity in functional assays

  • Combine with Lewis x antibodies to simultaneously detect the enzyme and its products

• Metastasis investigations:

  • Track FUT4 expression in primary tumors versus metastatic sites

  • Correlate FUT4 levels with EMT markers in invasive tumor fronts

• Therapeutic targeting validation:

  • Evaluate FUT4 expression reduction following experimental therapeutics

  • Use FUT4 antibodies as potential delivery vehicles for targeted therapies

What experimental designs effectively measure the impact of FUT4-mediated fucosylation on cancer cell behavior?

Effective experimental designs for measuring FUT4's impact on cancer cell behavior include:

• Genetic manipulation approaches:

  • CRISPR-dCas9-VPR system for transcriptional activation of FUT4 in cells lacking expression

  • shRNA-mediated knockdown of FUT4 in high-expressing cancer cells

  • Tetracycline-controlled transcriptional regulation for inducible expression

• Functional assays:

  • Boyden chamber invasion assays with Matrigel coating

  • Single-cell migration tracking using live-cell imaging systems

  • Cell adhesion assays with E-selectin, L-selectin, and other adhesion molecules

  • In vivo extravasation assays using fluorescently labeled cells

• Molecular pathway analysis:

  • RNA-seq to identify FUT4-mediated transcriptomic changes

  • Signaling pathway activation measurement (EGF, TGFβ, MAPK pathways)

  • EMT marker assessment (E-cadherin, SNAIL, SLUG)

• Glycomic profiling:

  • MALDI-MS mapping followed by LC-MS²/MS³ analysis of N-glycans

  • Immunoprecipitation-mass spectrometry to identify fucosylated proteins

  • Lectin binding assays to detect specific glycan structures

• In vivo metastasis models:

  • Tail vein injection for experimental metastasis

  • Subcutaneous xenograft models to assess spontaneous metastasis

  • Intracardiac injection for organotropic metastasis

How can researchers effectively integrate OCT4 antibody detection with other pluripotency markers to establish reliable stem cell characterization?

For comprehensive stem cell characterization, researchers should:

• Design multi-parameter analyses:

  • Co-staining protocols for OCT4 with SOX2, NANOG, and KLF4

  • Flow cytometry panels including surface markers (SSEA-3, SSEA-4, TRA-1-60)

  • Sequential immunoprecipitation to detect protein complexes

• Establish quantitative correlations:

  • Measure relative expression levels of multiple pluripotency factors

  • Create normalization standards across different antibody affinities

  • Develop scoring systems for pluripotency marker combinations

• Implement functional validation:

  • Correlate antibody staining with differentiation potential

  • Link marker expression to transcriptional activity using reporter systems

  • Assess self-renewal capacity in relation to marker levels

• Apply computational approaches:

  • Machine learning algorithms to identify marker expression patterns

  • Single-cell analysis to detect heterogeneity within populations

  • Trajectory mapping to identify cells transitioning between states

• Temporal analysis:

  • Time-course studies during differentiation or reprogramming

  • Live-cell imaging with antibody fragments or alternative detection methods

  • Correlation of marker dynamics with developmental milestones

What are the most common sources of variability in OCT4 antibody experiments and how can they be addressed?

Common sources of variability in OCT4 antibody experiments include:

• Antibody specificity issues:

  • Solution: Use antibodies specifically validated for OCT4A detection

  • Validate with multiple antibodies targeting different epitopes

• Cell culture conditions affecting OCT4 expression:

  • Solution: Standardize seeding density, passage number, and culture media

  • Monitor for spontaneous differentiation that reduces OCT4 expression

• Sample preparation variables:

  • Solution: Standardize fixation protocols (time, temperature, buffer composition)

  • Optimize antigen retrieval methods for tissue samples

• Detection system sensitivity:

  • Solution: Calibrate imaging settings across experiments

  • Use quantitative standards for western blot normalization

• Cross-reactivity with OCT4 pseudogenes or related proteins:

  • Solution: Perform RNA-seq to confirm absence of pseudogene expression

  • Include negative control samples known to express related POU-domain proteins

• Batch-to-batch antibody variation:

  • Solution: Purchase larger antibody lots for long-term studies

  • Validate each new antibody batch against previous results

What quality control measures should be implemented when validating new batches of OCT4 or FUT4 antibodies?

Comprehensive quality control for new antibody batches should include:

• Specificity validation:

  • Western blot analysis comparing with previous batches

  • Positive control testing (embryonic stem cells for OCT4, cancer cell lines for FUT4)

  • Negative control testing (differentiated cells, knockout lines)

• Sensitivity assessment:

  • Titration experiments to determine optimal concentrations

  • Detection limit determination using serially diluted samples

  • Signal-to-noise ratio comparison with previous batches

• Application-specific validation:

  • Cross-application testing (WB, IF, FC, IP) as applicable

  • Protocol optimization for each application

  • Comparison of staining patterns with published results

• Documentation:

  • Record lot numbers, validation dates, and results

  • Create standardized validation protocols

  • Maintain a database of antibody performance characteristics

• Functional correlation:

  • For OCT4: correlation with pluripotency phenotypes

  • For FUT4: correlation with fucosylation activity/Lewis x expression

  • Expression patterns in developmentally or pathologically relevant samples

How should researchers interpret conflicting results between OCT4 antibody detection and mRNA expression data?

When faced with conflicting results between antibody detection and mRNA expression:

• First, verify technical aspects:

  • Check primer specificity for OCT4A vs. OCT4B isoforms

  • Confirm antibody specificity for OCT4A protein

  • Examine RNA quality and protein extraction efficiency

• Consider biological explanations:

  • Post-transcriptional regulation affecting protein levels

  • Protein stability differences across experimental conditions

  • Heterogeneity within cell populations

• Perform additional validation:

  • Use multiple antibodies targeting different OCT4 epitopes

  • Employ alternative mRNA detection methods (digital PCR, RNA-seq)

  • Implement single-cell analysis to address population heterogeneity

• Functional assessment:

  • Evaluate pluripotency characteristics independent of markers

  • Perform OCT4 knockdown/knockout experiments

  • Test differentiation potential correlations

• Consider false positive scenarios:

  • Cross-reactivity with OCT4 pseudogenes at protein level

  • Cross-reactivity with other POU-domain proteins

  • Non-specific antibody binding to highly expressed proteins

As documented in research with testis-derived cells, antibody positivity without corresponding mRNA expression strongly suggests false-positive antibody signals and requires comprehensive validation .

How can FUT4 antibodies be used to develop targeted therapeutic approaches for cancer treatment?

FUT4 antibodies can facilitate targeted cancer therapeutic development through:

• Target validation and patient stratification:

  • Immunohistochemical analysis of tumor biopsies to identify high FUT4-expressing tumors

  • Correlation studies linking FUT4 expression to treatment response

  • Prognostic assessment to identify patients likely to benefit from FUT4-targeted therapies

• Direct therapeutic applications:

  • Antibody-drug conjugates delivering cytotoxic payloads to FUT4-expressing cells

  • Bi-specific antibodies linking FUT4-expressing cancer cells to immune effector cells

  • Antibody-based blocking of FUT4 enzymatic activity

• Combination therapy design:

  • Identification of synergistic pathways with FUT4 (EGF, TGFβ, MAPK signaling)

  • Evaluation of FUT4 inhibition with conventional chemotherapies

  • Assessment of FUT4 targeting with immune checkpoint inhibitors

• Resistance mechanism studies:

  • Monitoring FUT4 expression changes during treatment

  • Identification of compensatory glycosylation pathways

  • Development of strategies to overcome resistance

• Biomarker development:

  • Antibody-based detection of circulating FUT4 or Lewis x in liquid biopsies

  • Correlation of FUT4 levels with minimal residual disease

  • Monitoring treatment efficacy through glycomic changes

Research has shown that FUT4 promotes lung cancer invasion, migration, and metastasis, making it a promising therapeutic target for lung adenocarcinoma .

What novel applications are emerging for OCT4 antibodies beyond traditional pluripotency research?

Emerging applications for OCT4 antibodies include:

• Cancer stem cell identification and targeting:

  • Detection of OCT4-expressing cancer stem cell populations

  • Correlation with treatment resistance and disease recurrence

  • Development of OCT4-targeted elimination strategies

• Neurodevelopmental studies:

  • Investigation of OCT4 expression in neural progenitor cells

  • Mapping OCT4 distribution in developing brain regions

  • Correlation with neurodevelopmental disorders

• Regenerative medicine applications:

  • Quality control for clinical-grade pluripotent stem cells

  • Monitoring differentiation protocols in real-time

  • Validating reprogramming efficiency in various cell types

• Epigenetic research:

  • Combined OCT4 ChIP-seq with histone modification mapping

  • Investigation of OCT4 pioneering factor activity

  • Analysis of OCT4 binding site accessibility during cellular transitions

• Synthetic biology platforms:

  • OCT4-based biosensors for pluripotency assessment

  • Engineered OCT4 variants with modified capabilities

  • OCT4-driven genetic circuits for controlling cell fate

How can integrated glycoproteomic and transcriptomic approaches improve our understanding of FUT4 function in disease progression?

Integrated glycoproteomic and transcriptomic approaches provide powerful insights into FUT4 function through:

• Comprehensive pathway mapping:

  • RNA-seq identification of FUT4-regulated gene networks

  • Glycoproteomic identification of FUT4 target proteins

  • Integration of these datasets to identify key functional nodes

• Temporal dynamics analysis:

  • Time-course studies capturing FUT4-mediated changes

  • Correlation of glycosylation changes with transcriptional responses

  • Identification of early versus late events in FUT4-mediated pathways

• Cell-type specific profiling:

  • Single-cell RNA-seq with glycoproteomic analysis

  • Spatial transcriptomics combined with imaging mass spectrometry

  • Cell-specific FUT4 function across tumor microenvironments

• Context-dependent function assessment:

  • Comparative analysis across different cancer types

  • Evaluation of FUT4 function in primary versus metastatic sites

  • Investigation of microenvironmental influences on FUT4 activity

• Therapeutic target prioritization:

  • Identification of critical downstream effectors of FUT4

  • Cross-reference with druggable target databases

  • Stratification of patients based on integrated molecular profiles

Studies in lung adenocarcinoma have employed this integrated approach, revealing that FUT4 activates membrane trafficking machinery and enhances oncogenic signaling via aberrant fucosylation of multiple cellular targets .