PTF1A Antibody, FITC conjugated

What is PTF1A and why is it significant in pancreatic research?

PTF1A (Pancreas Specific Transcription Factor, 1a) is a key basic helix-loop-helix (bHLH) transcription factor critical for pancreatogenesis and maintenance of acinar cell identity. It plays an essential role in transcribing digestive enzymes in adult pancreatic tissue and maintaining the differentiated state of acinar cells. The significance of PTF1A extends beyond normal development, as its expression has been implicated in preventing tumorigenesis, making it a valuable target for both developmental biology and cancer research applications. Recent studies have demonstrated that homozygous Ptf1a deletion in adult acinar cells causes severe apoptosis through endoplasmic reticulum stress mechanisms .

What epitope does the FITC-conjugated PTF1A antibody recognize, and how does this influence experimental design?

The FITC-conjugated PTF1A antibody specifically recognizes amino acids 224-325 of the human PTF1A protein. This region contains functional domains critical for PTF1A's transcriptional activity. When designing experiments, researchers should consider that this epitope recognition influences detection sensitivity in different experimental contexts. The antibody has been validated for human samples with over 95% purity (Protein G purified), making it suitable for applications requiring high specificity . Researchers should note that experimental designs involving protein truncations or mutations within this amino acid region may affect antibody binding and subsequent detection.

What are the optimal storage conditions for maintaining FITC-conjugated PTF1A antibody activity?

For maximum retention of fluorescent signal and antibody functionality, store the FITC-conjugated PTF1A antibody at -20°C or -80°C and avoid repeated freeze-thaw cycles which can compromise both the antibody binding capacity and the fluorescent properties of the FITC conjugate. The antibody is typically provided in a storage buffer containing 50% Glycerol and 0.01M PBS (pH 7.4) with 0.03% Proclin 300 as a preservative . When planning long-term studies, aliquoting the antibody upon receipt is recommended to minimize freeze-thaw cycles. Exposure to light should be minimized as FITC is susceptible to photobleaching, which can reduce signal intensity in imaging applications.

How can the FITC-conjugated PTF1A antibody be optimally used for tracking acinar cell fate in pancreatic tissue?

For effective tracking of acinar cell fate using FITC-conjugated PTF1A antibody, implement the following methodology:

• Fresh tissue preparation: Process pancreatic tissue samples immediately after collection, fixing in 10% neutral buffered formalin for 24 hours.

• Permeabilization protocol: After PBS washing, permeabilize with 0.3% Triton X-100 in PBS for 30 minutes at room temperature to facilitate antibody penetration.

• Blocking step: Block with 5% normal serum in PBS for 1 hour to minimize non-specific binding.

• Antibody application: Apply the FITC-conjugated PTF1A antibody at a 1:200 dilution in blocking buffer and incubate overnight at 4°C.

• Co-staining strategy: For differentiation studies, combine with markers such as CPA1 (1:100) for mature acinar cells or CA19.9 for ductal cells .

This methodological approach enables researchers to distinguish between acinar cells maintaining PTF1A expression versus those undergoing dedifferentiation or acinar-to-ductal metaplasia, a critical process in pancreatic pathology studies.

What validation methods should be employed to confirm PTF1A antibody specificity in immunofluorescence applications?

To rigorously validate PTF1A antibody specificity for immunofluorescence applications, implement these methodological controls:

• Positive control validation: Use tissues with known PTF1A expression (adult pancreatic acinar cells) processed identically to experimental samples.

• Negative control assessment: Include tissue sections known to lack PTF1A expression (ductal cells) to confirm absence of non-specific binding.

• Peptide competition assay: Pre-incubate the antibody with excess recombinant PTF1A protein (224-325AA) to demonstrate signal reduction through competitive binding.

• Knockout validation: If available, use Ptf1a knockout tissues as demonstrated in conditional Elastase-CreERTM; Ptf1afloxed/floxed mouse models .

• Cross-reactivity testing: Confirm specificity by testing against related bHLH transcription factors to rule out cross-reactivity.

Documentation of these validation steps is essential for publication-quality immunofluorescence studies using this antibody.

How can FITC-conjugated PTF1A antibody be effectively used alongside UEA1 lectin for distinguishing acinar cell populations?

For dual labeling studies to distinguish acinar cell populations:

• Sequential labeling approach: First apply UEA1-FITC (which binds to α-linked fucose residues on chymotrypsin-positive acinar cells) at 1:200 dilution for 1 hour at room temperature.

• Washing protocol: Perform three 5-minute washes with PBS to remove unbound UEA1-FITC.

• Secondary labeling: Apply a different fluorophore-conjugated (e.g., AlexaFluor 594) secondary antibody against the primary PTF1A antibody to avoid spectral overlap with FITC.

• Temporal tracking method: UEA1-FITC incorporation can be detected up to 7 days after initial labeling, allowing for temporal tracking of acinar cell fate .

• Flow cytometry optimization: For FACS applications, use UEA1-FITC in combination with CA19.9 (duct marker) to specifically isolate and quantify acinar-derived, duct-like, and acinar-to-duct-like transdifferentiated cells .

This dual labeling technique provides robust identification of acinar cells and tracking of their differentiation state in both imaging and flow cytometry applications.

How can the FITC-conjugated PTF1A antibody be utilized in studying acinar-to-ductal metaplasia (ADM) in pancreatic tissue?

To investigate acinar-to-ductal metaplasia using FITC-conjugated PTF1A antibody:

• Time-course experimental design: Establish a defined timeline (1-6 days) for monitoring progressive loss of PTF1A expression during ADM.

• Multi-marker panel approach: Combine PTF1A detection with acinar markers (amylase, CPA1) and ductal markers (KRT19, CA19.9) to create a comprehensive differentiation profile.

• Quantitative image analysis: Implement digital image analysis to quantify the percentage of cells expressing different levels of PTF1A during transdifferentiation.

• In vitro ADM modeling: Culture isolated primary acinar cells with TGF-α or EGF to induce ADM, then monitor PTF1A expression changes using the FITC-conjugated antibody.

• Co-localization analysis: Perform pixel-based co-localization analysis between PTF1A and early ductal markers to identify cells in transition states.

This methodological framework allows researchers to track the progressive loss of acinar identity during ADM, a critical precursor to pancreatic neoplasia.

What are the methodological considerations for using FITC-conjugated PTF1A antibody in flow cytometric analysis of pancreatic cell populations?

For optimization of flow cytometric analysis with FITC-conjugated PTF1A antibody:

• Cell preparation protocol: Dissociate pancreatic tissue using collagenase P (0.2 mg/ml) for 20 minutes at 37°C, followed by gentle mechanical disruption.

• Fixation and permeabilization parameters: Use 2% paraformaldehyde for 15 minutes, followed by permeabilization with 0.1% saponin in PBS to maintain FITC signal intensity.

• Compensation strategy: Properly compensate for FITC spectral overlap with other fluorophores if using multi-parameter flow cytometry.

• Gating strategy design:

  • First gate: Forward/side scatter to identify cell populations

  • Second gate: Viability marker to exclude dead cells

  • Third gate: PTF1A-FITC positivity threshold based on negative control samples

• Sorting parameters for downstream applications: If sorting for further culture or molecular analysis, use a 100 μm nozzle at low pressure (20 psi) to maintain cell viability.

This methodology enables quantitative assessment of PTF1A expression across different pancreatic cell populations with high precision.

How can FITC-conjugated PTF1A antibody be utilized in investigations of unfolded protein response (UPR) pathway activation in acinar cells?

For investigating the relationship between PTF1A and UPR pathway activation:

• Stress induction protocol: Treat pancreatic acinar cells with ER stress inducers (thapsigargin at 1 μM or tunicamycin at 2 μg/ml) for 6-24 hours.

• Temporal analysis framework: Monitor PTF1A expression changes at 4, 8, 12, and 24 hours post-stress induction using the FITC-conjugated antibody.

• UPR branch-specific co-staining: Combine PTF1A-FITC detection with antibodies against:

  • ATF6 pathway: cleaved ATF6 (50 kDa fragment)

  • PERK pathway: phosphorylated eIF2α and ATF4

  • IRE1 pathway: spliced XBP1

• Apoptosis correlation analysis: Co-stain for CHOP and cleaved caspase-3 to correlate PTF1A loss with UPR-induced apoptosis .

• Live-cell imaging application: Utilize the FITC conjugation for real-time monitoring of PTF1A localization during ER stress responses.

This experimental approach provides mechanistic insights into how PTF1A deletion leads to UPR activation and subsequent apoptosis in acinar cells, as observed in conditional knockout models .

How does PTF1A expression modulate pancreatic ductal adenocarcinoma (PDAC) cell behavior, and how can this be studied using the FITC-conjugated antibody?

To investigate PTF1A's tumor-suppressive properties in PDAC:

• Inducible expression system: Establish doxycycline-inducible PTF1A expression in PDAC cell lines (such as Panc-1).

• Quantitative assessment of acinar differentiation markers: Following PTF1A induction, measure expression of digestive enzymes using RT-qPCR and western blotting.

• Proliferation analysis: Perform EdU incorporation assays pre- and post-PTF1A induction to quantify cell proliferation changes.

• Cancer stem cell quantification: Use flow cytometry with the FITC-conjugated PTF1A antibody in combination with CSC markers (CD24, CD44, ESA) to quantify stem cell population changes.

• Chemosensitivity testing: Evaluate gemcitabine sensitivity changes using MTT or similar viability assays following PTF1A induction.

Research has demonstrated that induced PTF1A expression in PDAC cells can re-establish acinar differentiation programs, decreasing tumor-associated properties including cell proliferation, cancer stem cell numbers, and increasing chemosensitivity to gemcitabine .

What methodological approaches can be used to study the role of PTF1A in ER stress and apoptosis in pancreatic acinar cells?

To investigate PTF1A's role in ER stress and apoptosis:

• Conditional knockout model: Utilize Elastase-CreERTM; Ptf1afloxed/floxed mouse models with tamoxifen-induced PTF1A deletion in adult acinar cells.

• ER stress evaluation protocol:

  • Ultrastructural analysis: Electron microscopy to visualize ER distension

  • UPR pathway activation markers: Western blot analysis for BiP/GRP78, phospho-PERK, phospho-eIF2α

  • Transcriptional analysis: RT-qPCR for stress-responsive genes (CHOP, ATF4, XBP1s)

• Apoptosis detection methods:

  • TUNEL assay for DNA fragmentation

  • Cleaved caspase-3 immunostaining

  • Annexin V flow cytometry for early apoptosis detection

• Rescue experiment design: Determine if chemical chaperones (4-PBA, TUDCA) can alleviate ER stress and prevent apoptosis in PTF1A-deleted acinar cells.

Studies have demonstrated that homozygous Ptf1a deletion causes severe acinar cell apoptosis through activation of the ATF6 and PERK pathways of the UPR, but not the IRE1 pathway, ultimately leading to CHOP-mediated cell death .

How can the interplay between PTF1A and other acinar transcription factors be investigated using co-localization studies with FITC-conjugated antibodies?

For studying PTF1A interactions with other transcription factors:

• Multi-color immunofluorescence protocol:

  • PTF1A-FITC (green channel)

  • MIST1 (red channel using Alexa Fluor 594)

  • Nuclear counterstain (DAPI, blue channel)

• Co-localization quantification methods:

  • Pearson's correlation coefficient for nuclear co-localization

  • Manders' overlap coefficient for partial co-localization analysis

  • Line scan analysis across nuclei to confirm signal overlap

• Proximity ligation assay (PLA) optimization: For detecting protein-protein interactions between PTF1A and potential binding partners within 40 nm proximity.

• Transcriptional network analysis: Combine with ChIP-seq data to correlate co-localization with co-regulated genes.

• Response to cellular stress: Monitor changes in co-localization patterns following exposure to various stressors (oxidative, ER stress, inflammation).

This methodological framework allows researchers to understand how PTF1A coordinates with other transcription factors like MIST1 to maintain the acinar differentiation program, with each factor regulating distinct aspects (PTF1A for digestive enzyme expression; MIST1 for vesicle trafficking genes) .

What are the common technical challenges when using FITC-conjugated PTF1A antibody, and how can they be addressed?

Common technical issues and their methodological solutions include:

• Signal fading during microscopy:

  • Use anti-fade mounting media containing DABCO or NPG

  • Minimize exposure time during image acquisition

  • Consider spectral imaging to separate autofluorescence from specific signal

• Background fluorescence in pancreatic tissue:

  • Implement extended blocking (2 hours) with 5% normal serum and 0.3% Triton X-100

  • Include 0.1% Sudan Black B in the blocking solution to quench lipofuscin autofluorescence

  • Optimize antibody concentration through titration experiments (1:100 to 1:500 dilutions)

• Weak nuclear signal detection:

  • Enhance nuclear permeabilization with 0.5% Triton X-100 for 30 minutes

  • Use heat-mediated antigen retrieval (citrate buffer pH 6.0) for formalin-fixed tissues

  • Extend primary antibody incubation to overnight at 4°C

• Insufficient specificity:

  • Pre-absorb the antibody with liver tissue homogenate to reduce non-specific binding

  • Include appropriate blocking peptides in control experiments

  • Compare results with alternative clone antibodies targeting different PTF1A epitopes

These methodological adjustments ensure optimal signal-to-noise ratio and specificity when working with FITC-conjugated PTF1A antibody in challenging pancreatic tissue samples.

How should researchers interpret contradictory results between PTF1A protein detection and mRNA expression analyses?

When facing discrepancies between protein and mRNA data:

• Temporal relationship assessment: Consider that mRNA changes typically precede protein changes by 4-6 hours; implement time-course experiments at 2-hour intervals.

• Post-transcriptional regulation analysis:

  • Evaluate miRNA regulators of PTF1A using prediction algorithms and validation assays

  • Assess protein stability through cyclohexamide chase experiments (blocking new protein synthesis)

  • Investigate ubiquitin-proteasome pathway involvement using proteasome inhibitors (MG132)

• Technical validation approach:

  • Compare multiple antibody clones targeting different PTF1A epitopes

  • Validate mRNA expression with multiple primer sets and reference genes

  • Implement absolute quantification (digital PCR) for both protein and mRNA

• Subcellular localization consideration: Distinguish between total, cytoplasmic, and nuclear PTF1A pools using fractionation techniques.

• Biological context interpretation: Consider that stress conditions may differentially affect mRNA stability versus protein degradation rates.

This systematic approach helps researchers resolve apparent contradictions between transcriptomic and proteomic data for PTF1A expression studies.