PTF1A Antibody, Biotin conjugated

What is PTF1A and why is it significant in developmental biology research?

PTF1A (Pancreas Specific Transcription Factor 1a) is a basic helix-loop-helix (bHLH) transcription factor that plays critical roles in cell fate determination across multiple organs. It binds to E-box consensus sequences (5'-CANNTG-3') and regulates downstream target gene expression . PTF1A is particularly important in pancreatic development where it determines whether cells allocated to pancreatic buds continue toward pancreatic organogenesis or revert to duodenal fates. It is essential for the formation of pancreatic acinar and ductal cells and maintains exocrine pancreas-specific gene expression, including elastase 1 (ELA1) and amylase . Beyond the pancreas, PTF1A functions in cerebellar development and, through regulation by FOXN4 and RORC, directs retinal progenitors toward horizontal and amacrine cell fates . Its significance extends to cancer research, as induced PTF1A expression in pancreatic ductal adenocarcinoma (PDAC) cells can re-establish acinar cell differentiation programs and reduce tumorigenic properties .

What are the technical specifications of PTF1A Antibody, Biotin conjugated?

The PTF1A Antibody, Biotin conjugated is a polyclonal antibody raised in rabbits against recombinant human PTF1A protein fragments. Based on the search results, the specifications are:

The antibody has been validated in various applications, demonstrating appropriate target specificity and performance in research settings .

How should PTF1A antibody be properly stored and reconstituted to maintain optimal activity?

Proper storage and reconstitution are critical for maintaining antibody activity and experimental reproducibility. For the PTF1A antibody, Biotin conjugated:

Storage Recommendations:

• Store lyophilized antibody at -20°C for up to one year from the date of receipt .

• After reconstitution, store at 4°C for short-term use (up to one month) .

• For long-term storage after reconstitution, aliquot and store at -20°C for up to six months .

• Avoid repeated freeze-thaw cycles as these can degrade antibody quality and reduce activity .

Reconstitution Protocol:

• Briefly centrifuge the antibody vial to ensure all material is at the bottom.

• Add 0.2 ml of distilled water to yield a concentration of 500 μg/ml .

• Gently mix by inversion or slow vortexing until completely dissolved.

• For long-term storage, prepare smaller working aliquots to avoid repeated freeze-thaw cycles.

The reconstituted antibody contains components that aid stability, including 4 mg Trehalose, 0.9 mg NaCl, 0.2 mg Na₂HPO₄, and 0.05 mg NaN₃ . These components help maintain protein structure and prevent microbial contamination during storage.

What is the recommended protocol for using PTF1A antibody in ELISA applications?

ELISA (Enzyme-Linked Immunosorbent Assay) is a primary application for the biotin-conjugated PTF1A antibody. The following protocol provides a methodological framework for researchers:

Indirect ELISA Protocol:

• Plate Coating:

  • Coat 96-well microplate with PTF1A protein standard or sample diluted in coating buffer (typically carbonate-bicarbonate buffer, pH 9.6).

  • Incubate overnight at 4°C.

• Blocking:

  • Wash wells 3-5 times with PBS-T (PBS + 0.05% Tween-20).

  • Block non-specific binding sites with 2-5% BSA or non-fat milk in PBS for 1-2 hours at room temperature.

• Primary Antibody:

  • Wash wells 3-5 times with PBS-T.

  • Add biotin-conjugated PTF1A antibody diluted in blocking buffer (recommended starting dilution: 1:1000).

  • Incubate for 1-2 hours at room temperature or overnight at 4°C.

• Detection:

  • Wash wells 3-5 times with PBS-T.

  • Add streptavidin-HRP conjugate diluted in blocking buffer (typically 1:2000-1:5000).

  • Incubate for 30-60 minutes at room temperature.

• Signal Development:

  • Wash wells 3-5 times with PBS-T.

  • Add TMB substrate solution and incubate for 15-30 minutes at room temperature in the dark.

  • Stop reaction with 2N H₂SO₄ or 1N HCl.

  • Measure absorbance at 450 nm with reference at 620 nm.

Critical Considerations:

• Always include a standard curve using recombinant PTF1A protein for quantitative analysis.

• Include negative controls (no antigen, no primary antibody) and positive controls.

• Optimize antibody concentration by testing serial dilutions to determine the optimal signal-to-noise ratio.

• For detecting endogenous PTF1A in complex samples, consider sample pre-clearing to reduce background.

How can I validate the specificity of PTF1A antibody in immunofluorescence studies?

Validating antibody specificity is crucial for ensuring reliable research findings. For PTF1A antibody in immunofluorescence applications:

Validation Protocol:

• Positive and Negative Control Tissues/Cells:

  • Use known PTF1A-expressing tissues (pancreatic tissue) or cell lines (PANC-1) as positive controls .

  • Use tissues/cells with confirmed absence of PTF1A expression as negative controls.

• Peptide Competition Assay:

  • Pre-incubate the PTF1A antibody with its immunizing peptide/recombinant protein.

  • Use this mixture in parallel with the standard antibody staining protocol.

  • A specific signal should be significantly reduced or eliminated in the peptide-blocked sample.

• RNAi or CRISPR Validation:

  • Compare staining in wild-type cells versus cells with PTF1A knockdown/knockout.

  • Signal should be reduced in proportion to the degree of knockdown or absent in knockout cells.

• Immunofluorescence Protocol for PTF1A:

  • Fix cells in 10% neutral buffered formalin .

  • Wash with PBS and permeabilize with 0.3% Triton X-100 in PBS .

  • Block with appropriate blocking solution (e.g., 10% goat serum) .

  • Incubate with PTF1A biotin-conjugated antibody (recommended dilution: 1:100-1:500).

  • Detect with avidin-conjugated fluorophore (e.g., Avidin-Alexa Fluor 488 or 594) .

  • Counterstain nuclei with DAPI .

  • Visualize using appropriate filter sets for the fluorophores used .

• Co-localization Studies:

  • Perform dual immunofluorescence with antibodies against known PTF1A-interacting proteins or co-expressed markers.

  • Expected co-localization patterns can confirm target specificity.

Example visualization from the literature shows PTF1A staining in pancreatic cells with nuclear localization, consistent with its function as a transcription factor .

How can PTF1A antibody be utilized to study transcriptional regulation in pancreatic development?

PTF1A antibody can be employed in multiple sophisticated approaches to investigate its transcriptional regulatory functions in pancreatic development:

Chromatin Immunoprecipitation (ChIP) Approaches:

• ChIP-seq Protocol Adaptation:

  • Cross-link cells/tissues with 1% formaldehyde.

  • Isolate and sonicate chromatin to ~200-500 bp fragments.

  • Pre-clear chromatin with protein A/G beads.

  • Immunoprecipitate PTF1A-bound chromatin using biotinylated PTF1A antibody coupled to streptavidin beads.

  • Wash, reverse cross-links, purify DNA, and prepare libraries for sequencing.

  • Analyze enriched regions for the E-box consensus sequence (5'-CANNTG-3') .

• ChIP-qPCR for Target Validation:

  • After ChIP, perform qPCR using primers specific to promoter regions of suspected PTF1A target genes such as digestive enzymes (PRSS2, CPA1) or acinar-specific transcription factors .

  • Compare enrichment to IgG control and non-target regions.

Transcriptional Co-Regulator Studies:

• Co-Immunoprecipitation (Co-IP):

  • Use biotinylated PTF1A antibody to pull down PTF1A complexes.

  • Analyze co-precipitated proteins by Western blot or mass spectrometry to identify transcriptional co-regulators.

• Proximity Ligation Assay (PLA):

  • Apply PLA technique using PTF1A biotin-conjugated antibody and antibodies against potential interacting transcription factors.

  • Visualization of interaction signals provides spatial information about PTF1A transcriptional complexes in cells.

Functional Validation Studies:

• Reporter Assays:

  • Clone promoter regions containing PTF1A binding sites into luciferase reporter vectors.

  • Measure reporter activity in response to PTF1A overexpression or knockdown.

  • Use site-directed mutagenesis of E-box sequences to confirm direct regulation.

• CRISPR Activation/Interference:

  • Target CRISPR activation or interference systems to PTF1A binding sites.

  • Measure effects on target gene expression and cellular phenotypes.

The combination of these techniques can provide a comprehensive understanding of PTF1A's transcriptional networks in pancreatic development and differentiation.

What methodological approaches can reveal PTF1A's role in pancreatic cancer progression?

Building on evidence that induced PTF1A expression in pancreatic ductal adenocarcinoma (PDAC) cells can reduce tumorigenic properties , researchers can employ several methodological approaches using PTF1A antibody:

Expression Analysis in Clinical Samples:

• Tissue Microarray Analysis:

  • Stain tissue microarrays containing normal pancreas, PanIN lesions, and PDAC samples of various grades.

  • Use biotin-conjugated PTF1A antibody with appropriate detection systems.

  • Quantify expression patterns across disease progression stages.

• Multiplex Immunofluorescence:

  • Perform co-staining of PTF1A with cancer stem cell markers (CD24/CD44) and proliferation markers.

  • Analyze correlation between PTF1A expression and tumorigenic properties.

Functional Studies:

• Inducible PTF1A Expression Systems:

  • Establish PDAC cell lines with doxycycline-inducible PTF1A expression as described in the literature .

  • Protocol:

    • Transduce PDAC cells with lentiviral vectors containing TetO-PTF1A.

    • Treat with doxycycline (1 μg/ml) to induce expression.

    • Confirm expression using biotin-conjugated PTF1A antibody.

    • Assess effects on:

      • Cell proliferation (measured by BrdU incorporation or Ki67 staining)

      • Cancer stem cell population (CD24+/CD44+ cells by flow cytometry)

      • Chemotherapy sensitivity (particularly to gemcitabine)

• Gene Expression Profiling:

  • Perform RNA-seq after PTF1A induction.

  • Analyze changes in:

    • Acinar differentiation genes (digestive enzymes)

    • Tumorigenic pathways

    • ATP-binding cassette efflux transporters (linked to drug resistance)

Mechanistic Studies:

• ChIP-seq:

  • Map PTF1A binding sites in normal pancreatic cells versus PDAC cells.

  • Identify differential binding patterns that may explain tumorigenic properties.

• Protein-Protein Interaction Analysis:

  • Use biotinylated PTF1A antibody for pull-down experiments.

  • Compare PTF1A interaction partners in normal versus cancer cells.

  • Identify cancer-specific interactions that might be therapeutic targets.

In vivo Models:

• Orthotopic Xenograft Models:

  • Implant PDAC cells with inducible PTF1A into mouse pancreas.

  • Induce PTF1A expression at different stages of tumor growth.

  • Monitor effects on tumor growth, metastasis, and response to therapy.

  • Analyze tumor sections with biotin-conjugated PTF1A antibody to confirm expression.

These approaches provide a comprehensive framework for understanding how PTF1A's loss contributes to pancreatic cancer progression and how its restoration might represent a therapeutic strategy.

What are common issues encountered when using PTF1A antibody and their solutions?

When working with PTF1A antibody, researchers may encounter several technical challenges. Here are methodological approaches to address them:

Issue 1: Weak or Absent Signal

Potential Causes and Solutions:

• Low antibody concentration: Titrate the antibody by testing concentrations from 1:100 to 1:2000. Western blot analysis has shown successful detection at 0.5 μg/mL .

• Insufficient antigen: PTF1A is normally expressed at low levels in many tissues. Consider using pancreatic tissue as a positive control, where PTF1A is known to be expressed .

• Improper sample preparation: Ensure proper fixation. For cell lines, 10% neutral buffered formalin fixation followed by 0.3% Triton X-100 permeabilization has proven effective .

• Degraded antibody: Avoid repeated freeze-thaw cycles. Store as recommended at -20°C and prepare working aliquots after reconstitution .

• Inefficient biotin-avidin interaction: Pre-block endogenous biotin using a biotin blocking kit, especially when working with tissues with high endogenous biotin levels.

Issue 2: High Background or Non-specific Binding

Potential Causes and Solutions:

• Insufficient blocking: Increase blocking time or concentration. 10% goat serum has been successfully used for PTF1A immunofluorescence .

• Cross-reactivity: Perform antibody validation using peptide competition assays with the immunogen peptide (recombinant human PTF1A protein, positions 224-325) .

• Excessive antibody concentration: Dilute the antibody further. For Western blot, 0.5 μg/mL has been effective .

• Contaminated buffers: Prepare fresh buffers and include 0.05% sodium azide to prevent microbial growth.

• Endogenous peroxidase activity: When using HRP-based detection systems, quench endogenous peroxidase activity with 0.3% H₂O₂ in methanol for 15-30 minutes before antibody incubation.

Issue 3: Inconsistent Results Between Experiments

Potential Causes and Solutions:

• Variable expression levels: PTF1A expression can vary with cell density and culture conditions. Standardize these parameters across experiments.

• Inconsistent sample preparation: Document and follow consistent protocols for fixation, permeabilization, and antigen retrieval.

• Batch variation: Record lot numbers of antibodies and reagents. Test new lots alongside previous ones before full implementation.

• Temperature variations: Perform critical incubation steps at controlled temperatures. For primary antibody incubations, overnight at 4°C often produces more consistent results than shorter incubations at room temperature .

How can I distinguish between specific and non-specific binding patterns when using PTF1A antibody?

Distinguishing specific from non-specific binding is crucial for accurate data interpretation:

Methodological Approach:

• Control Experiments:

  • Peptide competition: Pre-incubate the antibody with excess immunizing peptide. Specific signals should be eliminated or significantly reduced.

  • Isotype control: Use a non-relevant antibody of the same isotype (rabbit IgG for polyclonal PTF1A antibody) at the same concentration .

  • Knockout/knockdown controls: Compare staining in PTF1A-expressing versus PTF1A-depleted samples.

• Pattern Analysis:

  • Subcellular localization: PTF1A is primarily a nuclear protein (transcription factor). Predominant cytoplasmic staining may indicate non-specific binding .

  • Cell type specificity: In pancreatic tissue, PTF1A should be primarily detected in acinar cells, not in duct cells or islets. Non-selective staining across all cell types suggests non-specificity.

  • Signal intensity gradient: Specific binding typically shows a concentration-dependent pattern when antibody is titrated, while non-specific binding may not show clear dose-dependence.

• Multi-method Validation:

  • Cross-validation: Confirm results using multiple detection methods (e.g., immunofluorescence and Western blot).

  • Alternative antibodies: Compare staining patterns with different PTF1A antibodies targeting distinct epitopes.

  • mRNA correlation: Compare protein localization with mRNA expression (by in situ hybridization or RNA-seq of isolated cell populations).

• Technical Considerations:

  • Blocking optimization: Test different blocking agents (BSA, normal serum, commercial blockers) to identify the most effective for reducing background.

  • Wash stringency: Increase the number or duration of washes to remove weakly bound antibody.

  • Detection system selection: For biotin-conjugated antibodies, endogenous biotin can cause background. Consider biotin blocking steps or alternative detection systems.

• Quantitative Assessment:

  • Signal-to-noise ratio calculation: Measure specific signal intensity relative to background areas.

  • Threshold determination: Establish signal intensity thresholds based on negative controls to distinguish positive from negative staining.

How might PTF1A antibody be utilized in single-cell analysis techniques?

Single-cell technologies represent a frontier in biomedical research, and PTF1A antibody can be adapted for these approaches:

Single-Cell Protein Analysis:

• Mass Cytometry (CyTOF) Applications:

  • Protocol Adaptation:

    • Conjugate PTF1A antibody with rare earth metals instead of biotin.

    • Include in multiplexed antibody panels with other pancreatic lineage markers.

    • Perform intracellular staining after fixation and permeabilization.

    • Analyze using standard mass cytometry workflows.

  • Research Applications:

    • Profile heterogeneity in PTF1A expression across single cells during pancreatic development.

    • Correlate PTF1A levels with differentiation status markers in a high-dimensional manner.

    • Identify rare subpopulations with unique PTF1A expression patterns in pancreatic cancer.

• Single-Cell Western Blot:

  • Technical Approach:

    • Capture single cells in microwell arrays.

    • Perform in-situ cell lysis, protein separation, and immobilization.

    • Probe with biotin-conjugated PTF1A antibody followed by fluorescent streptavidin.

    • Quantify protein levels at single-cell resolution.

  • Research Value:

    • Measure absolute protein levels of PTF1A in individual cells.

    • Correlate with functional phenotypes in heterogeneous populations.

Integration with Genomic/Transcriptomic Data:

• CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing):

  • Methodological Approach:

    • Convert biotin-conjugated PTF1A antibody to oligonucleotide-tagged format.

    • Perform simultaneous protein and RNA profiling of single cells.

    • Bioinformatically integrate protein and transcript data.

  • Potential Insights:

    • Correlate PTF1A protein levels with mRNA expression of target genes.

    • Identify post-transcriptional regulation mechanisms.

    • Map transcriptional networks in PTF1A-expressing versus non-expressing cells.

• Spatial Transcriptomics with Protein Detection:

  • Technical Implementation:

    • Perform standard spatial transcriptomics with commercial platforms.

    • Add immunofluorescence layer using biotin-conjugated PTF1A antibody.

    • Integrate spatial protein data with spatial transcriptomic data.

  • Research Applications:

    • Map PTF1A protein distribution in tissue context.

    • Correlate with spatial expression patterns of target genes.

    • Identify tissue microenvironments that influence PTF1A expression.

What potential roles might PTF1A play in pancreatic cancer therapy resistance, and how can the antibody help investigate these mechanisms?

Research indicates that induced PTF1A expression can increase gemcitabine sensitivity in pancreatic cancer cells , suggesting its involvement in therapy resistance mechanisms:

Mechanistic Investigation Approaches:

• Drug Resistance Marker Correlation:

  • Methodological Framework:

    • Stain patient-derived xenografts or tissue microarrays for PTF1A and drug resistance markers (ABC transporters).

    • Perform multiplexed immunofluorescence with biotin-conjugated PTF1A antibody and antibodies against ABCB1, ABCG2, etc.

    • Quantify correlation between PTF1A levels and transporter expression.

  • Potential Insights:

    • Identify patient subgroups where PTF1A loss correlates with specific resistance mechanisms.

    • Develop predictive biomarkers for therapy response.

• Dynamic Resistance Development Models:

  • Experimental Design:

    • Establish gemcitabine-resistant PDAC cell lines through dose escalation.

    • Monitor PTF1A expression changes during resistance acquisition using the antibody.

    • Perform ChIP-seq with PTF1A antibody at different resistance stages.

    • Identify altered transcriptional targets during resistance development.

  • Expected Outcomes:

    • Map dynamic changes in PTF1A binding during resistance evolution.

    • Identify key regulatory networks that shift during resistance development.

• Therapeutic Restoration Strategies:

  • Experimental Approach:

    • Screen epigenetic modifiers that can restore PTF1A expression in resistant cells.

    • Validate PTF1A re-expression using the biotin-conjugated antibody.

    • Assess sensitization to chemotherapy after PTF1A restoration.

    • Perform transcriptome and ChIP-seq analysis to identify the molecular basis of sensitization.

  • Translational Potential:

    • Identify combinatorial approaches that restore PTF1A function and overcome resistance.

    • Develop biomarker panels to identify patients who might benefit from such approaches.

• Cancer Stem Cell (CSC) Regulation:

  • Technical Implementation:

    • Sort CD24+/CD44+ pancreatic CSCs .

    • Analyze PTF1A expression using flow cytometry with biotin-conjugated antibody.

    • Induce PTF1A expression in CSCs and assess effects on stemness, differentiation, and drug sensitivity.

    • Perform ChIP-seq to identify CSC-specific PTF1A targets.

  • Potential Discoveries:

    • Determine if PTF1A directly regulates stemness pathways.

    • Develop differentiation therapy approaches based on PTF1A restoration.

The methodological approaches outlined above provide a comprehensive framework for investigating PTF1A's role in therapy resistance and developing strategies to overcome resistance in pancreatic cancer.