POGLUT1 Antibody, FITC conjugated

What is POGLUT1 and what are its primary functions in cellular biology?

POGLUT1 (Protein O-glucosyltransferase 1) is a dual specificity glycosyltransferase that catalyzes the transfer of glucose and xylose from UDP-glucose and UDP-xylose, respectively, to serine residues within the consensus sequence C-X-S-X-P-C. The enzyme specifically targets extracellular EGF repeats of proteins including CRB2, F7, F9, and NOTCH2. POGLUT1 functions as a positive regulator of Notch signaling through O-glucosylation of Notch, which subsequently regulates muscle development.

Originally isolated from bone marrow cells of patients with myelodysplastic syndrome/acute myeloid leukemia, POGLUT1 has since been identified in multiple tissues including mammary glands, lymph nodes, intestine, liver, and spleen. Research indicates that POGLUT1 promotes cell proliferation in certain contexts and can counteract TGF-β1-mediated growth inhibition.

How does the FITC conjugation affect the application range of POGLUT1 antibodies?

The FITC (Fluorescein Isothiocyanate) conjugation to POGLUT1 antibodies enables direct visualization of POGLUT1 without requiring secondary antibody detection steps. This modification makes the antibody particularly valuable for:

• Flow cytometry applications

• Direct immunofluorescence microscopy

• Multiplexed imaging with compatible fluorophores

• Live cell imaging (under appropriate conditions)

When working with FITC-conjugated antibodies, researchers should consider that:

• The fluorophore has an excitation maximum at approximately 495 nm and emission maximum around 519 nm (green spectrum)

• FITC is pH-sensitive and may show reduced fluorescence at lower pH levels

• The fluorophore is susceptible to photobleaching, requiring appropriate controls and imaging protocols

• Background autofluorescence in the green channel may interfere with detection in certain tissue types

What are the key characteristics of the commercially available POGLUT1 Antibody, FITC conjugated?

The commercially available POGLUT1 Antibody, FITC conjugated is a polyclonal antibody raised in rabbit against recombinant Protein O-glucosyltransferase 1 protein (amino acids 132-392). It has the following specifications:

• Immunogen Species: Homo sapiens

• Clonality: Polyclonal

• Isotype: IgG

• Purification Method: Protein G purified (>95% purity)

• Conjugate: FITC (Fluorescein Isothiocyanate)

• Buffer Composition: 0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4

• Form: Liquid

The antibody recognizes POGLUT1, which is also known by several aliases including C3orf9, CLP46, KTELC1, MDS010, and Rumi, with UniProt ID Q8NBL1.

What are best practices for designing experiments with FITC-conjugated POGLUT1 antibodies?

When designing experiments using FITC-conjugated POGLUT1 antibodies, researchers should consider:

• Controls: Include appropriate negative controls (isotype control antibodies) and positive controls (cell lines known to express POGLUT1). Based on published research, BT474 breast cancer cells are suitable as they express detectable levels of POGLUT1.

• Fixation and Permeabilization: If studying intracellular localization of POGLUT1, optimize fixation methods (paraformaldehyde 4% is commonly used) and permeabilization conditions (Triton X-100 or methanol) to maintain protein epitope accessibility while preserving cellular architecture.

• Antibody Titration: Perform titration experiments to determine optimal antibody concentration, typically starting with manufacturer's recommendations and testing 2-fold dilutions.

• Autofluorescence Mitigation: Include unstained controls to assess and subtract tissue autofluorescence, particularly important when examining tissues like liver that naturally exhibit high green autofluorescence.

• Multicolor Panel Design: When designing multicolor panels, avoid fluorophores with spectral overlap with FITC (e.g., PE) or include appropriate compensation controls.

How can researchers validate the specificity of FITC-conjugated POGLUT1 antibodies?

Validation of antibody specificity is crucial for obtaining reliable results. For FITC-conjugated POGLUT1 antibodies, consider these validation approaches:

• Peptide Competition Assay: Pre-incubate the antibody with excess recombinant POGLUT1 protein (amino acids 132-392) before staining to demonstrate specific blocking of the antibody binding.

• POGLUT1 Knockdown/Knockout Controls: Use siRNA, shRNA, or CRISPR-Cas9 to create POGLUT1-depleted cells as negative controls.

• Western Blot Correlation: Perform parallel western blot analysis using unconjugated POGLUT1 antibodies to confirm the expression pattern observed in immunofluorescence or flow cytometry.

• Cross-Validation: Compare staining patterns obtained with different POGLUT1 antibodies targeting distinct epitopes.

• Recombinant Expression System: Overexpress POGLUT1 in a cell line with low endogenous expression to demonstrate specific staining increase.

What sample preparation protocols optimize detection of POGLUT1 using FITC-conjugated antibodies?

Optimal sample preparation depends on the experimental approach:

For Flow Cytometry:

• Harvest cells using gentle methods (avoid trypsin if possible)

• Fix cells with 2-4% paraformaldehyde for 10-15 minutes at room temperature

• Permeabilize with 0.1% Triton X-100 or 90% methanol if detecting intracellular POGLUT1

• Block with 5% normal serum from the same species as the secondary antibody

• Incubate with FITC-conjugated POGLUT1 antibody at optimized concentration

• Wash thoroughly with PBS containing 0.1% BSA

• Analyze promptly or store protected from light at 4°C

For Immunofluorescence Microscopy:

• Culture cells on glass coverslips or prepare tissue sections (4-8 μm)

• Fix samples with 4% paraformaldehyde for 15-20 minutes

• Permeabilize with 0.2% Triton X-100 for 10 minutes

• Block with 5-10% serum for 1 hour at room temperature

• Incubate with FITC-conjugated POGLUT1 antibody overnight at 4°C

• Wash extensively with PBS

• Counterstain nucleus with DAPI if desired

• Mount with anti-fade mounting medium

• Seal edges and store at 4°C protected from light

How can FITC-conjugated POGLUT1 antibodies be used to investigate POGLUT1's role in the TGF-β1 signaling pathway?

POGLUT1 has been shown to interact with the TGF-β1 signaling pathway, particularly affecting p16 expression and Smad3 phosphorylation. To investigate this role using FITC-conjugated POGLUT1 antibodies:

• Co-localization Studies: Perform dual immunofluorescence staining with FITC-conjugated POGLUT1 antibodies and antibodies against TGF-β1 pathway components (e.g., TGF-β receptors, Smad3) to assess potential co-localization.

• Phospho-Smad3 Correlation Analysis: Research has shown that POGLUT1 overexpression inhibits phosphorylated Smad3 (p-Smad3) expression in BT474 cells treated with TGF-β1. Design experiments with dual staining for POGLUT1 (using FITC-conjugated antibody) and p-Smad3 (using a compatible fluorophore) to quantify this inverse relationship at the single-cell level.

• Time-course Experiments: Stimulate cells with TGF-β1 and use FITC-conjugated POGLUT1 antibodies to track changes in POGLUT1 localization or expression levels over time.

• POGLUT1 Overexpression Studies: Create POGLUT1-overexpressing cell lines and use FITC-conjugated POGLUT1 antibodies to confirm overexpression while simultaneously measuring TGF-β1 pathway activation markers.

• Cell Cycle Analysis: Combine FITC-conjugated POGLUT1 antibody staining with cell cycle markers to investigate how POGLUT1 affects cell cycle progression in the context of TGF-β1 signaling, particularly at the G1/S checkpoint.

What methods can detect the impact of POGLUT1 on p16 expression using FITC-conjugated antibodies?

Research has demonstrated that POGLUT1 overexpression downregulates p16 expression in BT474 cells treated with TGF-β1. To investigate this relationship using FITC-conjugated POGLUT1 antibodies:

• Dual Immunofluorescence: Perform co-staining with FITC-conjugated POGLUT1 antibodies and p16 antibodies (conjugated to a spectrally distinct fluorophore) to quantify the inverse correlation between POGLUT1 and p16 expression at the single-cell level.

• Flow Cytometric Analysis: Use flow cytometry with FITC-conjugated POGLUT1 antibodies and differently labeled p16 antibodies to quantitatively assess the relationship between POGLUT1 and p16 expression across cell populations.

• Time-course Experiments: Treat cells with TGF-β1 and monitor changes in POGLUT1 and p16 expression over time using immunofluorescence or flow cytometry.

• Genetic Manipulation Studies: Compare p16 expression in wild-type and POGLUT1-overexpressing cells using FITC-conjugated POGLUT1 antibodies to confirm overexpression and anti-p16 antibodies to assess downregulation.

• Image Cytometry Analysis: Quantify fluorescence intensity of POGLUT1-FITC and p16 staining in individual cells to establish quantitative relationships.

How can researchers investigate the role of POGLUT1 in O-glucosylation of Notch using FITC-conjugated antibodies?

POGLUT1 is known to mediate O-glucosylation of Notch, acting as a positive regulator of Notch signaling. To investigate this function:

• Co-immunoprecipitation with Fluorescence Detection: Use FITC-conjugated POGLUT1 antibodies to detect POGLUT1 in immunoprecipitated Notch complexes, confirming their physical interaction.

• Confocal Microscopy Co-localization: Perform dual staining with FITC-conjugated POGLUT1 antibodies and antibodies against Notch receptors to assess co-localization in different cellular compartments.

• FRET Analysis: Where appropriate technical setups exist, use Förster Resonance Energy Transfer (FRET) between FITC-conjugated POGLUT1 antibodies and acceptor fluorophore-labeled Notch antibodies to detect close molecular proximity.

• Glycosylation Site Mutation Studies: Compare the binding pattern of FITC-conjugated POGLUT1 antibodies in wild-type cells versus cells expressing Notch with mutations in the consensus sequence C-X-S-X-P-C.

• Mass Spectrometry Validation: Use immunofluorescence with FITC-conjugated POGLUT1 antibodies to identify cells or tissues for subsequent glycoproteomic analysis of Notch O-glucosylation by mass spectrometry.

What are common technical challenges when using FITC-conjugated POGLUT1 antibodies and how can they be addressed?

Challenge 1: Background Fluorescence

• Solution: Include proper blocking steps (5-10% serum), optimize antibody concentration, include isotype controls, and consider adding 0.1-0.3% Triton X-100 to reduce non-specific binding.

Challenge 2: Photobleaching

• Solution: Minimize exposure to excitation light, use anti-fade mounting media containing anti-photobleaching agents, and capture images of control samples first.

Challenge 3: Fixation-induced Autofluorescence

• Solution: Use freshly prepared fixatives, consider alternative fixation methods (methanol instead of paraformaldehyde), or include a brief treatment with sodium borohydride (NaBH4) to reduce autofluorescence.

Challenge 4: Signal Variation Between Experiments

• Solution: Standardize all protocols, include calibration standards in each experiment, and normalize data to internal controls.

Challenge 5: Unexpected Subcellular Localization

• Solution: Validate findings using multiple detection methods, including unconjugated antibodies with secondary detection or antibodies targeting different epitopes.

How should FITC-conjugated POGLUT1 antibodies be stored and handled to maintain optimal performance?

To ensure optimal performance of FITC-conjugated POGLUT1 antibodies:

• Storage Conditions:

  • Store at -20°C in the dark

  • Avoid repeated freeze-thaw cycles (aliquot upon receipt)

  • The antibody is typically provided in 50% glycerol, 0.01M PBS, pH 7.4 with 0.03% Proclin 300 as preservative

• Handling Practices:

  • Minimize exposure to light at all stages

  • Allow antibody to equilibrate to room temperature before opening

  • Gently mix by inversion rather than vortexing

  • Return to storage promptly after use

  • Avoid introducing contaminants into the antibody stock

• Working Solution Preparation:

  • Prepare dilutions immediately before use

  • Use high-quality, filtered buffers

  • Centrifuge briefly before opening to collect liquid at the bottom

  • Consider adding protease inhibitors to working solutions

• Quality Control:

  • Test periodically with positive control samples

  • Monitor for changes in background or signal intensity over time

  • Record lot numbers and correlate with experimental outcomes

How can researchers accurately quantify POGLUT1 expression levels using FITC-conjugated antibodies?

Accurate quantification of POGLUT1 expression using FITC-conjugated antibodies requires:

• Flow Cytometry Quantification:

  • Use calibration beads with known quantities of fluorophore

  • Calculate molecules of equivalent soluble fluorochrome (MESF)

  • Include consistent positive and negative controls

  • Consider using quantitative flow cytometry standards

• Immunofluorescence Quantification:

  • Capture images using identical exposure settings

  • Include fluorescence intensity standards in each imaging session

  • Use software like ImageJ/FIJI with appropriate background subtraction

  • Report relative fluorescence units (RFU) or normalized values

• Controls for Quantification:

  • Include unstained, isotype, and single-stained controls

  • Use cells with known POGLUT1 expression levels as biological references

  • Correct for autofluorescence using unstained samples

• Standardization Approaches:

  • Normalize to housekeeping proteins or DNA content

  • Use internal reference cells in each experiment

  • Consider ratiometric approaches when examining changes over conditions

• Statistical Analysis:

  • Apply appropriate statistical tests for the experimental design

  • Consider the distribution of fluorescence intensity (normal vs. non-normal)

  • Present both mean/median values and measures of dispersion

How can FITC-conjugated POGLUT1 antibodies be used to study cancer cell proliferation mechanisms?

POGLUT1 has been implicated in promoting cell proliferation, particularly in cancer cells. To investigate its role:

• Correlation with Proliferation Markers: Perform dual staining with FITC-conjugated POGLUT1 antibodies and markers of proliferation (Ki-67, PCNA) to establish quantitative relationships.

• Cell Cycle Analysis: Combine POGLUT1-FITC staining with DNA content analysis to determine how POGLUT1 expression varies across cell cycle phases. Research indicates POGLUT1 functions primarily at the G1/S phase transition.

• Tumor Tissue Microarray Analysis: Apply FITC-conjugated POGLUT1 antibodies to tissue microarrays to assess expression patterns across multiple tumor samples and correlate with clinicopathological features.

• p16 Expression Studies: Investigate the inverse relationship between POGLUT1 and the tumor suppressor p16 using dual immunofluorescence approaches. Research shows POGLUT1 overexpression inhibits TGF-β1-induced p16 upregulation.

• Phospho-Smad3 Correlation: Examine how POGLUT1 expression levels correlate with phosphorylated Smad3, an important mediator of TGF-β1 antiproliferative effects. POGLUT1 overexpression has been shown to inhibit p-Smad3 expression in BT474 cells.

What experimental design best examines POGLUT1's relationship with Notch signaling using FITC-conjugated antibodies?

To investigate POGLUT1's relationship with Notch signaling:

• Co-localization Analysis: Perform dual immunofluorescence with FITC-conjugated POGLUT1 antibodies and antibodies against Notch receptors or downstream effectors (Hes1, Hey1).

• Notch Activation Studies: Induce Notch signaling using ligand stimulation or DAPT inhibition and monitor changes in POGLUT1 localization using FITC-conjugated antibodies.

• O-glucosylation Detection: Combine POGLUT1-FITC staining with antibodies that specifically recognize O-glucosylated Notch to assess correlation between enzyme presence and target modification.

• POGLUT1 Manipulation Experiments: Use genetic approaches to modulate POGLUT1 levels and examine effects on Notch target gene expression while confirming POGLUT1 status using FITC-conjugated antibodies.

• Developmental Studies: In appropriate model systems, use FITC-conjugated POGLUT1 antibodies to track expression during development in relation to Notch-dependent processes such as muscle development, as POGLUT1 regulates Notch signaling to coordinate muscle development.

• EGF Repeat Analysis: Focus on specific EGF repeats known to be O-glucosylated (particularly EGF6, EGF16, EGF26, and EGF27 in NOTCH1) when designing experiments to correlate POGLUT1 binding with functional outcomes.