POFUT2 Antibody, HRP conjugated

What is the molecular function of POFUT2 and why is it important in research?

POFUT2 catalyzes the reaction that attaches fucose through an O-glycosidic linkage to conserved serine or threonine residues specifically within thrombospondin type 1 repeats (TSRs) . This post-translational modification is essential for proper protein folding, stability, and function. POFUT2 is localized in the endoplasmic reticulum and exists in three isoforms (A, B, and C), each with distinct expression patterns that may contribute to functional diversity . Beyond its enzymatic role, POFUT2 may possess chaperone-like activity, aiding in the quality control and proper folding of glycoproteins, which is vital for maintaining cellular homeostasis . Research on POFUT2 is particularly relevant in understanding diseases where protein glycosylation is disrupted and in parasitic infections, as demonstrated in studies with Toxoplasma gondii where O-fucosylation of thrombospondin-like repeats plays crucial roles .

What applications are HRP-conjugated POFUT2 antibodies optimized for?

HRP-conjugated POFUT2 antibodies have been specifically optimized for multiple research applications including:

• Western Blotting (WB): Providing direct detection without requiring secondary antibodies, thus reducing background and cross-reactivity issues

• Immunohistochemistry with paraffin-embedded sections (IHC-P): Offering enhanced sensitivity for tissue section analysis

• Enzyme-Linked Immunosorbent Assay (ELISA): Enabling quantitative measurement of POFUT2 in complex biological samples

These direct HRP conjugates eliminate several steps in experimental workflows, reducing variability and potentially improving reproducibility. When employing these antibodies for Western blot applications, researchers typically use them at a concentration of approximately 2.5 μg/mL, whereas unconjugated primary antibodies would require an additional HRP-conjugated secondary antibody at dilutions between 1:50,000 and 1:100,000 .

What criteria should guide selection between polyclonal and monoclonal HRP-conjugated POFUT2 antibodies?

The selection between polyclonal and monoclonal HRP-conjugated POFUT2 antibodies should be based on the following methodological considerations:

When studying potentially novel POFUT2 isoforms or seeking broad epitope recognition, polyclonal antibodies may be preferable. For highly specific detection of known epitopes or when consistent long-term reproducibility is essential, monoclonal antibodies such as the G-1 clone offer advantages .

What storage and handling protocols optimize HRP-conjugated POFUT2 antibody performance?

To maintain optimal reactivity and stability of HRP-conjugated POFUT2 antibodies, researchers should follow these evidence-based protocols:

• Short-term storage (days): Store at 4°C in light-protected containers to prevent photobleaching of the HRP conjugate .

• Long-term storage: Store at -20°C, preferably in small working aliquots to minimize freeze-thaw cycles. The presence of 0.09% (w/v) sodium azide and 2% sucrose in typical storage buffers helps maintain antibody stability .

• Handling procedures:

  • Avoid repeated freeze-thaw cycles as they significantly reduce HRP enzymatic activity

  • Equilibrate to room temperature before opening to prevent condensation

  • Use sterile techniques when removing aliquots to prevent microbial contamination

  • Return to appropriate storage conditions immediately after use

  • Do not dilute the stock antibody until immediately before use

• Stability considerations: HRP activity diminishes over time, particularly after conjugation to antibodies. Using freshly prepared antibody dilutions for each experiment is recommended for consistent results .

How should I validate the specificity of HRP-conjugated POFUT2 antibodies?

Validating the specificity of HRP-conjugated POFUT2 antibodies requires a systematic approach incorporating multiple controls:

• Positive tissue/cell controls: Use tissues or cell lines known to express POFUT2, such as those with documented involvement in O-fucosylation pathways .

• Negative controls:

  • Primary antibody omission

  • Isotype controls (especially for monoclonal antibodies like G-1)

  • Tissues/cells from POFUT2 knockout/knockdown models when available

• Antigen blocking experiments: Pre-incubate the antibody with synthetic peptides corresponding to immunogen regions (for example, the C-terminal region peptide used for the antibody in search result ) .

• Western blot confirmation: Verify that the antibody detects a protein of approximately 49 kDa, which is the expected molecular weight of POFUT2 . Multiple bands may indicate detection of different isoforms or post-translational modifications.

• Cross-validation: Compare results with alternative POFUT2 antibodies targeting different epitopes, including both polyclonal antibodies targeting various regions and monoclonal antibodies like G-1 .

What methodological approaches enable differential detection of POFUT2 isoforms using HRP-conjugated antibodies?

Discriminating between the three known POFUT2 isoforms (A, B, and C) requires strategic selection of antibodies and experimental designs:

• Epitope-specific antibody selection: Different HRP-conjugated POFUT2 antibodies target distinct regions that may or may not be present in all isoforms. For instance:

  • C-terminal targeting antibodies may detect isoforms that share this region

  • Antibodies generated against specific peptide sequences unique to particular isoforms offer greater discrimination

• Migration pattern analysis: When performing Western blot analysis, careful optimization of gel percentage, running time, and molecular weight markers can resolve the subtle size differences between isoforms. The primary POFUT2 isoform is approximately 49 kDa, but other isoforms may present slight molecular weight variations .

• 2D gel electrophoresis: Combining isoelectric focusing with SDS-PAGE can separate isoforms based on both charge and size differences, providing improved resolution when using HRP-conjugated POFUT2 antibodies.

• Isoform-specific knockdown validation: Selectively silencing individual POFUT2 isoforms using siRNA/shRNA approaches followed by immunodetection can confirm antibody specificity for each isoform.

• Mass spectrometry correlation: Using immunoprecipitation with the HRP-conjugated antibody (after removing the HRP) followed by mass spectrometry can identify which isoforms are being captured, as demonstrated in studies examining POFUT2-modified proteins .

How do direct HRP-conjugated POFUT2 antibodies compare with two-step detection systems in sensitivity and specificity?

The choice between direct HRP-conjugated POFUT2 antibodies and unconjugated primary antibodies with separate HRP-conjugated secondary antibodies involves important technical trade-offs:

What optimization strategies improve immunohistochemical detection of POFUT2 using HRP-conjugated antibodies?

Optimizing immunohistochemical detection of POFUT2 requires attention to several methodological variables:

• Antigen retrieval optimization:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Enzymatic retrieval may be necessary for certain fixation conditions

  • Optimization of retrieval times based on tissue type and fixation duration

• Blocking protocol refinement:

  • Use 3-5% normal serum from the same species as the secondary antibody (if using amplification steps)

  • For direct HRP-conjugated antibodies, use protein blockers like BSA or commercial blockers specifically designed to reduce background with direct conjugates

  • Add 0.1-0.3% Triton X-100 for improved antibody penetration in tissue sections

• Signal amplification considerations:

  • For tissues with low POFUT2 expression, consider tyramide signal amplification (TSA) with HRP-conjugated antibodies

  • Biotin-free detection systems avoid endogenous biotin interference

• Counterstain selection:

  • Use light hematoxylin counterstaining to avoid obscuring HRP/DAB signal

  • Consider fluorescent counterstains for multichannel imaging when using converted fluorescent systems

• Incubation parameters:

  • Extend primary antibody incubation times (overnight at 4°C) for improved sensitivity

  • Optimize antibody concentration specifically for IHC-P applications, which may differ from the optimal concentration for Western blotting (2.5 μg/mL)

• Visualization system:

  • DAB (3,3'-diaminobenzidine) provides stable, permanent staining

  • AEC (3-amino-9-ethylcarbazole) offers alternative visualization with different contrasting properties

What experimental controls are essential when using HRP-conjugated POFUT2 antibodies to study protein O-fucosylation patterns?

When investigating O-fucosylation patterns mediated by POFUT2 using HRP-conjugated antibodies, comprehensive controls are necessary:

• Enzymatic modification controls:

  • Samples treated with O-glycosidase to remove O-linked glycans (which would eliminate O-fucose modifications)

  • Fucosidase treatment to specifically remove fucose residues

  • Comparison with samples from POFUT2 knockout/knockdown models

• Substrate validation controls:

  • Include known POFUT2 substrates containing thrombospondin type 1 repeats (TSRs) as positive controls

  • Include proteins lacking the POFUT2 consensus motif as negative controls

• Mass spectrometry correlation:

  • Parallel analysis using mass spectrometry to identify specific modification sites, as demonstrated in research examining thrombospondin modified sites

• Cross-comparison between antibody detection methods:

  • Complementary use of lectin staining specific for fucose residues

  • Comparison between different anti-POFUT2 antibodies targeting distinct epitopes

  • Correlation with metabolic labeling using fucose analogs

• Biological validation:

  • Functional studies examining protein stability, trafficking, or activity in the presence or absence of POFUT2-mediated modifications

  • Comparison with other O-fucosyltransferases like POFUT1, FUT10, or FUT11 to distinguish substrate specificities

How should researchers troubleshoot non-specific binding when using HRP-conjugated POFUT2 antibodies?

When encountering non-specific binding with HRP-conjugated POFUT2 antibodies, implement this systematic troubleshooting approach:

• Background identification and characterization:

  • Determine pattern (diffuse vs. specific structures)

  • Assess consistency across different sample types

  • Compare with isotype control or secondary-only controls

• Blocking optimization:

  • Increase blocking agent concentration (3-5% BSA or serum)

  • Extend blocking time (1-2 hours at room temperature)

  • Test alternative blocking agents (commercial blockers specifically designed for HRP-conjugated antibodies)

  • Add 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

• Antibody dilution refinement:

  • Prepare a dilution series of the HRP-conjugated POFUT2 antibody

  • Standard working concentration of 2.5 μg/mL for Western blot may need adjustment for other applications

  • Optimize incubation times and temperatures

• Washing protocol enhancement:

  • Increase number of washes (5-6 washes instead of 3)

  • Extended washing times (10-15 minutes per wash)

  • Add detergents (0.05-0.1% Tween-20) to washing buffers

  • Consider using PBS-T with varying ionic strengths

• Sample-specific considerations:

  • For tissues with high endogenous peroxidase activity, use additional quenching steps (3% H₂O₂ for 10-15 minutes)

  • For tissues with high background, consider pre-incubation with unconjugated host species immunoglobulins

  • For formalin-fixed samples, optimize antigen retrieval methods

• Storage and handling review:

  • Verify antibody has been stored according to manufacturer recommendations (4°C short-term, -20°C long-term)

  • Check for signs of degradation (precipitates, unusual coloration)

  • Confirm proper aliquoting to avoid freeze-thaw cycles

What methodological approaches enable reliable quantification of POFUT2 using HRP-conjugated antibodies?

Achieving accurate quantification of POFUT2 using HRP-conjugated antibodies requires rigorous standardization and careful technique:

• Western blot quantification:

  • Use graduated loading controls (GAPDH, β-actin) at multiple concentrations

  • Employ recombinant POFUT2 protein standards at known concentrations

  • Capture images within the linear dynamic range of detection

  • Use advanced analysis software with background subtraction capabilities

  • Standardize exposure times between experimental replicates

• ELISA optimization:

  • Develop standard curves using recombinant POFUT2 protein

  • Determine optimal coating concentrations of capture antibody

  • For sandwich ELISA, combine HRP-conjugated POFUT2 antibody (dilution 1:62500) with a second POFUT2 antibody targeting a different epitope

  • Include internal controls on each plate to normalize between experiments

• Immunohistochemistry quantification:

  • Use calibrated imaging systems with standardized acquisition parameters

  • Employ digital image analysis software for objective quantification

  • Include reference standards on each slide

  • Adopt systematic random sampling approaches for tissue analysis

  • Normalize to total cell count or tissue area

• Flow cytometry applications:

  • Establish fluorescence-minus-one (FMO) controls

  • Use calibration beads to standardize fluorescence intensity

  • Convert arbitrary units to molecules of equivalent soluble fluorochrome (MESF)

  • Include compensation controls when multiplexing with other fluorophores

How can researchers validate POFUT2 antibody specificity in CRISPR/Cas9 knockout or RNAi knockdown models?

Validating POFUT2 antibody specificity using genetic modification models involves several critical steps:

• CRISPR/Cas9 knockout validation strategy:

  • Design guide RNAs targeting conserved regions of POFUT2 gene

  • Confirm genetic modification via sequencing and/or PCR analysis

  • Assess complete protein loss using multiple anti-POFUT2 antibodies targeting different epitopes

  • Compare HRP-conjugated and unconjugated versions of the same antibody clone

  • Include rescue experiments by re-expressing POFUT2 (similar to approaches for FUT10/FUT11)

• RNAi knockdown validation approach:

  • Design multiple siRNA/shRNA sequences targeting different regions of POFUT2 mRNA

  • Establish dose-dependent knockdown efficiency

  • Correlate mRNA reduction (via qRT-PCR) with protein reduction (via Western blot)

  • Test antibody specificity across different knockdown levels

  • Include non-targeting control siRNA/shRNA

• Quantitative assessment methods:

  • Perform Western blot analysis using HRP-conjugated POFUT2 antibody at the recommended concentration (2.5 μg/mL)

  • Quantify signal reduction compared to wildtype/control samples

  • Evaluate effects on known POFUT2 substrates and downstream O-fucosylation patterns

• Cross-validation with orthogonal techniques:

  • Complement antibody-based detection with mass spectrometry analysis

  • Assess functional consequences of POFUT2 loss on protein O-fucosylation using lectin staining

  • Monitor effects on known POFUT2 substrates containing thrombospondin type 1 repeats (TSRs)

How can HRP-conjugated POFUT2 antibodies be employed in comparative glycobiology studies across species?

HRP-conjugated POFUT2 antibodies offer valuable tools for evolutionary and comparative glycobiology:

• Cross-species reactivity assessment:

  • While some POFUT2 antibodies are human-specific , others demonstrate cross-reactivity with mouse and rat POFUT2

  • Epitope conservation analysis across species can inform antibody selection

  • Validation in evolutionarily diverse models (vertebrates to invertebrates) may reveal conserved O-fucosylation mechanisms

• Methodology for comparative studies:

  • Standardize tissue preparation and fixation across species

  • Optimize antigen retrieval conditions specifically for each species

  • Use conserved housekeeping proteins as loading controls for quantitative comparisons

  • Apply phylogenetic analysis to correlate POFUT2 sequence conservation with antibody reactivity

• Parasite-host interaction studies:

  • Apply HRP-conjugated POFUT2 antibodies to study O-fucosylation in parasites like Toxoplasma gondii

  • Compare human and parasite POFUT2 using epitope-specific antibodies

  • Investigate POFUT2-mediated modifications in host-pathogen protein interactions

• Developmental biology applications:

  • Track POFUT2 expression across embryonic development in multiple species

  • Correlate with the expression of thrombospondin repeat-containing proteins

  • Investigate evolutionary conservation of O-fucosylation patterns in developmental processes

What are the methodological considerations for using HRP-conjugated POFUT2 antibodies in mass spectrometry-based proteomics workflows?

Integrating HRP-conjugated POFUT2 antibodies into mass spectrometry workflows requires careful attention to methodological details:

• Immunoprecipitation optimization:

  • Remove/inactivate HRP before mass spectrometry analysis to prevent interference

  • Use gentle elution conditions to preserve post-translational modifications

  • Include appropriate controls (IgG, isotype controls)

  • Validate efficiency using Western blot before proceeding to MS analysis

• Sample preparation for glycoproteomic analysis:

  • Enrich for O-fucosylated proteins using POFUT2 antibody immunoprecipitation

  • Optimize enzymatic digestion protocols to preserve glycopeptides

  • Consider complementary enrichment strategies (lectin affinity, hydrophilic interaction chromatography)

  • Use parallel glycosidase treatments to confirm glycan identity

• Mass spectrometry analysis strategies:

  • Employ collision-induced dissociation (CID) and electron transfer dissociation (ETD) methods for glycopeptide analysis, as demonstrated in TSR analysis

  • Monitor diagnostic fragment ions for fucose (dHex) and fucose-hexose disaccharides

  • Use low collision energy settings (10-15 eV) to preserve glycan modifications

  • Implement targeted methods for known POFUT2 substrates

• Data interpretation challenges:

  • Account for potential glycan rearrangements during MS analysis

  • Integrate glycopeptide and glycan databases into search algorithms

  • Validate MS/MS assignments with synthetic glycopeptide standards when possible

  • Correlate MS findings with antibody-based detection methods