PRNP Antibody, Biotin conjugated
Description
Antibody Characteristics
A. Antibody Type and Host
PRNP antibodies are available as polyclonal (rabbit) or monoclonal (mouse) variants. Polyclonal antibodies recognize multiple epitopes, offering broader reactivity, while monoclonal antibodies target specific epitopes for precise detection. Host species include rabbit, mouse, and others, with reactivity varying across human, rat, mouse, and primate models .
B. Biotin Conjugation
Biotin is covalently linked to the antibody’s Fc region, enabling binding to streptavidin-conjugated enzymes (e.g., HRP, alkaline phosphatase) or fluorescent probes. This amplification enhances signal detection in assays like ELISA, Western blotting, and immunohistochemistry (IHC) .
C. Immunogen and Epitope Specificity
Immunogens include recombinant PrP fragments (e.g., 23–64AA, 51–150AA). Monoclonal antibodies like PRIOC mAbs selectively bind oligomeric PrPSc (abnormal prion protein), distinguishing them from monomeric PrPC (cellular prion protein) .
Applications in Research
A. ELISA and Western Blotting
Biotin-conjugated PRNP antibodies amplify signal detection when paired with streptavidin-HRP or AP. For example, PRIOC mAbs enable sensitive detection of soluble oligomers in prion-infected cell lines .
B. Immunohistochemistry (IHC)
Used to localize PrP in tissue sections. For instance, anti-PRNP antibodies (e.g., PB9783) detect PrP in mouse and rat pancreas via biotin-streptavidin complex (SABC) with DAB chromogen .
C. Oligomer Detection
PRIOC mAbs specifically recognize aggregated PrPSc oligomers, enabling early diagnosis of prion diseases. In ScN2a neuroblastoma cells, these antibodies show large immunoreactive deposits, contrasting with membrane-bound monomers detected by anti-PrP controls .
D. Flow Cytometry
Biotinylated anti-PrP antibodies (e.g., 8G8) assess PrP expression in lymphocytes. Co-staining with CD19/CD3 markers reveals PrP co-localization with B/T cells .
Research Findings and Clinical Relevance
A. Oligomer Detection and Disease Diagnosis
PRIOC mAbs bind conformational epitopes exposed in PrPSc oligomers, enabling detection in blood and cerebrospinal fluid. This contrasts with monomeric antibodies, which fail to recognize oligomers .
C. Cross-Species Reactivity
Rabbit polyclonal antibodies (e.g., Bioss bs-4728R-Biotin) show broad reactivity across human, mouse, rat, cow, sheep, and horse, facilitating comparative studies .
Challenges and Considerations
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- An interaction site of peptide aptamer 8 in PrP was identified, and the complex was modeled in silico. This allowed the design of targeted mutations in PA8, potentially enhancing its binding properties. PMID: 29460268
- Research suggests that PrP protects cells against premature senescence induced by copper. PMID: 28800967
- These findings highlight a novel cellular response triggered by CsA treatment, leading to the secretion of misfolded PrP species from the cell. This mechanism may underlie the spread of toxic prions among cells and across tissues. PMID: 29127190
- Luman, a ubiquitous, non-canonical unfolded protein response (UPR), has been identified as a novel regulator of endoplasmic reticulum stress-induced PRNP expression. PMID: 28205568
- Studies have revealed that PrP enhances the responses to TNF-alpha, promoting proinflammatory cytokine production. This could contribute to inflammation and tumorigenesis. PMID: 28900035
- Asparagine and glutamine residues in the bank vole PrP enable prion conversion of human and rabbit PrPC. PMID: 28931606
- In contrast to western populations, the diverse phenotypical presentations of D178N mutants of PRNP in Chinese individuals were not solely determined by the 129 genotypes. PMID: 29569252
- The octarepeat region within the PrP peptide significantly influences the effects of redox on the biochemical phenotypes of PrP, highlighting the importance of the number of octarepeats in the biological functions of PrP. PMID: 29393338
- Genetic prion diseases (gPrDs) are caused by autosomal-dominant mutations in the prion protein gene (PRNP). PMID: 29478593
- All these data suggest that hypoxiamediated PrPC plays a crucial role in angiogenesis. This review summarizes the characteristics of PrPC, which is produced by HIF1alpha in hypoxia, in relation to angiogenesis. PMID: 28901450
- This study is the first to demonstrate that tauroursodeoxycholic acid protects MSCs against ER stress via the Akt-dependent PrP(C) and Akt-MnSOD pathway. PMID: 28004805
- The stabilization mechanism of specific binding compounds can be summarized as follows: they stabilize either the flexible C-terminal of alpha2 and the hydrophobic core, or only the hydrophobic core, or the overall structure of PrP(C) through high binding affinity. N159 and Q160 play a significant role in the specific binding of these compounds, all of which interact similarly with L130, P158, N159, Q160,H187, T190, T191. PMID: 28795797
- Disease-associated mutations provide valuable insights into possible key structural determinants underlying the misfolding of PrPC. (review) PMID: 28838676
- This study reports a novel p.S17G mutation in a clinically diagnosed LOAD patient, suggesting that the PRNP mutation is present in Chinese Alzheimer's disease patients. However, M129V polymorphism is not a risk factor for Alzheimer's disease or frontotemporal dementia in the Chinese Han population. PMID: 27910931
- These data indicate that the overexpression of PLK3-mediated degradation of abnormal PrP is largely dependent on the chaperone-mediated autophagy pathway. PMID: 27344333
- Biochemical characteristics of the valine-to-isoleucine substitution at codon 180 (V180I) in the PRNP gene were investigated in autopsied brains of patients with genetic Creutzfeldt-Jakob disease. Findings indicate that abnormal prion proteins in the neocortex are associated with toxicity resulting in severe spongiosis and that V180I is not a polymorphism, but is an authentic pathogenic mutation associated with specific biochemical characteristics. PMID: 29382530
- This research explores the mechanism of unfolding of the human prion protein. PMID: 28030950
- This paper provides an overview of the increasingly complex picture of prion protein proteolysis and sheds light on physiological and pathological roles associated with these cleavages. PMID: 28693923
- The coordination bonds between the Methionine-Lysine-Histidine (Ac-MKH-NHMe) tripeptide model associated with the fifth metal binding site, which triggers the beta-sheet formation of human prion protein, and divalent metal cations such as Mn(2+), Cu(2+) and Zn(2+) were studied. PMID: 27611644
- Importantly, flies expressing human PrP exhibiting a robust eye phenotype will allow for genetic screens to uncover novel mechanisms mediating PrP neurotoxicity. PMID: 28415023
- Research indicates that the PrP gene has an IRES-dependent translation initiation mechanism, and the IRESs inside the prion protein gene were successfully identified. PMID: 29107182
- The kinetics of prion replication occur in a prion protein codon 129 genotype-dependent manner, reflecting the genotype-dependent susceptibility to clinical variant Creutzfeldt-Jakob disease found in patients. PMID: 29141869
- The modulation of HOP-PrP(C) engagement or the decrease of PrP(C) and HOP expression may represent a potential therapeutic intervention in glioblastoma. PMID: 28412969
- A strong overexpression of PrP(C) is observed in human Merlin-deficient mesothelioma cell line TRA and in human Merlin-deficient meningiomas. PrP(C) contributes to increased proliferation, cell-matrix adhesion, and survival in schwannoma cells acting via 37/67 kDa non-integrin laminin receptor (LR/37/67 kDa). PMID: 28692055
- The homozygous state of position 129 in the PRNP is not a risk factor for MSA. No other variants in the PRNP gene were associated with an increased risk for MSA. PMID: 27793473
- Transgenic Creutzfeldt-Jakob disease (CJD) mice, expressing the mouse PrP (moPrP) homolog of human PrP D178N/V129 (moPrP D177N/V128), closely reproduce essential features of CJD. The mutant PrPs expressed in these mice are misfolded but unable to self-replicate. They accumulate in different compartments of the neuronal secretory pathway, impairing the membrane delivery of ion channels essential for neuronal function. PMID: 26864450
- Several nuclear PrP(c) partners have been identified, including gamma-catenin, one of its desmosomal partners, beta-catenin and TCF7L2, the main effectors of the canonical Wnt pathway, and YAP, one effector of the Hippo pathway. PMID: 27216988
- A rare mutation in PRNP leading to an exchange of amino acid from glutamic acid (E) to alanine (A) at codon 196 (E196A) is associated with Creutzfeldt-Jacob disease. PMID: 27310471
- This article discusses a framework for investigating the hypothesis that PrPC may be involved in major depression associated with neurodegenerative conditions, with a focus on the Transmissible Spongiform Encephalopathies (TSEs, or Prion Diseases) and Alzheimer's Disease (AlzD). PMID: 27057694
- Expert commentary: Computational approaches provide novel insights into prion-like protein functions, their regulation, and their role in disease. PMID: 28271922
- Findings show that a reduction of PFN-1 expression by elevated levels of PrP(c) may contribute to protective effects PrP(c)-overexpressing SH-SY5Y cells confer against STS-induced apoptosis. PMID: 28102851
- Research shows that sPrPc is involved in the processes of HIV neuropathogenesis and contributes to inflammation and neuronal damage. PMID: 28533442
- Copper(II) interaction with the Human Prion 103-112 fragment and its mutants has been studied with various techniques. The studied human prion fragment contains both histidine and methionine residues, while methionine residues are systematically replaced or displaced in the studied mutants. PMID: 28260678
- These data indicate that the disruption of the PrP(C)-HOP complex could be a potential therapeutic target for modulating the migratory and invasive cellular properties that lead to metastatic Colorectal cancer (CRC). PMID: 27112151
- A computational approach was used to elucidate in detail the aggregation propensity of PrP protein systems including wild type, wild type treated at different [Ca2+] or E200K mutant. Models for the self-assembly of either the E200K mutated or Ca2+-bound PrPC were sketched and discussed. PMID: 27959938
- The results have unveiled a novel molecular pathway driven by interactions between prion protein (PrP) and Notch1 in the progression of pancreatic ductal adenocarcinoma (PDAC), supporting a critical tumor-promoting role of Notch1 in PrP-expressing PDAC tumors. PMID: 27639164
- The present findings unveil particular neuropathological and neuroinflammatory profiles in Fatal familial insomnia(FFI) and novel characteristics of natural prion protein in FFI, altered PrPres and Scrapie PrP (abnormal and pathogenic PrP) patterns, and region-dependent putative capacity of PrP seeding. PMID: 27056979
- The data indicate a four-rung beta-solenoid structure as a key feature for the architecture of infectious mammalian prions. PMID: 27606840
- Distinctive properties of plaque-type dura mater graft-associated Creutzfeldt-Jakob disease PrPSc proteins in cell-protein misfolding cyclic amplification. PMID: 26878132
- Molecular insights obtained through MD (molecular dynamics) simulations suggested that each bispidine-based peptidomimetic differently engages a conserved Tyr 169 residue at the alpha2-beta2 loop of HuPrP and affects the stability of alpha2 and alpha3 helices. PMID: 27803245
- These data identify a network of proteins implicated in PrP(C) trafficking and demonstrate the power of this assay for identifying modulators of PrP(C) trafficking. PMID: 28341739
- Active compounds do not alter total or cell-surface levels of PrP(C), and do not bind to recombinant PrP in surface plasmon resonance experiments. However, at high concentrations, they inhibit PrP(Sc)-seeded conversion of recombinant PrP to a misfolded state in an in vitro reaction (RT-QuIC). PMID: 27803163
- Data provided molecular details about the interaction between HuPrP and the NCAM fibronectin domain, and revealed a new role of PrP(C) N terminus as a dynamic and functional element responsible for protein-protein interaction. PMID: 27535221
- This work sheds light on the amyloid core structures underlying prion strains and how I138M, I139M, and S143N affect prion protein aggregation kinetics. PMID: 27576687
- Data suggest that the second and third helices (H2 and H3) of the C-terminal region of the prion protein serve as Achilles heels of prion protein stability. The separation of B1-H1-B2 and H2-H3 domains appears to play a key role as well. (H1, H2, H3 denote the 3 alpha-helices; B1, B1 denote the 2 beta-sheets. Studies involved molecular dynamic simulations using nuclear magnetic resonance data obtained for N-terminal and C-terminal domains.) PMID: 28102071
- Prion protein-derived cell-penetrating peptide cytotoxicity is modulated by pH but independent of amyloid formation. PMID: 27818203
- This research investigates the effect of familial Creutzfeld-Jacob disease prion genes on prion protein conformation and secondary structure. PMID: 27107654
- The two cases reported here of sporadic Creutzfeldt-Jakob disease belonged to the same family and carried the most common point mutation of the PRNP gene observed in Italy. PMID: 26268049
- The protonation state of histidine 111 regulates the aggregation of the evolutionary most conserved region of the human prion protein. PMID: 27184108
- This study demonstrated that Prion Protein-Hemin Interaction Upregulates Hemoglobin Synthesis. PMID: 26836195
Q&A
What is PRNP and why is it a significant research target?
PRNP (prion protein) is a protein encoded by the PRNP gene, also known as CD230 or PRP. It has significant research importance due to its role in prion diseases. The prion protein exists in multiple conformational states, with the normal cellular form (PrP^C) capable of converting to a disease-associated form (PrP^Sc). This protein transformation is central to transmissible spongiform encephalopathies, making PRNP detection crucial for neurological disease research .
The protein typically has a calculated molecular weight of 27,661 Da, though it often appears at approximately 80 kDa in Western blot analyses due to post-translational modifications . Antibodies against PRNP are essential tools for studying protein localization, interaction networks, and pathological changes in prion diseases.
What advantages does biotin conjugation offer for PRNP antibody applications?
Biotin conjugation provides several methodological advantages for PRNP antibody applications:
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Signal amplification: The tetravalent structure of avidin/streptavidin molecules allows multiple biotin-binding opportunities, significantly enhancing detection sensitivity in low-abundance PRNP samples .
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Versatility of detection systems: Biotin-conjugated antibodies can be detected using various avidin/streptavidin-conjugated reporter enzymes (HRP, AP) or fluorophores, allowing flexibility in experimental design .
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Stability: The biotin-streptavidin bond is one of the strongest non-covalent interactions (Kd = 10^-15 M), providing stable detection complexes that withstand stringent washing conditions .
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Compatibility with proximity labeling: Biotin-conjugated PRNP antibodies can be used in proximity labeling approaches to identify protein interaction partners in situ .
What criteria should be considered when selecting a biotin-conjugated PRNP antibody?
When selecting a biotin-conjugated PRNP antibody, researchers should evaluate:
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Specificity: Verify that the antibody has been validated for specific recognition of PRNP without cross-reactivity to other proteins. Look for antibodies that have undergone rigorous validation processes, such as those designated as premium antibodies (e.g., Picoband) with minimal background .
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Epitope location: Consider whether the antibody targets an epitope in the N-terminal, middle region, or C-terminal of PRNP, as this affects detection of different PRNP conformations or fragments .
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Species reactivity: Confirm the antibody's reactivity with the target species. For example, some antibodies are specific to human PRNP while others detect mouse PRNP .
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Application compatibility: Ensure the antibody has been validated for your specific application (Western blot, ELISA, IHC, IP) .
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Biotin conjugation method: If purchasing a pre-conjugated antibody, inquire about the biotin:antibody ratio and conjugation chemistry used, as these factors affect performance .
How can I validate the specificity of a biotin-conjugated PRNP antibody?
Validation of biotin-conjugated PRNP antibody specificity should include:
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Positive and negative controls: Use tissues or cell lines known to express or lack PRNP expression respectively.
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Blocking peptide experiments: Pre-incubate the antibody with the immunizing peptide to demonstrate signal elimination in positive samples .
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Western blot analysis: Verify a single band at the expected molecular weight (typically observed at approximately 80 kDa for PRNP despite a calculated weight of 27,661 Da) .
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Knockout/knockdown validation: Test the antibody in PRNP knockout tissues or knockdown cell lines to confirm absence of signal.
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Cross-reactivity testing: Test against closely related proteins to ensure specificity, especially important since the search results indicate "no significant cross-reactivity or interference" should be observed with high-quality antibodies .
How can biotin-conjugated PRNP antibodies be utilized in ELISA assays?
For ELISA applications using biotin-conjugated PRNP antibodies:
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Sandwich ELISA protocol:
The standard ELISA workflow involves approximately 1-hour incubations for each step at 37°C with three washes between steps, as outlined in the assay procedure below:
| Step | Reagent | Incubation | Temperature |
|---|---|---|---|
| 1 | Standards or samples | 2 hours | 37°C |
| 2 | Working Biotin Conjugate Antibody | 1 hour | 37°C |
| 3 | Working Streptavidin-HRP | 1 hour | 37°C |
| 4 | Substrate Solution | 15-20 min | 37°C (dark) |
| 5 | Stop Solution | - | - |
This method can achieve detection sensitivity for PRNP as low as 0.078 ng/mL, with a detection range of 0.16-10 ng/mL .
What is the Biotinylation by Antibody Recognition (BAR) method and how can it be applied to PRNP research?
The Biotinylation by Antibody Recognition (BAR) method represents an advanced application for PRNP proximity research:
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Methodology: BAR uses a primary antibody (such as anti-PRNP) to target the protein of interest in fixed, permeabilized samples. An HRP-conjugated secondary antibody then catalyzes the formation of free radicals from hydrogen peroxide and phenol biotin, resulting in biotinylation of proteins in proximity to PRNP .
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Application to PRNP research: This method enables identification of proteins that interact with or localize near PRNP in situ, providing insights into PRNP's protein interaction network under both normal and pathological conditions .
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Advantages over traditional methods: Unlike conventional co-immunoprecipitation, BAR allows for harsh solubilization conditions after biotinylation, improving recovery of membrane-associated proteins like PRNP .
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Quantitative analysis: When combined with Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC), BAR provides quantitative contrasting of specific interactions versus nonspecific background .
Based on comparative studies with other interactome methods, BAR has demonstrated high coverage (81%) of high-confidence interactors defined by multiple datasets, making it valuable for comprehensive PRNP interaction studies .
What are the key differences between Avidin-Biotin Complex (ABC) and Labeled Streptavidin-Biotin (LSAB) methods for PRNP detection?
Both ABC and LSAB are signal amplification methods that leverage the biotin-avidin/streptavidin interaction, but they differ in several important aspects:
The choice between ABC and LSAB for PRNP detection should be based on the specific requirements of sensitivity versus background concerns. For detecting low levels of PRNP expression, the ABC method may provide greater signal amplification, while LSAB might offer cleaner results with less optimization .
How can I minimize background when using biotin-conjugated PRNP antibodies in tissue samples?
Background reduction when using biotin-conjugated PRNP antibodies requires addressing several potential sources of non-specific signal:
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Block endogenous biotin: Pre-treat tissue sections with avidin followed by biotin blocking solution, particularly important for biotin-rich tissues like liver, kidney, and brain where PRNP research is often conducted .
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Optimize antibody concentration: Titrate the biotin-conjugated PRNP antibody to determine the optimal concentration that maximizes specific signal while minimizing background .
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Reduce endogenous peroxidase activity: For HRP-based detection systems, pre-treat samples with hydrogen peroxide in methanol to quench endogenous peroxidase activity .
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Use appropriate blocking solution: Include proteins (BSA, normal serum) in blocking and antibody diluent buffers to reduce non-specific binding .
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Optimize incubation parameters: Adjust antibody incubation temperature and duration; lower temperatures (4°C) with longer incubations often yield better signal-to-noise ratios for PRNP detection .
These optimizations are particularly important for neurological tissue samples where PRNP detection specificity is crucial for accurate assessment of prion-related pathology.
How can I determine the linearity and recovery rates when developing a biotin-conjugated PRNP antibody-based assay?
Establishing assay linearity and recovery rates is essential for quantitative PRNP detection:
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Linearity assessment: Prepare serial dilutions (1:2, 1:4, 1:8, 1:16) of samples spiked with known PRNP concentrations and calculate the percentage of measured concentration to expected concentration. Acceptable linearity typically falls within 80-120% across the dilution range .
Sample linearity data for PRNP detection in different matrices:
| Dilution | Cell Culture Media Recovery (%) | Serum Recovery (%) |
|---|---|---|
| 1:2 | 101 (88-114) | 95 (80-105) |
| 1:4 | 92 (89-107) | 95 (84-99) |
| 1:8 | 91 (88-119) | 102 (88-105) |
| 1:16 | 107 (90-118) | 112 (80-114) |
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Recovery testing: Spike known quantities of recombinant PRNP into sample matrices and calculate percent recovery. For PRNP detection, expect recovery rates of approximately 80-84% in cell culture media and 90-117% in serum samples .
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Assay precision: Determine intra-plate precision (CV<10%) by testing samples 20 times on one plate and inter-plate precision (CV<15%) by testing across multiple plates .
These parameters provide critical validation of assay performance before applying biotin-conjugated PRNP antibodies to experimental or clinical samples.
Is it possible to conjugate existing PRNP antibodies with biotin for custom applications?
Yes, existing non-conjugated PRNP antibodies can be biotinylated for custom applications . The process requires:
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Antibody preparation: Ensure the antibody is in an amine-free buffer at approximately 1-10 mg/mL.
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Biotin reagent selection: Choose an appropriate biotinylation reagent:
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NHS-biotin derivatives for amine coupling (most common)
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Maleimide-activated biotin for thiol coupling
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Photoactivatable biotin for non-specific attachment
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Optimization of biotin:antibody ratio: Typically 3-8 biotin molecules per antibody provides optimal signal without compromising binding affinity.
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Purification: Remove excess unbound biotin using dialysis, gel filtration, or desalting columns.
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Validation: Test the biotinylated PRNP antibody against the original non-biotinylated version to ensure retention of specificity and sensitivity.
Commercial biotinylation kits are available that simplify this process, providing all necessary reagents and protocols for successful conjugation while preserving antibody activity.
How can biotin-conjugated PRNP antibodies contribute to interactome studies of prion proteins?
Biotin-conjugated PRNP antibodies offer powerful approaches for characterizing the PRNP interactome:
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Proximity-dependent biotinylation: Using methods like BAR, researchers can identify proteins in close proximity to PRNP under various physiological and pathological conditions .
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Comparative interactome analysis: Biotin-conjugated PRNP antibodies enable systematic comparison of interacting partners between normal (PrP^C) and disease-associated (PrP^Sc) prion protein conformations.
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Cross-dataset validation: Studies using biotin-conjugated PRNP antibodies have demonstrated high coverage (81%) of high-confidence interactors defined across multiple datasets, suggesting robust methodology for interactome characterization .
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Subcellular localization-specific interactions: By combining with subcellular fractionation, biotin-conjugated PRNP antibodies can reveal compartment-specific interaction networks.
This approach has particular relevance for understanding mechanistic aspects of prion conversion and identifying potential therapeutic targets that could modulate PRNP interactions.
What methodological considerations are important when using biotin-conjugated PRNP antibodies for distinguishing between PrP^C and PrP^Sc conformations?
Distinguishing between normal cellular prion protein (PrP^C) and the disease-associated conformation (PrP^Sc) requires specialized approaches:
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Epitope accessibility: Some epitopes are masked or exposed differently between PrP^C and PrP^Sc. Select biotin-conjugated antibodies targeting epitopes that differentially recognize these conformations .
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Sample preparation: Employ proteinase K digestion protocols, as PrP^Sc is partially resistant to proteinase K while PrP^C is completely digested.
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Conformation-dependent immunoassays: Develop sandwich ELISA formats using biotin-conjugated antibodies that recognize conformation-specific epitopes.
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Differential extraction methods: Combine with extraction protocols that separate PrP^C (detergent-soluble) from PrP^Sc (detergent-insoluble) fractions before antibody application.
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Signal validation: Include appropriate controls (brain samples from prion-infected and non-infected animals) to validate the specificity of conformation-dependent detection.
These methodological considerations are critical for accurate assessment of prion pathology and disease progression monitoring using biotin-conjugated PRNP antibodies.
