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| Product Name | 5'-Biotin-TEG Oligonucleotides, HPLC Purified, Custom Sequence, Affinity Grade |
| Catalog No. | PRIPRO-0038 |
| Description | Custom oligonucleotides with 5'-biotin modification via a triethylene glycol (TEG) spacer arm, HPLC-purified for the highest purity and consistent biotinylation. The biotin moiety, covalently attached through a 15-atom TEG linker to the 5'-phosphate of the oligonucleotide, provides a flexible, hydrophilic spacer that extends the biotin away from the DNA duplex, ensuring optimal accessibility for streptavidin, avidin, or NeutrAvidin binding. These affinity probes are widely used for: streptavidin-coated surface immobilization (microplates, magnetic beads, biosensor chips, microarrays); electrophoretic mobility shift assays (EMSA) with streptavidin-induced supershift; DNA pull-down and affinity purification of DNA-binding proteins; construction of DNA origami and nanostructures; and biotin-streptavidin bridge systems in diagnostic assay development. |
| Intended Use | High-affinity capture and immobilization probes for: streptavidin/avidin/NeutrAvidin-coated 96-well plates (ELISA-based DNA detection); streptavidin magnetic beads (DNA pull-down, target enrichment, template preparation for NGS); surface plasmon resonance (SPR) and biolayer interferometry (BLI) biosensor chips (streptavidin-coated); DNA microarray printing on streptavidin-coated slides; electrophoretic mobility shift assays (EMSA/supershift with streptavidin); DNA affinity chromatography for transcription factor and DNA-binding protein purification; DNA-directed immobilization in biosensor and nanotechnology applications; proximity ligation assays (PLA) and proximity extension assays (PEA) requiring biotinylated oligonucleotides. |
| Principle / Technology | Biotin (vitamin B7, vitamin H) is a 244 Da bicyclic molecule that binds to streptavidin (a 60 kDa homotetrameric protein from Streptomyces avidinii) with extraordinary affinity (Kd approximately 10^-14 to 10^-15 M), one of the strongest non-covalent interactions known in biology. The TEG spacer (15-atom linker, approximately 20 Angstroms extended length) between biotin and the 5'-phosphate of the oligonucleotide provides rotational flexibility and distance to overcome steric hindrance from the oligonucleotide duplex and the streptavidin binding pocket, enhancing capture efficiency compared to biotin without a spacer or with short spacers. The TEG linkage is stable under standard biochemical conditions and is compatible with thermal denaturation and reannealing of the DNA duplex. |
| Detection Method | Customer provides target sequence (5' to 3', 15-60 bases); 5'-Biotin-TEG added during the final coupling step of automated DNA synthesis using biotin-TEG phosphoramidite (DMT-biotin-TEG-CEP); extended coupling time (3-6 minutes) for complete biotin incorporation; standard ammonium hydroxide deprotection (55 C, 8-16 h); DMT-ON or DMT-OFF purification by reversed-phase HPLC (DMT-ON: separation based on hydrophobic DMT group; DMT-OFF: ion-pairing or ion-exchange); final product desalted, lyophilized. HPLC-purified probes include QC documentation with chromatogram and mass spectrum. |
| Sample Type | Customer-specified sequence (standard IUPAC: A, C, G, T); recommended length 15-50 bases for optimal synthesis and purification; avoid internal biotin-dT when 5'-biotin-TEG is used (both can be included upon request for dual-biotin probes). |
| Performance Range / Specifications | Oligonucleotide length: 15-50 bases (up to 100 upon request); biotin incorporation: >=95% (ratio of biotin-positive to total oligonucleotide); purity: >=95% by HPLC; binding capacity: >=90% of HPLC-purified biotin-oligo captured by streptavidin magnetic beads at saturating bead concentration; spacer: TEG (15 atoms, approximately 20 Angstroms). |
| Sensitivity / LOD | Biotinylated oligonucleotide detection: <10 fmol detected by streptavidin-HRP chemiluminescence in dot blot; <1 fmol captured on streptavidin biosensor chip detectable by SPR. |
| Specificity | Biotin-streptavidin binding is highly specific; biotinylated oligonucleotide binding to streptavidin is resistant to: high salt (up to 2 M NaCl), detergents (up to 1% SDS, 1% Triton X-100), denaturants (up to 6 M urea), and organic solvents (up to 30% DMF or DMSO). Elution of biotinylated DNA from streptavidin requires: boiling in 95% formamide with 10 mM EDTA, or competitive displacement with excess free biotin (10-50 mM, requires 30-60 min at 65 C). |
| Reaction Conditions / Protocol | Capture of biotinylated oligonucleotide on streptavidin-coated surface: incubation 15-60 min at RT in binding buffer (PBS or TBS with 0.05% Tween-20); elution: (1) thermal: 95 C 5 min in water or formamide, or (2) competitive: 10-50 mM free biotin, 30-60 min at 65 C; stability of biotin-streptavidin complex: weeks at 4 C, months at -20 C, survives PCR thermal cycling. |
| Components / Formulation | 5'-Biotin-TEG oligonucleotide (lyophilized, specified scale and purification); QC Documentation: RP-HPLC chromatogram, MALDI-TOF mass spectrum, UV quantitation (A260), Certificate of Analysis, Biotin-streptavidin binding application guide. |
| Storage Conditions | Lyophilized: stable at -20 C for 24 months; reconstituted in TE or water (100 uM) at -20 C for 12 months; avoid repeated freeze-thaw cycles; protect from nuclease contamination. |
| Shelf Life | 24 months from date of synthesis at -20 C lyophilized. |
| Package Specifications | 50 nmol, 200 nmol, 1 umol (desalted); 50 nmol, 200 nmol (HPLC purified). |
| Product Form | White to off-white lyophilized pellet; reconstitution yields clear colorless solution; biotin not visible. |
| Quality Control | Each probe: (1) RP-HPLC: >=95% purity (DMT-ON or DMT-OFF); (2) MALDI-TOF MS: mass within 0.1% of calculated; (3) UV-Vis quantitation by A260; (4) Biotin binding activity: >=90% captured by streptavidin magnetic beads (M-280 Dynabeads) from 1 pmol input; (5) Functional test: captured probe retains hybridization capability (tested with complementary labeled target). |
| Key Features | 5'-Biotin with TEG spacer for optimal streptavidin accessibility; HPLC purified >=95%; verified streptavidin binding >=90%; TEG spacer minimizes steric hindrance; resistant to harsh elution conditions; custom sequence; application guide included. |
| Purity | >=95% full-length biotinylated product by HPLC; no detectable free biotin or biotin-TEG phosphoramidite; biotin incorporation >=95%. |
| Concentration | Lyophilized: specified nmol; reconstitute to 100 uM in TE or nuclease-free water; working concentration 10-500 nM for most capture applications; verify concentration by A260 measurement. |
| Activity / Unit Definition | >=90% capture efficiency on streptavidin magnetic beads (Dynabeads M-280) at 1 pmol input; functional after capture: >70% hybridization efficiency with complementary oligonucleotide on bead surface. |
| Molecular Weight | Calculated from sequence composition plus biotin-TEG moiety (biotin-TEG adds approximately 465 g/mol to 5'-hydroxyl oligonucleotide). |
| Source / Origin | Oligonucleotide: automated phosphoramidite synthesis; biotin-TEG phosphoramidite (DMT-biotin-TEG-CEP) from commercial supplier; all synthesis reagents anhydrous grade; streptavidin binding verification performed with commercial streptavidin beads. |
| pH Range / Optimal pH | Biotin-streptavidin binding stable at pH 4.0-10.0; optimal binding pH 7.0-8.5; oligonucleotide stable at pH 6.0-9.0; biotin stable under standard biochemical conditions. |
| Shipping Conditions | Ambient temperature (lyophilized); stable at ambient for 4 weeks. |
| Expiration Date / Stability | 24 months at -20 C lyophilized; reconstituted: 12 months at -20 C; working dilution: 3 months at 2-8 C protected from nuclease contamination. |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic use. |
| Compatibility | Compatible with: streptavidin-coated magnetic beads (Dynabeads M-270/M-280/MyOne Streptavidin C1/T1), streptavidin-coated polystyrene microplates, streptavidin agarose and Sepharose resins, streptavidin biosensor chips (Biacore SA, ForteBio SA), streptavidin-coated quantum dots, and NeutrAvidin-coated surfaces. Compatible with streptavidin-HRP, streptavidin-AP, streptavidin-fluorophore conjugates for detection. For DNA pull-down, the TEG spacer allows efficient capture even with short proximity to the DNA duplex. Not compatible with avidin at low pH (avidin has high non-specific binding at acidic pH). The biotin-streptavidin interaction is insensitive to high salt (up to 2 M), allowing stringent wash conditions. |
| Recommended Buffer System | Reconstitution: TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) or nuclease-free water; Binding buffer: PBS pH 7.4 + 0.05% Tween-20, or TBS pH 7.4 + 0.05% Tween-20, or 10 mM Tris-HCl pH 7.5, 1 M NaCl, 1 mM EDTA. |
| Application Notes / Precautions | Centrifuge tube before opening to collect all material. Reconstitute to 100 uM in TE; aliquot to avoid repeated freeze-thaw cycles. Biotinylated oligonucleotide concentration can be verified by HABA-avidin dye displacement assay. For magnetic bead immobilization: wash beads, incubate with biotinylated oligonucleotide (typically 10-50 pmol per 50 uL beads) in binding buffer for 15-30 min at RT with rotation, wash to remove unbound. Test capture efficiency by measuring A260 of supernatant before and after immobilization. For EMSA/supershift: incubate biotinylated probe with nuclear extract, then add streptavidin (0.5-2 ug per reaction) for supershift. Avoid using excessive streptavidin which causes all probe to supershift and obscures specific complexes. Store biotinylated probes at -20 C; working solution stable for several freeze-thaw cycles. |
| Batch-to-Batch Consistency | HPLC purity >=95% for every synthesis; streptavidin capture efficiency >=90% for every lot; MALDI-TOF mass within 0.1% of calculated; biotin incorporation >=95%. |
For research use only, not for clinical use.
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