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| Product Name | Biotin Protein-Protein Interaction Pull-Down Kit, Streptavidin Magnetic Beads, NHS-Biotin Labeling |
| Catalog No. | PTR-HMM-0093 |
| Description | Complete protein-protein interaction pull-down kit combining NHS-biotin labeling of the bait protein with streptavidin magnetic bead capture for identification of protein binding partners from cell and tissue lysates. The kit includes all reagents for biotinylation of the bait protein via primary amine-reactive NHS-PEG4-biotin (long-chain, water-soluble), removal of excess biotin by desalting spin columns, incubation of biotinylated bait with prey lysate, capture of bait-prey complexes on streptavidin magnetic beads, stringent washing to remove non-specific binders, and elution for downstream analysis by SDS-PAGE, western blot, or mass spectrometry. The magnetic bead format eliminates centrifugation steps and provides rapid, clean separations with low background binding. The PEG4 spacer reduces steric hindrance and preserves bait protein function compared to short-linker biotin reagents. |
| Intended Use | Identification and validation of protein-protein interactions: bait protein (biotinylated) used to pull down interacting partners from cell or tissue lysates; identification of binding partners by mass spectrometry (LC-MS/MS); confirmation of suspected interactions by western blot; mapping interaction domains using bait protein fragments or mutants; competitive disruption assays with small molecule inhibitors or peptides; and interaction studies in different cellular conditions (treated vs untreated, wild-type vs mutant). |
| Principle / Technology | Step 1 — Bait biotinylation: purified bait protein (typically recombinant) is labeled with NHS-PEG4-biotin, which reacts with primary amines (N-terminus and lysine epsilon-amino groups) forming stable amide bonds. Excess biotin is removed to prevent competition. Step 2 — Pull-down: biotinylated bait is incubated with cell/tissue lysate containing potential prey proteins. Step 3 — Capture: streptavidin magnetic beads are added; the high-affinity biotin-streptavidin interaction (Kd ~10^-15 M) captures bait protein along with any interacting proteins. Step 4 — Wash: beads are washed stringently to remove non-specific binders. Step 5 — Elution: proteins are eluted in Laemmli buffer by boiling, releasing bait-prey complexes for SDS-PAGE, western blot, or in-gel digestion for mass spectrometry. |
| Detection Method | 1) Buffer exchange bait protein into amine-free buffer (PBS pH 7.4); 2) Add 20-fold molar excess NHS-PEG4-biotin (prepared fresh in DMSO), incubate 30-60 min on ice or 2 h at 4 C; 3) Remove excess biotin using desalting spin column; 4) Confirm biotinylation by HABA assay or western blot with streptavidin-HRP; 5) Incubate biotinylated bait (1-10 ug) with prey lysate (0.5-2 mg total protein) for 1-4 h at 4 C with rotation; 6) Add 50 uL streptavidin magnetic beads slurry, incubate 30-60 min at RT with rotation; 7) Wash beads 3-5x with Wash Buffer; 8) Elute in 30-50 uL 1x Laemmli buffer at 95 C for 5-10 min; 9) Analyze by SDS-PAGE/western blot or MS. |
| Sample Type | Bait protein: purified recombinant protein (0.1-1 mg/mL, >80% purity recommended, amine-free buffer). Prey lysate: cell or tissue lysate (0.5-2 mg total protein, RIPA or NP-40 based lysis buffer with protease inhibitors). Include a no-bait control (biotin only) and a no-biotin control (unlabeled bait) for specificity assessment. |
| Performance Range / Specifications | Biotinylation efficiency: 1-3 biotin molecules per protein molecule (typical for NHS-ester labeling); bait capture efficiency on beads: >90% of biotinylated bait captured; prey protein detection: depends on interaction affinity (Kd 10^-6 to 10^-12 M typically detectable); bead binding capacity: >2 nmol biotin per mL settled beads. |
| Sensitivity / LOD | Detection of protein-protein interactions with affinity as weak as Kd ~10^-5 M; identification of low-abundance interacting proteins (0.1-1% of bait) by mass spectrometry when combined with appropriate controls and sufficient starting material (1-5 mg lysate); detection of 10-50 fmol biotinylated protein on western blot with streptavidin-HRP. |
| Specificity | Streptavidin magnetic beads specifically capture biotinylated proteins via the extraordinarily strong biotin-streptavidin interaction; non-specific binding to beads: typically <0.1% of input lysate protein; specificity confirmed by comparing with no-biotin-bait control and no-bait (biotin-only) control; competition with excess free biotin (2 mM) can serve as additional specificity control by eluting streptavidin-captured proteins. |
| Reaction Conditions / Protocol | Biotinylation: 30-60 min on ice; prey incubation: 1-4 h at 4 C; bead capture: 30-60 min at RT; washes: 3-5 rounds, 5 min each; elution: 5-10 min at 95 C; total protocol: 4-8 hours (excluding overnight incubation if desired). |
| Components / Formulation | NHS-PEG4-Biotin (lyophilized, 1 mg, 2 vials), Anhydrous DMSO (0.5 mL), Desalting Spin Columns (2 mL, 10 columns), Streptavidin Magnetic Beads (1 mL settled beads, 10 mg/mL slurry), Lysis Buffer (50 mL), Wash Buffer (100 mL), Biotin Quantitation Kit (HABA/avidin, sufficient for 50 assays), Elution Buffer (5x Laemmli, 5 mL), Magnetic Separation Rack (1 unit, for 1.5-2 mL tubes), Protocol. |
| Storage Conditions | NHS-PEG4-Biotin at -20 C desiccated; DMSO at RT; Desalting Spin Columns at 2-8 C; Streptavidin Beads at 2-8 C (do not freeze); Lysis Buffer, Wash Buffer, HABA reagents at 2-8 C; Elution Buffer at -20 C; Magnetic Rack at RT. |
| Shelf Life | 12 months from date of manufacture; NHS-biotin: 24 months at -20 C desiccated. |
| Package Specifications | 1 kit (sufficient for 10 pull-down experiments plus controls). |
| Product Form | Lyophilized NHS-biotin; liquid buffers and bead suspension; magnetic separation rack. |
| Quality Control | Each lot: NHS-biotin purity >95% by HPLC, reactive NHS content >90% by hydrolysis assay; streptavidin bead binding capacity >2 nmol biotin/mL beads; bead non-specific protein binding <0.1% of input BSA; desalting column >95% removal of small molecules (<1,000 Da); magnetic rack pull force tested. |
| Key Features | Complete kit (biotinylation + pull-down + detection); NHS-PEG4-biotin with long spacer; magnetic bead format (no centrifugation); HABA biotin quantitation included; desalting columns for excess biotin removal; magnetic separation rack included; optimized for MS and western blot. |
| Purity | NHS-PEG4-Biotin: >95% by HPLC; Streptavidin: >98% by SDS-PAGE; all buffers: analytical grade, 0.2 um filtered. |
| Concentration | NHS-PEG4-Biotin: reconstitute at 10 mM in DMSO; Beads: 10 mg/mL slurry (approximately 50% settled beads); magnetic beads: 1 um diameter, iron oxide core with polymer coating. |
| Activity / Unit Definition | NHS-PEG4-Biotin: reactive NHS ester content >90% of theoretical; Streptavidin beads: biotin binding >2 nmol/mL settled beads; HABA/avidin reagent: absorbance change at 500 nm linear with biotin concentration. |
| Molecular Weight | NHS-PEG4-Biotin: 588.67 g/mol; Streptavidin monomer: approximately 13.3 kDa (tetramer ~53 kDa); Biotin: 244.31 g/mol. |
| Source / Origin | NHS-PEG4-Biotin: synthetic; Streptavidin: recombinant from S. avidinii expressed in E. coli; Magnetic beads: synthetic iron oxide core with hydrophilic polymer shell; all reagents: animal-free except where noted. |
| pH Range / Optimal pH | Biotinylation pH 7.2-8.0 (optimal pH 7.4 in PBS); prey incubation pH 7.0-8.0; bead capture pH 7.0-8.0; elution pH 6.8 (Laemmli buffer). |
| Shipping Conditions | Cold pack (2-8 C) for beads, desalting columns, and buffers; NHS-biotin on dry ice; magnetic rack ambient. |
| Expiration Date / Stability | 12 months overall; NHS-biotin: 24 months at -20 C; reconstituted NHS-biotin in DMSO: use immediately (hydrolyzes within hours in DMSO solution); beads: 12 months at 2-8 C (do not freeze — freezing damages magnetic bead coating). |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic use. |
| Compatibility | Compatible with purified recombinant proteins from E. coli, yeast, insect, and mammalian expression systems. NHS-biotin is amine-reactive: bait protein must be in amine-free buffer (PBS, HEPES, bicarbonate — NOT Tris, glycine, or ammonium-containing buffers). Biotinylation is compatible with most buffer additives: NaCl up to 500 mM, glycerol up to 10%, DTT up to 1 mM, EDTA up to 5 mM. Detergent compatibility: Triton X-100, NP-40, CHAPS, Tween-20 at standard concentrations (0.1-1%) do not interfere. SDS >0.05% denatures bait protein and is incompatible with biotinylation. Compatible with protease inhibitor cocktails (EDTA-free preferred — EDTA may interfere with some protease inhibitors). Prey lysates in RIPA or NP-40 buffer are compatible after dilution to <0.1% SDS. |
| Recommended Buffer System | Biotinylation Buffer: PBS pH 7.4 (amine-free); Lysis Buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, protease inhibitors (add fresh); Wash Buffer: PBS pH 7.4, 0.1% Tween-20; Elution: 1x Laemmli buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 5% beta-mercaptoethanol, 0.01% bromophenol blue). |
| Application Notes / Precautions | NHS-PEG4-biotin is moisture-sensitive: warm vial to RT before opening to prevent condensation; prepare fresh solution in anhydrous DMSO immediately before use. NHS ester hydrolysis half-life in aqueous solution at pH 7.4: approximately 30 min; work quickly and keep on ice. A 20-fold molar excess of biotin reagent over bait protein is recommended; calculate based on bait protein molecular weight and concentration. For bait proteins with few lysine residues or N-terminal modification, increase biotin excess or use a longer incubation. Confirm biotinylation by HABA assay (displacement of HABA from avidin by biotin causes A500 decrease) before proceeding to pull-down. Always include three critical controls: beads + lysate (no bait), beads + bait + lysate + 2 mM free biotin (competition), and beads + unlabeled bait + lysate. For MS, use at least 2-3 mg lysate protein and 5-10 ug bait; combine triplicate pull-downs for higher coverage. |
| Batch-to-Batch Consistency | NHS-biotin purity >95%, reactive NHS >90%; bead binding capacity >2 nmol biotin/mL; non-specific binding <0.1%; desalting column protein recovery >95%, small molecule removal >95%. |
For research use only, not for clinical use.
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