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| Product Name | Anti-FLAG Magnetic Beads |
| Catalog No. | MAGBEA-0012 |
| Description | Anti-FLAG monoclonal antibody-conjugated magnetic beads for the rapid, high-specificity immunoprecipitation and purification of FLAG-tagged recombinant proteins. The anti-FLAG M2 monoclonal antibody is covalently coupled to the bead surface in an oriented manner that preserves the antigen-binding paratope, providing high-capacity capture of FLAG-tagged (DYKDDDDK) fusion proteins from cell lysates, culture supernatants, and in vitro translation mixtures. The mild competitive elution with 3× FLAG peptide preserves protein complex integrity and enzymatic activity. |
| Intended Use | Immunoprecipitation, pull-down, and small-scale purification of FLAG-tagged (DYKDDDDK) recombinant proteins from mammalian, bacterial, insect, and yeast expression systems for biochemical characterization, protein interaction studies, enzymatic assays, and structural biology sample preparation. |
| Principle / Technology | Monoclonal anti-FLAG M2 antibody (mouse IgG1) is covalently immobilized on the bead surface through oriented coupling via the Fc region or carbohydrate moiety, leaving the antigen-binding sites accessible. The M2 antibody recognizes the FLAG epitope (DYKDDDDK) with high specificity at the N-terminus, C-terminus, or internal positions of fusion proteins. Binding is calcium-independent for the M2 clone. After capturing FLAG-tagged proteins and washing away contaminants, the tagged protein is eluted under native conditions using 3× FLAG peptide (MDYKDHDGDYKDHDIDYKDDDDK) competition, preserving protein complexes, activity, and conformation. |
| Detection Method | DLS for bead size; BCA for antibody density; FLAG-BAP (bacterial alkaline phosphatase) binding assay for functional capacity; SDS-PAGE and Western blot for eluted protein purity; mass spectrometry for protein identity confirmation; enzymatic activity assay of eluted proteins; anti-FLAG Western blot for tag integrity |
| Sample Type | FLAG-tagged (DYKDDDDK epitope) recombinant proteins expressed in E. coli, S. cerevisiae, P. pastoris, insect cells (Sf9, Sf21, High Five), and mammalian cells (HEK293, CHO, HeLa); compatible with N-terminal FLAG, C-terminal FLAG, and 3× FLAG fusion constructs; cell lysates, conditioned medium, nuclear extracts, and in vitro translation products |
| Performance Range / Specifications | Bead diameter: 1-2 µm; anti-FLAG antibody density: 2-5 µg antibody per mg beads (2-5 mg/mL settled beads); FLAG-tagged protein binding capacity: 0.5-2 mg FLAG-BAP per mL settled beads (protein-dependent); binding affinity (Kd): ~10^-9 M for FLAG peptide; elution efficiency: >90% with 100-200 µg/mL 3× FLAG peptide; non-specific binding: <2% of total bound protein; magnetic separation: <10 seconds |
| Sensitivity / LOD | Detectable FLAG protein binding from lysates with <10 ng/mL tagged protein; effective immunoprecipitation from lysate volumes as small as 50 µL; single-step purification from crude lysate yielding >80% purity by SDS-PAGE; detection of low-abundance FLAG-tagged transcription factors (<100 copies per cell) |
| Specificity | M2 antibody specifically recognizes DYKDDDDK sequence; minimal cross-reactivity with endogenous mammalian proteins (confirmed by proteomic analysis of mock IP eluates); no binding to HA, myc, His6, GST, V5, or other common epitope tags; position-independent recognition (N-terminal, C-terminal, internal FLAG); calcium-independent binding (M2 clone) |
| Reaction Conditions / Protocol | Equilibrate beads with TBS or PBS containing 150 mM NaCl; add cell lysate (pre-cleared by centrifugation at 14,000g for 10 minutes); incubate at 4°C for 1-2 hours or room temperature for 30-60 minutes with end-over-end rotation; magnetically separate; wash beads 3-5 times with TBS containing 0.1-0.5% NP-40 or Triton X-100; wash once with TBS without detergent; elute with 100-200 µg/mL 3× FLAG peptide in TBS for 30 minutes at 4°C or 15 minutes at room temperature; magnetically separate; collect eluate containing purified FLAG protein; total protocol: 1.5-3 hours |
| Components / Formulation | Anti-FLAG M2 monoclonal antibody (mouse IgG1) covalently conjugated to superparamagnetic beads, supplied as 25-50% slurry in PBS pH 7.4 with 0.02% sodium azide; 3× FLAG peptide not included but available separately; blocking and wash buffers not included |
| Storage Conditions | 2-8°C in PBS with sodium azide; do not freeze (freezing denatures antibody); protect from light; store upright; gentle vortex before use; avoid prolonged storage in detergent-containing buffers |
| Shelf Life | 12 months at 2-8°C; anti-FLAG antibody activity >85% at 12 months; antibody leaching <0.1% of total antibody; no significant reduction in binding capacity through shelf life; opened product: 6 months with proper handling |
| Package Specifications | 0.2 mL, 0.5 mL, 1 mL, 5 mL settled bead volume (25-50% slurry); sufficient for 5-100 immunoprecipitation reactions depending on scale; trial packs for method development; custom packaging available |
| Product Form | Dark brown bead suspension; homogeneous after gentle vortexing; supernatant clear after magnetic separation; no visible antibody precipitate or bead aggregation; uniform particle appearance by microscopy |
| Quality Control | Per lot: anti-FLAG antibody density, FLAG-BAP binding capacity and linearity, non-specific binding, antibody leaching, endotoxin, sterility, elution efficiency with 3× FLAG peptide, eluted protein purity; functional validation by IP-Western of FLAG-GFP from HEK293 lysate |
| Key Features | Monoclonal M2 antibody for consistent specificity; calcium-independent binding; mild competitive elution preserves protein activity and complexes; high capacity for small-scale purification; magnetic format for easy handling and rapid processing; validated for proteomics and protein interaction studies; oriented antibody coupling for maximum antigen accessibility |
| Purity | Anti-FLAG M2 antibody >95% by SDS-PAGE; no protease contamination; endotoxin <0.1 EU/mg beads; sodium azide concentration verified; no microbial contamination |
| Concentration | 25-50% slurry (v/v); antibody density 2-5 µg/mg beads; exact binding capacity in lot-specific certificate; bead count approximately 10^9-10^10 particles per mL settled beads |
| Activity / Unit Definition | FLAG-BAP binding capacity: 0.5-2 mg/mL settled beads; binding constant ~10^-9 M; elution efficiency >90% with 3× FLAG peptide; binding capacity maintained >85% through shelf life; regenerable for multiple uses (2-3 times) |
| Molecular Weight | Anti-FLAG M2 IgG: ~150 kDa (intact IgG); FLAG peptide (DYKDDDDK): ~1,012 Da; 3× FLAG peptide: ~2,861 Da; bead molecular weight not defined |
| Source / Origin | Anti-FLAG M2 monoclonal antibody from mouse hybridoma (proprietary cell line); magnetic beads are synthetic polymer-magnetite composite; M2 antibody is purified by Protein A affinity chromatography; all raw materials traceable; animal-derived component (antibody) with traceability |
| pH Range / Optimal pH | Binding: pH 6.5-8.5 (optimal pH 7.0-7.5); elution: pH 7.0-7.5 (competitive elution, no pH change); bead stability: pH 3-11; antibody stability: pH 5-9 for extended exposure; avoid pH <3 (antibody denaturation); 3× FLAG peptide active across pH 6-9 |
| Shipping Conditions | Cold pack shipping at 2-8°C; insulated packaging; do not freeze; overnight or 2-day delivery recommended; non-hazardous; include temperature indicator for shipment monitoring |
| Expiration Date / Stability | 12 months at 2-8°C; antibody activity >85% at expiry; real-time stability at 0, 3, 6, 9, 12 months; no increase in antibody leaching; sodium azide preservative effective through expiry; after opening: 6 months with sterile technique |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic applications; mouse monoclonal antibody; appropriate for biochemical and cell biology research; ISO 9001 manufacturing; certificate of analysis per lot |
| Compatibility | Compatible with standard lysis buffers: RIPA, NP-40, Triton X-100 (up to 1%), CHAPS, octyl glucoside; compatible with 150-500 mM NaCl; avoid SDS >0.1% (antibody denaturation); reduce DTT to <1 mM during binding; TBS preferred over PBS for elution; 3× FLAG peptide compatible with most enzymatic assays |
| Recommended Buffer System | Storage: PBS pH 7.4, 0.02% NaN3; binding/wash: TBS (50 mM Tris-HCl pH 7.4, 150 mM NaCl) with optional 0.1% NP-40; elution: TBS + 100-200 µg/mL 3× FLAG peptide; peptide can be removed by dialysis or desalting column if needed |
| Application Notes / Precautions | Pre-clear lysate before bead incubation to reduce non-specific binding; optimize bead-to-lysate ratio (0.5-2 µL settled beads per µg target protein typically); for low-abundance targets, increase lysate volume and incubation time; competitive elution may be incomplete for very high-affinity binders—extend elution to 1 hour; 3× FLAG peptide can be reused 2-3 times for cost saving; DYKDDDDK recognition requires Asp at position 1; for N-terminal FLAG, ensure correct processing after Met removal |
| Batch-to-Batch Consistency | Antibody density CV <15% inter-lot; FLAG-BAP binding capacity CV <12%; elution efficiency >90% across all lots; antibody leaching <0.1% per lot; endotoxin <0.1 EU/mg all lots; each lot functionally tested with FLAG-GFP IP-Western |
For research use only, not for clinical use.
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