Thermo Scientific Pierce NHS-Activated Magnetic Beads enable covalent, amine-based conjugation of proteins to magnetic beads in a simple mix-and-go format for use in custom affinity purification experiments.
The activated magnetic beads contain N-hydroxy-succinimide (NHS) functional groups that react with primary amines forming stable amide linkages. Once they are covalently attached, the immobilized proteins are highly resistant to leaching from the bead surface. When prepared beads are used in experiments, nonspecific binding is negligible because nonreacted NHS-ester groups are thoroughly blocked during the coupling procedure. Pierce NHS-Activated Magnetic beads can be coupled and processed either manually with a magnetic stand or with automated platforms such as the Thermo Scientific KingFisher Instruments.
- High capacity – at least four times higher binding capacity than NHS-activated magnetic beads from other suppliers
- Easy to use – immobilize in a simple one-step reaction with minimal hands-on time
- Safe – no hazardous chemicals needed (e.g., sodium cyanoborohydride and cyanogen bromide)
- Ligand-compatible – use to immobilize with nearly any primary amine-containing compound or affinity ligand
- Low non-specific binding – the bead surface is pre-blocked and any nonreacted NHS-ester groups are fully quenched
- Protocol-compatible – protein coupling to the beads and downstream applications can be performed manually or by automation (e.g., Thermo Scientific KingFisher Instruments)
- Immobilization of ligands for the purification of recombinant proteins
- Immobilization of antibodies for immunoprecipitations and co-immunoprecipitations free of antibody contamination in the eluates
- Immobilization of secondary antibody for the affinity purification of antibody subtypes from ascites, serum and cell culture supernatant
Pierce NHS-Activated Magnetic Beads offer a convenient way to conjugate any desired protein to a magnetic bead surface. The process does not require hazardous chemicals or lengthy reaction schemes. The beads are first incubated with protein for 1 to 2 hours in an amine free buffer at pH 7 to 9 to allow for covalent coupling through NHS-ester chemistry. The beads are subsequently washed and then any remaining active NHS-ester groups are quenched. The amine reactive chemistry and subsequent quench are completed in 3 to 4 hours. Coupled protein does not leach from the bead surface. In addition, the beads exhibit very low non-specific binding due to effective quenching and a proprietary blocking agent that coats the surface of the base particle. Following conjugation the prepared magnetic beads are typically used in affinity purification procedures.
Properties of Thermo Scientific Pierce NHS-Activated Magnetic Beads.
||N-hydroxysuccinimide (NHS) functional groups on a blocked magnetic bead surface
||Superparamagnetic (no magnetic memory)
||10mg/mL in DMAC
||≥26μg of rabbit IgG/mg of beads
|Reaction scheme for conjugation of protein on to Pierce NHS-Activated Magnetic Beads.
||Significantly better coupling capacity with Thermo Scientific Pierce NHS-Activated Magnetic Beads. Rabbit IgG (1mg/mL) was coupled in PBS for 2 hours at pH 7.2 to 3mg each of Pierce NHS-Activated Magnetic Beads (Our Beads) and NHS Mag Sepharose™ (GE Life Sciences) (Their Beads). Negative control beads (–) were prepared by quenching or blocking using respective manufacturer protocols. Bound protein was measured using the Pierce 660 nm Protein Assay (Part No. 22660) by subtracting the amount of protein in the flow through from the amount loaded. The Pierce Beads coupled more than four times as much protein as the equivalent amount of NHS Mag Sepharose™ Beads.
||Better immunoprecipitation results with Thermo Scientific Pierce NHS-Activated Magnetic Beads. Anti-FOXP2 antibody (5μg) was coupled to 25μL of Pierce NHS-Activated Magnetic Beads and an equivalent amount of NHS Mag Sepharose™ (GE Life Sciences). The two sets of prepared beads were then used to immunoprecipitate FOXP2 from 0.5mg aliquots of the same 293T (human epithelial kidney) cell lysate. The eluates were resolved by SDS-PAGE and analyzed by Western blot for FOXP2.
Tech Tip #43: Protein stability and storage
Tech Tip #55: Remove BSA and gelatin from antibody solutions using Melon Gel
Tech Tip #75: Measure protein bound to Pierce NHS-Activated Magnetic Beads
Pierce Direct Magnetic IP/Co-IP Kit
Pierce NHS-Activated Agarose
Other Pierce Magnetic Beads
Thermo Scientific KingFisher Magnetic Particle Processors