Thermo Scientific Pierce NHS-Activated Agarose is a high-quality, amine-reactive, beaded-agarose resin for rapid and stable immobilization of proteins, peptides and other ligands via primary amines.
NHS-activated agarose is crosslinked, 6% beaded agarose resin that contains N-hydroxysuccinimide (NHS) functional groups. The activated resin reacts with primary amines to form stable amide linkages that covalently immobilize antibodies or other proteins for use in affinity purification procedures. The resin is available as a slurry in acetone and as a dry powder. Pierce NHS-Activated Agarose has a coupling capacity greater than 30mg/mL for the slurry and greater than 25mg/mL for the reconstituted dry resin.
- Easy to use – immobilize in a simple one-step reaction with minimal hands-on time
- Rapid and efficient – greater than 85% coupling for most proteins within 30 minutes
- Innovative format – the dry agarose concentrates the sample, making it ideal for immobilizing dilute proteins
- Safe – No hazardous chemicals needed (e.g., sodium cyanoborohydride, cyanogen bromide)
- Versatile – affinity resin is adaptable to column and batch affinity chromatography techniques and FPLC applications
- Compatible – use with any primary amine-containing compound
- Reusable – the leak-resistant chemistry means you can reuse the affinity resin
- High binding capacity – coupling capacity of >30mg/mL (slurry) or >25mg/mL (dry resin)
- Rapid Immobilization of goat anti-mouse and anti-rabbit antibodies in order to purify IgG produced in animals or hybridomas.
- Bulk immobilization of Protein A for purification of monoclonal antibodies
- Immobilization of ligands for purification of recombinant proteins
Pierce NHS-Activated Agarose resin uses reliable NHS-ester chemistry and does not require hazardous chemicals for immobilization. Other amine-reactive supports, such as periodate-oxidized resins, use toxic sodium cyanoborohydride to stabilize the reaction linkage to primary amines and take 4 to 6 hours to complete. Traditional methods such as cyanogen bromide-activated supports also couple amines; however, this chemistry results in nonspecific binding and constant slow leakage of the coupled ligand. Reactions with Pierce NHS-Activated Agarose are complete in less than one hour and yield much more stable linkages.
The NHS-Activated Agarose coupling reaction is performed in an amine-free buffer at pH 7-9. Protein coupling efficiency is typically greater than 80%, regardless of the ligand’s molecular weight or pI. Once a ligand is immobilized, the prepared resin can be used for multiple affinity purification procedures. The crosslinked beaded agarose has fast linear flow potential, making it useful for gravity-flow and low- to medium-pressure applications.
The Pierce NHS-Activated Agarose resin is available in an anhydrous acetone slurry and as dry powder. The unique dry form does not require storage in or removal and disposal of the acetone solvent. In addition, the dry resin is ideal for coupling reactions with dilute samples because it concentrates the sample as the resin swells, reducing the volume of the starting material and resulting in highly efficient ligand immobilization.
||Superior coupling capacity with Thermo Scientific Pierce NHS-Activated Agarose. Human IgG was applied at 9mg to 0.1mL resin. The coupling was carried out in PBS at pH 7.2 for 30 minutes. Protein recovered in the flow-through and washes represent unbound IgG after 30 minutes of coupling. The concentration of unbound IgG was determined using Pierce 660nm Protein assay (Part No. 22660). The amount of bound IgG was calculated by subtracting the unbound IgG value from the total amount added. Results, displayed as mg/mL, demonstrate that Pierce NHS Activated Agarose has a higher coupling capacity than the NHS-activated Sepharose™ 4 Fast Flow (GE Healthcare).
|Thermo Scientific Pierce NHS-Activated Agarose more effectively couples dilute samples than competing products. Rabbit IgG (0.5mg) was applied to 0.25mL of activated agarose in PBS at pH 7.2 for 30 minutes. The protein concentration of the flow-through and washes (uncoupled IgG) was determined using the Pierce 660nm Protein Assay. The bound IgG was calculated by subtracting the amount of uncoupled IgG from the amount added. The coupling efficiencies of both dry and slurry forms of Pierce NHS-Activated Agarose were higher than NHS-activated Sepharose™ 4 Fast Flow (GE Healthcare) and Affi-Gel™ 10 Gel (Bio-Rad).
||Specific protein purification using IgG coupled to NHS-activated agarose. Human IgG (25mg) was immobilized to 1mL of Pierce NHS-Activated Agarose. The affinity resin was used to purify secreted recombinant Protein G from E. coli. A final yield of 25mg of protein was obtained. Lane 1: bacterial pellet, Lane 2: MW marker, Lane 3: culture supernatant, Lane 4: flow-through, Lane 5-12: elutions, Lane 13: boiled resin following elutions.
||NHS agarose is effective for immunoprecipitation of MAP kinase from HeLa cell lysate. Anti-MAP kinase antibody (80µg, Millipore) was immobilized on 0.1mL of Thermo Scientific Pierce NHS-Activated Agarose. The affinity resin was incubated with 1.5mg of HeLa cell lysate overnight at 4°C. MAP kinase was eluted with 0.2mL of IgG Elution Buffer (Part No. 21004). Elutions were diluted 1:1 with reducing SDS sample buffer, heated for 5 minutes at 100°C, separated on a Pierce Precise Gel (Part No. 25224) and transferred to nitrocellulose membrane. The membrane was probed with anti-MAP kinase antibody (1µg/mL) and detected with goat-anti rabbit-HRP (0.1µg/mL, Part No. 31460) and Thermo Scientific SuperSignal West Dura Chemiluminescent Substrate (Part No. 34075).
Review of affinity purification
Review of covalent immobilization methods
Amine-reactive crosslinker chemistries
NHS-Activated Magnetic Beads
Empty Spin and Centrifuge Affinity Columns
All Affinity Supports for Ligand Immobilization