PureCube NHS Activated Agaroses is available as off-the-shelf item and as a bulk product. Our careful in-house production provides low lot-to-lot variation, making activated agaroses also suitable for commercial production. PureCube NHS Activated Agarose carries a N-hydroxy-succinimide (NHS) functionality. Proteins can bind covalently via amine groups, forming a peptide bond.
PureCube NHS Activated Agaroses provide:
||Coupling proteins to active NHS Groups
||Activated NHS groups that bind to free amine groups of proteins
|High binding capacity
||30 mg / ml
||Delivered as a 50 % suspension
||Affinity agarose with activated NHS groups
- PBS Buffer, pH 7.2, 250 mL
- Quenching Buffer, pH 7.4, 250 mL
- Agarose Storage Buffer, pH 6.5, 250 mL
- Centrifuge for 15 mL tubes
- Centrifuge tubes (15 mL)
- End-over-end mixer or thermomixer
Note: It is highly recommended to withdraw resin under protective gas and after equilibration to room temperature in order to avoid condensation of humidity and hydrolysis. Furthermore, it is recommended to directly react the resin with the target molecule after withdrawal, because hydrolysis will reduce activated groups directly after contact with humidity.
|A.||NHS Coupling protocol|
- Transfer 2 mL PureCube NHS-Activated Agarose suspension (corresponding to 1 mL bed volume) into a 15 mL centrifuge tube. Tip: The coupling reaction can be linearly scaled up and down, by increasing or decreasing the amounts of buffers and solutions described in this protocol.
- Spin the tube at 500 x g to pellet the agarose. Remove the supernatant.
- Wash the agarose with 2 mL PBS. Pellet the agarose and remove the supernatant. Important: Once PBS is added, work quickly to avoid hydrolysis of the NHS groups.
- . Prepare a solution of 2.5 mL PBS containg the protein to be coupled to the agarose. The exact protein amount needs to be optimized, and 5 to 15 mg protein is a good starting point. Tip: When coupling a particular protein for the first time, try 3-5 different protein concentrations to make sure you are offering enough protein in the reaction but not wasting any protein.
- Add the protein solution to the agarose and mix by vortexing.
- Depending on the temperature stability of the protein, incubate at room temperature or 4°C for 2 h on an end-overend shaker or thermoshaker.
- Spin the tube at 500 x g to pellet the agarose. Remove the supernatant and analyze the supernatant in a spectrophotometer. Record absorption at 280 nm to monitor coupling efficiency. Tip: Monitoring absorbance at 280 nm tells you about the coupling efficiency of the protein (compare A280 of the original protein solution to the supernatant in step 6 to determine % coupling). It also helps you identify the optimal amount of protein required for efficient coupling.
- Add 5 mL PBS buffer to the agarose pellet, mix by vortexing, and spin at 500 x g. Remove the supernatant.
- Repeat step 7.
- Wash four times with 5 mL double distilled water each.
- Add 5 mL Quenching Buffer and incubate again for 1 h at room temperature or for 4 hours at 4°C. Note: The quenching step ensures that no free NHS groups are left on the agarose matrix that might interfere with subsequent assays.
- Wash four times with 5 mL PBS each, and twice with 5 mL double distilled water each.
- Resuspend the coupled Agarose in 2 mL Agarose Storage buffer, yielding a 50% suspension. Store at 4°C.