PureCube NHS Activated MagBeads carry a N-hydroxy-succinimide (NHS) functionality. Proteins can bind covalently via amine groups, forming a peptide bond. Our careful in-house production provides low lot-to-lot variation, making activated MagBeads also suitable for commercial production. PureCube NHS Activated MagBeads are delivered as a 25% suspension.
PureCube NHS Activated MagBeads provide:
||Covalent coupling of biomolecules via amine groups
||Affinity to free amine groups of e.g. proteins
||20 mg / mL
|Carboxylic group densits
||higher than 15 µmol/ml
||Delivered as a 25 % suspension
|A.||NHS coupling protocol|
- Transfer 1 mL PureCube NHS-Activated MagBeads into a 2mL microcentrifuge 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.
- Place the tube on a magnetic stand and allow the beads to separate. Remove the supernatant.
- Wash the beads once with 1 ml PBS. Allow the beads to separate and remove the supernatant. Important: Once PBS is added, work quickly to avoid hydrolysis of the NHS groups.
- Prepare a solution of 625 µL PBS containg the protein to be coupled to the MagBeads. The exact protein amount needs to be optimized, and 1 to 3 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 MagBeads 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.
- Place the tube on a magnetic stand and allow the beads to separate. 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 1.5 mL PBS buffer to the MagBeads, mix by vortexing, and separate on a magnetic stand. Remove the supernatant.
- Repeat step 7.
- Wash four times with 1.5 mL double distilled water each.
- Add 1.2 mL Quenching Buffer and incubate again for 1 h at room temperature or for 4 hours at 4°C.
- Wash four times with 1.5 mL PBS each, and twice with 1.5 mL double distilled water each.
- Resuspend the coupled MagBeads in 1 mL MagBead Storage buffer, yielding a 25% suspension. Store at 4°C.