PureCube Carboxy MagBeads carry a free carboxyl group that can be activated by adding N-hydroxy-succinimide (NHS). Proteins can then 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 Carboxy MagBeads are delivered as a 25% suspension.
PureCube Carboxy MagBeads provide:
- Ready to use matrix
- Reproducible, long-lasting material for coupling via EDC/NHS
- Ideal for pull-down experiments
- Low unspecific binding
- Also available as Carboxy Agarose
||Coupling of biomolecules via the crosslinking agents EDC and NHS
||Covalent coupling via EDC and NHS to amine groups
||Delivered as a 25 % suspension
|Carboxy Groups density
|| higher than 15 µmol/ml
||Activated Carboxy (COOH) groups
- Sodium dihydrogen phosphate
- Sodium chloride
- Sodium acetate trihydrate
- Sodium hydroxide (NaOH)
- Acetic acid
- N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC)
- N-Hydroxysuccinimide (NHS)
- Magnetic holder for microcentrifuge tubes (for separation of magnetic beads)
- Microcentrifuge tubes (2 mL)
- pH meter
- End-over-end mixer or thermomixer
|A.||Carboxy coupling protocol:|
- Important: After NHS activation, it is highly recommended to directly react the resin wit target molecule, because hydrolysis will reduce activated groups directly after activation and removal of EDC/NHS.
- Transfer 1 mL PureCube Carboxy 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. Resuspend the magnetic beads with 750 µL Phosphate Buffer.
- Dissolve 63 mg NHS in 250 µL Phosphate Buffer, add it to the agarose suspension, and mix by vortexing. Note: NHS and EDC should always be prepared fresh. Equilibrate the two chemicals to room temperature before weighing, and store the powders under protective gas (nitrogen). Add the two chemicals immediately one after another to prevent hydrolysis of the NHS-activated matrix.
- Dissolve 63 mg EDC in 250 µL Phosphate Buffer, add it to the agarose suspension, and mix by vortexing.
- Incubate at room temperature for 1 h on an end-over-end shaker or in a thermoshaker.
- 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 double distilled water to the MagBeads, mix by vortexing, and separate on a magnetic stand. Remove the supernatant.
- Repeat step 10 five times.
- Add 1.2 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 MagBeads that might interfere with subsequent assays.
- . 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.