Coupling of proteins to PureCube Carboxy MagBeads

This protocol delineates a coupling procedure for proteins to PureCube Carboxy MagBeads using the crosslinking reagents EDC and NHS. Proteins are coupled covalently and can be used for different applications, e.g. purification of interaction partners. Amounts given in this protocol are for 1 mL Carboxy MagBead suspension, which contains 250 µL magnetic beads. This reaction can be linearly scaled up or down using appropriate magnetic holders. Magnetic holders for a wide range of volumes are available e.g. from Sepmag (www.sepmag.eu).

Buffer compositions - Carboxy Activated coupling purification protocol

Phosphate buffer, pH 6.0, 250 mL

Component	Final concentration	Molecular weight (g/mol)	Stock concentration	Amount needed for buffer

NaH₂PO₄ dihydrate	25 mM	156.01	n.a.	975 mg
Instructions: Dissolve sodium phosphate in 200 mL water, adjust the pH to 6.0 with NaOH. Add water to a total volume of 250 mL.

PBS Buffer, pH 7.2, 250 mL

Component	Final concentration	Molecular weight (g/mol)	Stock concentration	Amount needed for buffer

NaH₂PO₄ dihydrate	150 mM	156.01	n.a.	5.85 g
NaCl	100 mM	58.44	n.a.	1.463 g
Instructions: Dissolve components in 200 mL water, adjust the pH to 7.2 with NaOH. Add water to a total volume of 250 mL.

Quenching Buffer, pH 7.4, 250 mL

Instructions: Dissolve component in 200 mL water, adjust the pH to 7.4 with HCl. Add water to a total volume of 250 mL.

Component	Final concentration	Molecular weight (g/mol)	Stock concentration	Amount needed for buffer

Ethanolamine	1 M	61.08	n.a.	15.27 g

Agarose Storage Buffer, pH 6.5, 250 mL

Instructions: Dissolve sodium acetate in 150 mL water, adjust the pH to 6.5 with acetic acid. Add 48 mL water and 51 mL ethanol to yield a total volume of 250 mL.

Component	Final concentration	Molecular weight (g/mol)	Stock concentration	Amount needed for buffer

Sodium acetate trihydrate	20 mM	136.08	n.a.	674 mg
Ethanol	20 % (v/v)	-	n.a.	51 mL


A.	Carboxy coupling protocol:
	Important: After Carboxy 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.

Carboxy coupling protocol:

Important: After Carboxy 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.