Coupling of proteins to PureCube NHS-Activated MagBeads

 

This protocol delineates a coupling procedure for proteins to PureCube NHS-Activated MagBeads. 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 NHS-Activated 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 - NHS Activated coupling coupling protocol

PBS Buffer, pH 7.2, 250 mL

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount 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

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount needed for buffer
Ethanolamine 1 M 61.08 n.a. 15.27 g
NaCl 100 mM 58.44 n.a. 1.463 g
Instructions: Dissolve component in 200 mL water, adjust the pH to 7.4 with HCl. Add water to a total volume of 250 mL.

Agarose Storage Buffer, pH 6.5, 250 mL

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount needed for buffer
Sodium acetate trihydrate 20 mM 136.08 n.a. 674 mg
Ethanol 20 % (v/v) - 100 % (v/v) 51 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.
   
A.NHS coupling protocol
 
 
  1. 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.
  2. Place the tube on a magnetic stand and allow the beads to separate. Remove the supernatant.
  3. 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.
  4. 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.
  5. Add the protein solution to the MagBeads and mix by vortexing.
  6. 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.
  7. 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.
  8. Add 1.5 mL PBS buffer to the MagBeads, mix by vortexing, and separate on a magnetic stand. Remove the supernatant.
  9. Repeat step 7.
  10. Wash four times with 1.5 mL double distilled water each.
  11. Add 1.2 mL Quenching Buffer and incubate again for 1 h at room temperature or for 4 hours at 4°C.
  12. Wash four times with 1.5 mL PBS each, and twice with 1.5 mL double distilled water each.
  13. Resuspend the coupled MagBeads in 1 mL MagBead Storage buffer, yielding a 25% suspension. Store at 4°C.
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