Coupling of proteins to PureCube Carboxy Agarose

 

This protocol delineates a coupling procedure for proteins to PureCube Carboxy Agarose 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 2 mL of a 50% Carboxy Agarose suspension, corresponding to 1 mL agarose bed volume. The process can be linearly scaled up and down from 100 µL to several 100 mL bed volume. Separation of Agarose and supernatants in low mL scale can be done by centrifugation, or, more conveniently, using PureCube 1-step batch Columns. See the separate protocol available for a combination of these products. For larger scales, use of special equipment like glass vaccum tank filters, is recommended.

Buffer compositions - Carboxy Activated coupling purification protocol

Phosphate buffer, pH 6.0, 250 mL

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount 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

 
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

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

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount 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.

 
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) - n.a. 51 mL
   
A.Carboxy Coupling protocol
 
 
  1. 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.
  2. . Spin the tube at 500 x g to pellet the agarose. Remove the supernatant. Resuspend the agarose in 3 mL Phosphate Buffer
  3. . Dissolve 250 mg NHS in 1 mL 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.
  4. Dissolve 250 mg EDC in 1 mL Phosphate Buffer, add it to the agarose suspension, and mix by vortexing.
  5. Incubate at room temperature for 1 h on an end-over-end shaker or in a thermoshaker.
  6. 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.
  7. Add the protein solution to the agarose and mix by vortexing.
  8. . 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.
  9. 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.
  10. Add 5 mL double distilled water to the agarose pellet, mix by vortexing, and spin at 500 x g. Remove the supernatant
  11. Repeat step 10 five times
  12. 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.
  13. Wash four times with 5 mL PBS each, and twice with 5 mL double distilled water each.
  14. Resuspend the coupled Agarose in 2 mL Agarose Storage buffer, yielding a 50% suspension. Store at 4°C.
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