Coupling of proteins to PureCube NHS-Activated Agarose

This protocol delineates a coupling procedure for proteins to PureCube NHS-Activated Agarose. 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% NHS-Activated Agarose suspension, corresponding to 1mL 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 - NHS Activated coupling coupling protocol

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

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

Ethanolamine	1 M	61.08	n.a.	15.27 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

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)	-	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
	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. Spin the tube at 500 x g to pellet the agarose. Remove the supernatant. Wash the agarose with 2 mL PBS. Pellet the agarose and remove the supernatant. Important: Once PBS is added, work quickly to avoid hydrolysis of the NHS groups. 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. Add the protein solution to the agarose 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 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. Add 5 mL PBS buffer to the agarose pellet, mix by vortexing, and spin at 500 x g. Remove the supernatant. Wash four times with 5 mL double distilled water each. 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. Wash four times with 5 mL PBS each, and twice with 5 mL double distilled water each. Resuspend the coupled Agarose in 2 mL Agarose Storage buffer, yielding a 50% suspension. Store at 4°C.

NHS Coupling protocol

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.
Spin the tube at 500 x g to pellet the agarose. Remove the supernatant.
Wash the agarose with 2 mL PBS. Pellet the agarose and remove the supernatant.
Important: Once PBS is added, work quickly to avoid hydrolysis of the NHS groups.
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.
Add the protein solution to the agarose 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
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.
Add 5 mL PBS buffer to the agarose pellet, mix by vortexing, and spin at 500 x g. Remove the supernatant.
Wash four times with 5 mL double distilled water each.
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.
Wash four times with 5 mL PBS each, and twice with 5 mL double distilled water each.
Resuspend the coupled Agarose in 2 mL Agarose Storage buffer, yielding a 50% suspension. Store at 4°C.