Carboxy Agarose is available as off-the-shelf item and as a bulk product. Our careful in-house production provides low lot-to-lot variation, making activated agaroses also suitable for commercial production. PureCube Carboxy Agarose carries a free carboxyl group that can be activated by adding N-hydroxy-succinimide (NHS). Proteins can then bind covalently via amine groups, forming a stable peptide bond.
PureCube Carboxy Agarose provides:
||Coupling proteins to active Carboxy groups via EDC and NHS
||crosslinking reagents EDC and NHS.
|Carboxy group density
||higher than 15 µmol/ml
||Delivered as a 50 % suspension
||Affinity agarose with activated Carboxy groups
- Phosphate buffer, pH 6.0, 250 mL
- PBS Buffer, pH 7.2, 250 mL
- Quenching Buffer, pH 7.4, 250 mL
- Agarose Storage Buffer, pH 6.5, 250 mL
- Centrifuge for 15 mL tubes
- Centrifuge tubes (15 mL)
- End-over-end mixer or thermomixer
Note: After NHS activation, it is highly recommended to directly react molecule, because hydrolysis will reduce activated groups directly after activation and removal of EDC/NHS.
|A.||Carboxy 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. Resuspend the agarose in 3 mL Phosphate Buffer
- . 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.
- Dissolve 250 mg EDC in 1 mL 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 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 double distilled water to the agarose pellet, mix by vortexing, and spin at 500 x g. Remove the supernatant
- Repeat step 10 five times
- 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.