PureCube Epoxy Activated 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 Epoxy Activated Agarose carries an epoxy functionality. Proteins can bind covalently via free amines or thiol groups.
PureCube Epoxy Activated Agarose provides:
||Coupling proteins via their amine, thiol and hydroxyl groups
||Affinity to amine, thiol and hydroxyl groups
||up to 6 mL/min
|Epoxy Group Density
||higher than 20µmol/ml
||Delivered as a 50 % suspension
||Affinity agarose with activated Maleimide groups
- Coupling Buffer I (PBS) pH 7.2, 250 mL
- Coupling Buffer II pH 8.3, 250 mL
- Quenching Buffer, pH 7.4, 250 mL
- Alternative Quenching Buffer, pH 7.4, 250 mL
- Agarose Storage Buffer, pH 6.5, 250 mL
- Centrifuge tubes (15 mL)
- Centrifuge for 15 mL tubes
- End-over-end mixer or thermomixer
|A.||Epoxy coupling protocol|
- Transfer 1 mL PureCube Epoxy-Activated Agarose suspension (corresponding to 1 mL bed volume) into a 2 ml 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.
- Spin the tube at 500 x g to pellet the agarose. Remove the supernatant.
- Wash the Activated Agarose four times with 1 ml double distilled water, then wash once with 1 ml coupling buffer I or II.
- Resuspend the Agarose in 500 µl coupling buffer and incubate for 10 minutes at 20 or 37°C, depending on the stability of your protein.
- Add the protein to be coupled to the mix. The exact protein amount needs to be optimized, and 5 to 10 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.
- Incubate at 20-37°C for 24 hours on an end-over-end shaker or thermoshaker. Tip: Choose the highest temperature the protein is stable at. Coupling efficiency increases with temperature, incubation time, and protein concentration.
- 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.
- Wash the agarose five times with 1 ml double distilled water and once with 1 ml Storage buffer, yielding a 50% suspension. Store at 4°C.
- . Optional: Add 500 µl Quenching Buffer and incubate again for 8 h at 20-37°C. Wash five times with 1 mL double distilled water each and once with 1 ml Storage buffer. Note: The quenching step ensures that no free carboxy groups are left on the agarose matrix that might interfere with subsequent assays.
- . Resuspend the coupled Agarose in 1 mL Storage buffer, yielding a 50% suspension. Store at 4°C.