Protocols for SDR HyperD® Solvent-Detergent Removal Chromatography Resin

• Empty plastic small volume column with porous PE frits (Pierce, disposable polypropylene column, Part No. 29922).
• Degassed 50% (v/v) slurry of the SDR HyperD resin (see note below).
• Degassed buffer such as Phosphate Buffered Saline.

Note: To prepare a 50% (v/v) slurry, it may be necessary to use a clean spatula to remove some of the packed media and transfer to a graduated glass cylinder containing degassed buffer. To avoid contaminating the bulk media, DO NOT add any media back to the storage bottle. Thoroughly mix and allow settling. Note the volume of the settled resin. Decant the supernatant and add a volume of buffer equal to the volume of settled resin to make a 50% (v/v) slurry. 

Packing the Column

1. Equilibrate column, degassed 50% gel slurry, and degassed buffer solution to room temperature.
2. Secure a bottom cap on the column tip and clamp the column upright in a laboratory stand.
3. Add a sufficient volume of degassed buffer to the column to fill it up to the reservoir (wide-mouth) portion, and gently tap the end and side of the column to dislodge any air bubbles.
4. Float a porous disc on top of the liquid within the column.
5. Using the reverse end of a Pasteur pipette or reverse end of a serum separator (Pierce, Product No. 69710), push the disc evenly to the bottom of the column.
6. Decant most of the liquid from the empty column, being sure to avoid getting air bubbles in the tip region of the column below the inserted disc. Place the column back in its stand with bottom cap still in place.
7. Add sufficient volume of degassed gel slurry to obtain the desired settled gel volume of 1-2 mL.
8. Allow gel to settle in the column for at least 5 minutes.
9. Position a second porous disc on top of the settled gel bed by floating it on the liquid within the column and pushing it down to just above the settled gel. Leave 1-2 mm of space between the top of the gel bed and the top disc; do not compress the gel bed.
10. Wash the inside top part of the column with buffer to remove residual gel that may have remained along the sides during packing.
11. The packed column is now ready for storage or use.

Notes:

• Store the packed column upright at 4 °C with the gel bed submerged under 1-2 mL of buffer and a top column cap securely in place. Sodium azide added to the storage buffer to a concentration of 0.02% will discourage microbial growth.
• Always remove the top cap before the bottom cap to avoid drawing air bubbles down into the gel bed.
• Prevent air bubbles from forming in the gel bed by using only degassed buffer and sample solutions. Degassing involves subjecting a solution to vacuum to “boil” off excess dissolved air. Excessive degassing (vacuum too strong or too long) can lead to evaporation of water or solvent from the solution. Note: check the final volume after degassing and if necessary add more solvent or water to return to original volume.

Detergent Removal in a Drip Column Format

1. Prepare a 1-2 mL column as described above (scale up or down as needed).
2. Wash the SDR HyperD media with 5 column volumes (CV) of water or buffer to remove the 20% ethanol storage buffer.
3. Allow the liquid to drain from the column and load the sample up to a 2 mL volume onto the column.
4. Collect the column effluent in 1 mL fractions and measure absorbance @ 280 nm to locate the protein peak. Note: protein rapidly elutes from the column and should be found in the first three fractions. Some slight dilution of the sample will occur during elution. If necessary, the sample can be re-concentrated with a centrifugal ultrafiltration device, such as a Pall Microsep™ device with a 10K cut-off ultrafiltration membrane.
5. After protein has eluted, discard the resin from the column.

Note: If re-use is desired, the retained detergents can be eluted with 10 CV of PBS/ethanol (50/50 mix), followed by 10 CV of 95% ethanol, followed by requilibration with water or buffer. If sanitization or depyrogenation is required, two methods are recommended by BioSepra:

1. Alcohol/acid treatment. Wash with at least 3 CV of degassed 20% (v/v) ethanol containing 1M Acetic acid. Allow the resin to soak in the presence of the solution (recycle effluent manually or using a pump) for an exposure time of 1 h. Note: monitor volume during degassing. Follow by requilibration with water or buffer
2. Diethyl pyrocarbonate/ethanol treatment. Wash with 2 CV of PBS in 50% (v/v) ethanol and 5% (v/v) diethyl pyrocarbonate. Allow the resin to soak in the presence of the solution (recycle effluent manually or using a pump) for an exposure time of 1 h. Wash the column with 3 CV of 3M pyrogen free sterilized NaCl/1M acetic acid to remove all pyrogens. Follow by requilibration with sterile pyrogen free water or buffer.

• Nanosep GHP devices
• Collection tubes 
• 50% (v/v) slurry of the SDR HyperD resin (see note below)
• Appropriate buffer, such as Phosphate buffered saline.
• Vacuum flask and rubber stopper with a hole bored to form a "loose" fit with the Nanosep spin device. 
• A low vacuum source at about 200 mmHg.
• Vacuum is not necessary if a centrifuge will be used for preparing the column for sample addition and elution.

Note: To prepare a 50% (v/v) slurry, it will be necessary to use a clean spatula to remove some of the packed media and transfer to a graduated glass cylinder containing buffer. To avoid contaminating the bulk media, DO NOT add any media back to the storage bottle. Thoroughly mix and allow settling. Note the volume of the settled resin. Decant the supernatant and add a volume of buffer equal to the volume of settled resin to make a 50% (v/v) slurry.

Packing the Spin Column

1. Prepare 50% (v/v) SDR HyperD resin slurry as described above.
2. Wash this slurry with 5 column volumes (CV) of water or buffer to remove the 20% ethanol preservative.
3. Remove the spin device from the Nanosep collection tube and place in the rubber stopper on the vacuum flask. Apply a low vacuum. Alternatively, place the Nanosep device and collection tube into an appropriately-sized centrifuge and spin at 800-900 x g for 2 min.
4. Mix the resin slurry and quickly pipet 0.4 mL of the slurry to the Nanosep device. Rapidly follow with a second and third volume of slurry. Note: in between each addition of the slurry allow the resin bed to settle. This can be done in a microfuge with repeated centrifugation steps.
5. After final addition, allow the vacuum to remove the liquid from the resin bed which should fill the Nanosep device.
6. Replace the spin device in the collection tube until sample addition. Note: because we have removed all the preservative from the resin in these devices, they should be used immediately or stored at 4 °C for no more than one week.

Detergent Removal in a Spin Column Format

1. Prepare the spin column as described above.
2. Centrifuge the spin column in a swinging bucket rotor at 800-1000 x g for 2 min to remove excess fluid from the packed bed.
3. Remove the filtrate from the collection tube.
4. Very carefully pipet the sample (0.05 - 0.2 mL; maximum volume depends on detergent capacity vs. resin volume and device size) onto the top of the dry packed bed in the Nanosep device. Replace in the collection tube.
5. Centrifuge the spin column in a swinging bucket rotor at 1000 x g for 4 min to pass the sample through the SDR resin bed.
6. Filtrate in the collection tube will be detergent depleted.