Cleaning the Sephacryl Column

Cleaning the Sephacryl Column

Regular Cleaning

1. Wash the column with one-half column volume of 0.2 M NaOH at a flow rate of 15 cm/h (0.5 ml/min for 16/60 or 1.3 ml/min for 26/60) to remove most proteins nonspecifically bound to the medium.
2. After cleaning, immediately equilibrate the column with at least two column volumes of buffer. Further equilibration is necessary if your buffer contains detergent. Wait until the UV baseline has stabilized before applying next sample.

More Rigirous Cleaning

16/60 (120mL CV) Total distilled water needed: 12 CV = 1440 mL

Wash the column at a flow rate of 10 cm/h (0.3 ml/min for 16/60 or 0.8 ml/min for 26/60) at room temperature with the following solutions:

1. One-quarter of a column volume 0.5 M NaOH (removal of hydrophobic proteins or lipoproteins), followed by four column volumes of distilled water.
2. One-half column volume 30% isopropanol (removal of lipids and very hydrophobic proteins), followed by four column volumes distilled water.
3. To remove precipitated proteins, digest the protein with one column volume pepsin (1 mg/ml in 0.1 M acetic acid, 0.5 M NaCl) overnight at room temperature. (1.5CV on protocol so that there is still pepsin in the column after it flows thru)
4. Wash with one-half column volume 0.2 M NaOH at 15 cm/h (0.5 ml/min for 16/60 or 1.3 ml/min for 26/60) to remove trace amounts of enzyme remaining in the system, followed by four column volumes distilled water.

If a new purification is to be run, equilibrate the column after cleaning with at least five column volumes of buffer.

Note: HiPrep columns cannot be opened or refilled.

http://webhome.auburn.edu/~duinedu/manuals/Sephacryl_new.pdf