Bitan:SpinColumns

1. Choose and order appropriate Zeba desalting spin columns. Note, for sample volumes between 130-200µL, use 2-mL columns.

2. Similarly, select the appropriate sized collection tube. For 10mL spin columns, use 50mL FalconTM conical tubes. For 5mL and 2mL spin columns, use 15mL FalconTM conical tubes. For 0.5mL spin columns, use 2mL microcentrifuge tubes

3. Snap off and discard the column’s bottom closure. Loosen the cap and place in appropriate size collection tube.

4. Centrifuge column(s) for 2 minutes at 1000xg at 4˙C, in order to remove the storage solution. For 0.5mL columns, centrifuge for 1 minute at 1500xg at 4˙C. After centrifugation, resin should appear white and compacted.

5. Place a mark on the side of the column where the compacted resin is slanted upward. Place column in centrifuge with the mark facing outward in all subsequent centrifugation step.

6. Add appropriate volume either 1xPBS or 1xTBS buffer to the column. Determine appropriate buffer based on original sample buffer matrix.

6a) For 0.5mL columns, add 300µL of buffer

6b) For 2mL columns, add 1mL of buffer

6c) For 5mL columns, add 2.5mL of buffer

6d) For 10mL columns, add 5mL of buffer

7. As before, centrifuge the column 2 minutes at 1000xg at 4˙C. Discard flow-through from the collection tubes. For 0.5mL columns, centrifuge for 1 minute at 1500xg at 4˙C.

8. Repeat steps 6 through 7 two additional times. Blot the bottom of the column with a KimWipe to remove excess liquid.

9. Obtain a new collection tube and add appropriate volume of 10 x Protease and Phosphatase inhibitor in 1X buffer solution to tube. Inhibitors are used to prevent proteins from precipitating out of solution. In order to determine appropriate volume, use the following equation: Original Sample Vol. (µL) ÷ 9 = Protease Inhibitor Vol. (µL)

10. Transfer columns to a new, clean collection tube for sample recovery. Use a clean pipette tip and apply the entire sample on top of the resin bed.

11. (Optional) To ensure maximal protein recovery from low-volume samples, apply a stacker of the same buffer to the resin bed after the sample has fully absorbed. For 0.5mL columns and volume <70µL, apply 15µL stacker buffer, for 2mL columns and volume <350µL, apply 40µL stacker buffer, for 5mL columns and volume <750µL, apply 100µL stacker buffer, For 10mL columns and volume <1500µL, apply 200µL stacker buffer.

12. Perform final centrifugation to collect the flow-through (samples). Centrifuge column for 2 minutes at 1000xg at 4˙C, or 1500xg at 4˙C for 0.5mL tubes. Discard spin column after use.

13. Transfer sample to a new siliconized microcentrigue tube and freeze at -20˙C.

Solutions: See buffer preparations page for preparation of 1x PBS or 1x TBS. Follow manufacturer instructions to prepare 10x protease inhibitor and 10x phosphatase inhibitor solution in 1x PBS buffer.