Knight:Microcon filtration of proteins

Overview

This protocol is used to concentrate a protein sample by filtration as well as to change the buffer in which the sample resides.

Note that although this method should work, I find that I lose most of my protein using this approach. It also seems to concentrate the salts in my sample. Try Knight:Centrifuge desalting instead.

Materials

• Microcon filter
  • In general, the molecular weight cutoff for your column should be about half the molecular weight of your protein of interest.
• Tabletop centrifuge
• Knight:Protein DNA binding buffer
• Passivation solution
  • 1% BSA in PBS (recommended by Millipore)
  • 1% powdered milk in distilled water (recommended by Millipore)

Procedure

The following steps assume use of a YM-10 column. For a YM-3 column, increase spin times to 100 mins.

Read literature that comes with the Microcon filter carefully. It contains a lot of useful information.

Passivation

Passivation is optional. Supposedly it increases recovery of dilute protein from these columns because nonspecific binding sites of the column are blocked.

Procedure specified by Millipore

1. Assemble Microcon column onto tube.
2. Add 500 μL passivation solution to Microcon column. Cap and soak at least 2 hours or overnight at room temperature.
3. Uncap and rinse all devices thoroughly with tap water.
4. To remove residuals, add 500 μL distilled water to each device and cap.
5. Spin 30 mins in table top centrifuge at 12,000 g. Spin at room temperature.
6. Discard flow-through.
7. Add 500 μL distilled water to each device and cap.
8. Spin 30 mins in table top centrifuge at 12,000 g. Spin at room temperature.
9. Discard flow-through.
10. Invert Microcon column in tube.
11. Spin for 3 mins at 1000 g. Spin at room temperature.
12. Discard flow-through.
13. If not using the Microcon columns immediately, add distilled water to assembled sample reservoirs, cap and refrigerate at 4°C to prevent the membrane from drying out and cracking.

Modified procedure

Avoids rinse with tap water.

1. Assemble Microcon column onto tube.
2. Add 500 μL passivation solution to Microcon column. Cap and soak at least 2 hours or overnight at room temperature.
3. Discard passivation solution.
4. Rinse column with distilled water.
5. To remove residuals, add 500 μL distilled water to column and cap.
6. Spin 30 mins in table top centrifuge at 12,000 g. Spin at room temperature.
7. Discard flow-through.
8. Add 500 μL distilled water to column and cap.
9. Spin 30 mins in table top centrifuge at 12,000 g. Spin at room temperature.
10. Discard flow-through.
11. Add 100μL distilled water to column and cap.
12. Invert Microcon column in tube.
13. Spin for 3 mins at 1000 g. Spin at room temperature.
14. Discard flow-through.
15. If not using the Microcon columns immediately, add distilled water to assembled sample reservoirs, cap and refrigerate at 4°C to prevent the membrane from drying out and cracking.

Concentration and buffer exchange

1. Assemble the Microcon column onto the tube (if you haven't done the passivation step).
2. Add entire protein sample volume to Microcon filter.
3. Spin 30 mins in table top centrifuge at 12,000 g. Spin at room temperature.
4. Discard flow-through.
5. Add 500 μL Knight:Protein DNA binding buffer to Microcon column.
6. Spin 30 mins in table top centrifuge at 12,000 g. Spin at room temperature.
7. Discard flow-through.
8. Add 500 μL Knight:Protein DNA binding buffer to Microcon column.
9. Spin 30 mins in table top centrifuge at 12,000 g. Spin at room temperature.
   The above two wash steps changes the buffer.
10. Discard flow-through.
11. Move Microcon filter to new eppendorf tube.
12. Add 10 μL Knight:Protein DNA binding buffer to Microcon column.
13. Invert Microcon column in tube.
    The column won't be very stable but it should remain in place for the next step.
14. Spin for 3 mins at 1000 g. Spin at room temperature.
15. Discard Microcon column. The liquid in the tube should contain your purified sample.

Notes

• If you find a large volume is being retained in the column, this is usually an indication that you loaded a lot of material in the column. It is not necessary to add 20μL of Knight:Protein DNA binding buffer prior to the elution spin.
• The spin steps should be carried out at room temperature rather than 4°C otherwise longer spin times are needed for complete spin through of the sample.
• Passivation may improve recovery of material. See the protocol note (html, pdf) for details.
• Even though they are not supposed to, sometimes these columns can end up concentrating your buffers. This may explain why the dye front runs oddly when loading samples from the microcon. The salt content has been increased. (from Kathleen).
• These columns tend to bind an absolute amount of protein, not a fraction of what you send through it. Hence, larger scale preps tend to be preferable. (from Kathleen).