- Use 25 µL of CD nanobeads-streptavidin [25fmol]
- Add 150 µL of wash buffer
- Add 30 µL of 6.8µM Biotin-3HB-Spycatcher3 [200pmol]
- Leave O/N in cold room
- Add biotin to 1 mM and mix well
- Remove aggregation by Minispin 10 sec, (You may see small ppt)
- Transfer soluble beads to new tube
- Spin 16000g 20min 4ºC, Remove the supernatant on the handmade magnetic rack
- Resuspend beads with 200 µL of wash buffer
- Spin 16000g 20min 4ºC, Remove the supernatant on the handmade magnetic rack
- Resuspend beads with 200 µL of wash buffer
- Add 200pmol N-tag mono SPY-avidin tetramer
- Leave O/N in the cold room
- Remove aggregation by Minispin 10 sec, transfer soluble beads to a new tube
- Spin 16000g 20min 4ºC, Remove the supernatant on the handmade magnetic rack
- Resuspend beads with 200 µL of wash buffer
- Spin 16000g 20min 4ºC, Remove the supernatant on the handmade magnetic rack
- Resuspend beads with 200 µL of wash buffer
- Add 800pmol Biotin-60nm SAH-Spycatcher3
- Leave 12 hrs in cold room
- Add biotin to 1 mM and mix well
- Mini-spin 10 sec, transfer soluble beads to a new tube
- Spin 16000g 20min 4ºC, Remove the supernatant on the handmade magnetic rack
- Resuspend the beads with 200 µL of wash buffer
- Spin 16000g 20min 4ºC, Remove the supernatant on the handmade magnetic rack
- Resuspend the beads with 200 µL of wash buffer
- Add 800pmol SPY-GFP enhancer
- Leave O/N in the cold room
- Spin 16000g 20min 4ºC, Remove the supernatant on the handmade magnetic rack
- Resuspend the beads with 200 µL of wash buffer
- Spin 16000g 20min 4ºC, Remove the supernatant on the handmade magnetic rack
- Resuspend the beads with 250 µL of wash buffer (For storage at -20ºC , resuspend beads with 50% glycerol containing wash buffer)