Biopanning Assay

Unfixed chalcopyrite:

1. Pre-sieve chalcopyrite with 40um size cell strainer. Then autoclave by Dry-5 times settings
2. Incubate chalcopyrite (40um in size, 30mg) in culture of Caulobacter crescentus for 2hrs, shaking at low rpm.
3. Filter chalcopyrite + Caulobacter crescentus using a 40um Falcon™ Cell Strainers
4. Washing steps:
   • 1st round of panning: Wash strained chalcopyrite with 5ml 1x Tris-buffered saline (TBS) containing 0.1% (v/v) Tween-20, pH 7.2. Plate cells.
   • 2nd round of panning: Wash strained chalcopyrite with 5ml 1x Tris-buffered saline (TBS) containing 0.2% (v/v) Tween-20, pH 7.2.
   • 3rd round of panning: Wash strained chalcopyrite with 5ml 1x Tris-buffered saline (TBS) containing 0.3% (v/v) Tween-20, pH7.2.
   • 4th round panning: Wash strained chalcopyrite with 5ml 1x Tris-buffered saline (TBS) containing 0.4% (v/v) Tween-20, pH7.2.
   • 5th round panning: Wash strained chalcopyrite with 5ml 1x Tris-buffered saline (TBS) containing 0.5% (v/v) Tween-20, pH7.2
5. Elution steps
   • Treat with elution buffer 1 (EB1) containing 50mM Tris-HCl pH7.5, 250mM NaCl for 15 minutes. Collect Elution.
   • Treat with elution buffer 2 (EB2) containing 50mM Tris-HCl pH7.5, 500mM NaCl for 15 minutes. Collect Elution.

Tris-buffered saline (TBS) (1X)

50 mM Tris-Cl, pH 7.5
150 mM NaCl
To prepare, dissolve 6.05 g Tris and 8.76 g NaCl in 800 mL of H2O. Adjust pH to 7.5 with 1 M HCl and make volume up to 1 L with H2O. TBS is stable at 4°C for 3 mo.
Then add tween.

Tris-buffered saline (TBS) (1X)

50 mM Tris-Cl, pH 7.5
250 mM NaCl
To prepare, dissolve 6.05 g Tris and 14.61 g NaCl in 800 mL of H2O. Adjust pH to 7.5 with 1 M HCl and make volume up to 1 L with H2O. TBS is stable at 4°C for 3 mo.

Tris-buffered saline (TBS) (1X)

50 mM Tris-Cl, pH 7.5
500 mM NaCl
To prepare, dissolve 29.22 g Tris and 14.61 g NaCl in 800 mL of H2O. Adjust pH to 7.5 with 1 M HCl and make volume up to 1 L with H2O. TBS is stable at 4°C for 3 mo.