Proteomic Analysis of Proteins Secreted by Streptococcus pyogenes - Bacterial Pathogenesis: Methods and Protocols - page 17

13. Transfer 750 μL of the solution into four 1.5-mL microcentrifuge tubes ( see
Note 14 ).
14. Vortex well and incubate at -20 °C for 30 min.
15. Centrifuge at 25,000 × g for 5 min at 4 °C.
16. Remove the supernatant fluid and discard it in a hazardous waste container.
17. Add 1.5 mL of ice-cold ethanol to each tube.
18. Vortex well. The pellet will disperse but may not completely dissolve.
19. Incubate at -20 °C for 30 min.
20. Vortex well and centrifuge at 25,000 × g for 5 min at 4 °C.
21. Pour off supernatant fluid. Invert tubes and let any remaining ethanol evaporate.
22. Resuspend the pellet with 250 μL of 0.04 M Tris-HCl (pH 7.5).
23. Vortex well and combine the solutions into one microcentrifuge tube.
24. Add 24,000 U/mL of DNase, 75,000 U/mL of RNase, and 10 μL of 10× DNase
buffer ( see Note 15 ). Incubate for2hat4°C.
25. Add 100% cold TCA to a final concentration of 10% (v/v) and invert the tube
several times to mix.
26. Add ice-cold acetone to a final concentration of 5% (v/v) and invert the tube
several times to mix ( see Note 13 ).
27. Incubate at -20 °C for 1 h. Invert the tube several times every 20 min.
28. Centrifuge at 25,000 × g for 20 min at 4 °C.
29. Discard supernatant fluid.
30. Add 1.5 mL of ice-cold ethanol to the pellet and resuspend the pellet by
vortexing.
31. Vortex well and incubate at -20 °C for 30 min.
32. Centrifuge at 25,000 × g for 5 min at 4 °C.
33. Remove supernatant fluid and discard in a hazardous waste container.
34. Add 1.5 mL of ice-cold ethanol to the tube.
35. Vortex well. The pellet will disperse but may not completely dissolve.
36. Incubate at -20 °C for 30 min.
37. Centrifuge at 25,000 × g for 5 min at 4 °C.
38. Pour off supernatant fluid. Invert tubes and let any remaining ethanol evaporate.
39. Resuspend the pellet in SR solution containing 75 m M DTT ( see Note 16 ).
40. Centrifuge samples at 25,000 × g for 5 min at 4 °C and transfer the supernatant
fluid to a clean microcentrifuge tube. Proceed to the next step or store the sample
at 4 °C until the protein concentration can be determined.
41. Determine the protein concentration by using the 2-D quant kit (GE Healthcare
Life Sciences) ( see Note 17 ).
42. Store sample at 4 °C.
3.2. Isoelectric Focusing
1. Remove protein samples from 4 °C storage and equilibrate to room temperature.
2. Vortex well. Dilute the samples to a concentration of 0.8 mg/mL in a final
volume of 125 μL with SR solution containing 75 m M DTT. Add 1.875 μL of
IPG buffer (pH 4-7) ( see Note 18 ). Vortex well and mix samples on a platform
rocker at room temperature for at least 1 h.