E. Coli lysate separation

Transcription

1 ZY12 E. Coli lysate separation SEC, IEX-SAX, Reversed-Phase

2 ZY12 E. Coli lysate separation Size exclusion chromatography Zenix SEC-15 (3 µm, 15 Å, 7.8 x 3 mm); Zenix SEC-3 (3 µm, 3 Å, 7.8 x 3 mm); Two columns in tandem 2

3 Separation of E.coli lysate Bio-rad # Zenix SEC-15 Column: Zenix SEC-15 (3 µm, 15 Å, 7.8 x 3 mm); Mobile phase: 15mM Sodium Phosphate Buffer, ph=7. Flow rate: 1 ml/; Detector: UV 28, 214 nm; Column temperature: 25 ; Sample: 2.7 mg/ml Bio-rad E. coli lysate; Injection volume: 1 ml UV 28 nm ZY UV 214 nm

4 Separation of E.coli lysate Bio-rad # Zenix SEC-3 Column: Zenix SEC-3 (3 µm, 3 Å, mm); Mobile phase: 15mM Sodium Phosphate Buffer, ph=7. Flow rate: 1 ml/; Detector: UV 28 nm; Column temperature: 25 ; Sample: 2.7 mg/ml Bio-rad E. coli lysate; Injection volume: 1 ml ZY UV 28 nm UV 214 nm

5 Overylay of SEC-15 and SEC-3 for E. coli lysate separation-uv28 nm Column: Zenix SEC-15 (3 µm, 15 Å, mm); Zenix SEC-3 (3 µm, 3 Å, mm); Mobile phase: 15mM Sodium Phosphate Buffer, ph=7. Flow rate: 1 ml/; Detector: UV 28 nm; Column temperature: 25 ; Sample: 2.7 mg/ml Bio-rad E. coli lysate; Injection volume: 1 ml 3 ZY SEC-3 SEC

6 Overylay of SEC-15 and SEC-3 for E. coli lysate separation-uv214 nm Column: Zenix SEC-15 (3 µm, 15 Å, 7.8 x 3 mm); Zenix SEC-3 (3 µm, 3 Å, 7.8 x 3 mm); Mobile phase: 15 mm Sodium Phosphate Buffer, ph=7. Flow rate: 1 ml/; Detector: UV 214 nm; Column temperature: 25 ; Sample: 2.7 mg/ml Bio-rad E. coli lysate; Injection volume: 1 ml 5 ZY SEC-3 SEC-15 6

7 SEC-15 and SEC-3 in tandem for E. coli lysate separation Column: Zenix SEC-15 (3 µm, 15 Å, 7.8 x 3 mm) and Zenix SEC-3 (3 µm, 3 Å, 7.8 x 3 mm); Mobile phase: 15 mm Sodium Phosphate Buffer, ph=7. Flow rate: 1 ml/; Detector: UV 28 nm, 214 nm; Column temperature: 25 ; Sample: 2.7 mg/ml Bio-rad E. coli lysate; Injection volume: 2 ml ZY UV 28 nm UV 214 nm

8 SEC-15 and SEC-3 in tandem for E. coli lysate separation-uv214 nm Loading test Column: Zenix SEC-15 (3 µm, 15 Å, 7.8 x 3 mm) and Zenix SEC-3 (3 µm, 3 Å, 7.8 x 3 mm); Mobile phase: 15 mm Sodium Phosphate Buffer, ph=7. Flow rate:.8 ml/; Detector: UV 214 nm; Column temperature: 25 ; Sample: 2.7 mg/ml Bio-rad E. coli lysate; Injection volume: 5, 1, 2 ml 5 µl 5 1 µl 2 µl 4 ZY

9 SEC-15 and SEC-3 in tandem for E. coli lysate separation-uv28 nm Loading test Column: Zenix SEC-15 (3 µm, 15 Å, 7.8 x 3 mm) and Zenix SEC-3 (3 µm, 3 Å, 7.8 x 3 mm); Mobile phase: 15 mm Sodium Phosphate Buffer, ph=7. Flow rate:.8 ml/; Detector: UV 28 nm; Column temperature: 25 ; Sample: 2.7 mg/ml Bio-rad E. coli lysate; Injection volume: 5, 1, 2 ml 5 µl 3 1 µl 2 µl 25 ZY

10 ZY12 E. Coli lysate separation Ion exchange chromatography Proteomix SAX-NP3 (3 µm, 4.6 x 5 mm)

11 Separation of E. coli lysate with Proteomix SAX columns Column: Proteomix SAX-NP3 (3 µm, 4.6 x 5 mm); Mobile phase: A: 2 mm Tris, ph 9.; B: A +.5 M NaCl; Gradient: -1% B (3 ) Flow rate: 1 ml/; Detector: UV 28 nm; Column temperature: ambient; Sample: 2.5 mg/ml Custom source E. coli lysate; Injection volume: 1, 2, 5 ml ZY12 11

12 ZY12 E. Coli lysate separation Reversed phase chromatography PolyRP-1 (5 µm, 1 Å, 4.6 x 1 mm) 12

13 E. Coli lysate separation on PolyRP-1 ZY12 Column: PolyRP-1 (5 µm, 1 Å, 4.6 x 1 mm); Mobile phase: A:.1% TFA in water; B:.1% TFA in 1% ACN; Flow rate: 1. ml/; Detector: UV 21 nm; Column temperature: 8 C; Sample: Bio-rad E. coli lysate diluted in.1% TFA, filter before injection (1.3 mg/ml) ; Injection volume: 3 ml Zoom view Time() %B

14 Bio-rad E. Coli lysate separation on PolyRP-1 ZY12 Column: PolyRP-1 (5 µm, 1 Å, 4.6 x 1 mm); Mobile phase: A:.1% TFA in water; B:.1% TFA in 1% ACN; Flow rate: 1. ml/; Detector: UV 21 nm; Column temperature: 8 C; Sample: See chromatogram 35 Blue: direct sample injection, 2.7 mg/ml, 1 ml Red: dilute with.1% TFA, 1.3 mg/ml, 3 ml Time() %B