1702-pET30b-A/WSN/33 M15/88K

Cloning of 1702-pET30b-A/WSN/33 M15/88K

Purpose: Cloning of 1702-pET30b-A/WSN/33 M15/88K

Date: 2013-3-18---2013-3-20

Notebook:0

Page:

Clone ID:1702

Experimenters: yingying

Results

cloning

Colony Screening

Date: 2013-3-20

Make miniprep and digest with Nde1 + Xho1

Result intepretation/Summary

• <br>

Protein Purification

3/28/2013

M H H H H H H SSLLTEVETYVLSIVPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGN
K
DPNNMDKAVKLYSKLKSEITFHGAKEIALSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQMLE HHHHHH

pI/Mw: 8.47 / 20194.21

All buffers contain 1~5 mM beta-ME to avoid aggregation.

1: Transform Nico21(DE3)/pLysN with plasmid, grow 4 L in LB/Kan/Chl,  induce with 0.1 M IPTG o/n 37 oC.

2. Capture using 20 ml Ni column, wash extensively with 20 mM Tris-HCl, pH7.5, 20 mM Imidazole, 300 mM NaCl, 1 mM beta-ME

3. Elute with 20 mM Tris-HCl, pH7.5, 250 mM Imidazole, 300 mM NaCl, 1 mM beta-ME

4. Dilute 5 folds with 20 mM Tris-HCl, pH7.5, 1 mM beta-ME to salt conc <=50 mM NaCl.

5. Load to 20 ml SP Sepharose FF. Wash with 20 mM Tris-HCl, pH7.5, 50 mM NaCl, 1 mM beta-ME

6. Elute with linear gradient of 20 mM Tris-HCl, pH7.5, 1 mM beta-ME, from 50 mM NaCl to 1 M over 250 ml. Protein eluted at ~200 mM NaCl.

7. Conc fractions containing major peak using spin column.

8. Load to gel filtration column BioSil TSK 250 equilibrated with 55 mM KH2PO4/K2HPO4, 0.2 M NaCl, 1 mM beta-ME, pH4.0., elute with the same buffer at 2.5 ml/min

9. Collect 5 ml fractions. protein eluted between fraction 32~34.

10. Combine fractions 32~34. Add 1 M  TECP to final conc 2 mM (100 ul of 1 M to 50 ml). Store at 4 oC.

11. Use 15 ul for SDS-PAGE (marker is fermentas Page-Ruler Plus, 10, 15, 25 (red), 35,  55, 70(red), 100, 130, 250 kDa

Yield: 3 tubes:

1. 45 ml, 1.4 mg/ml, total 63 mg

2: 40 ml, 0.8 mg/ml,  total 32 mg

3: 45 ml, 0.5 mg/ml, total  22.5 mg

total: 117 mg.

--Paul.xu 18:21, 28 March 2013 (EST)