Crm at 18:12, 3 May 2017

2017-05-03T18:12:12Z

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= MS 3 20 2017 =
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1. Grow 2295 yPolyA in LB/K/C

== Expression ==

1. Take out 2295 yPolyA protein expression stock and inoculate 200 ml LB//Kan/Chl.

2. Grow at 37 ºC o/n.

3. Split the overnight culture  into 8 x 1L LB medium, add 1 ml of 10,000X Kan and 1 ml of 10,000X Chl, 20 ml of 20X50 solution'''.'''

4. Grow at 37 ºC  2~4 hours.

5. When cell density OD600 is ~0.8, add 0.5 ml of 1 M IPTG to 1 L culture and induce at 25 ºC(Winter 25ºC, Summer 20ºC) O/N. 

6. In the morning, spin down cells, combine. Weigh cell pellet, record weight is 45.45g. Store cell pellet at -20 ºC.

== Purification ==

1. Add 800 ml '''Buffer A'''  to cell pellet, resuspend cell pellet using a spatula

2. Homogenize the cell suspension with a homogenizer.

3. 2295 yPolyA is not able to incubate at 75 ºC.

4. Spin at 6000 rpm for 15 min at 4 oC using high speed centrifuge, collect supernatant. 

5. Add cold 400 ml '''Buffer A''' to pellet. Homogenize again. 

6. Spin at 6000 rpm for 15 min at 4 oC using high speed centrifuge, collect supernatant. 

7. Transfer supernatant to ultracentrifuge tubes.  Cool ultraspeed rotor with dry ice for 5 min.

8. Spin at 25,000 rpm for 30 min at 4 oC using ultra speed centrifuge, collect clarified supernatant.

9. Load supernatant to 20 ml Ni column in cold room, flow rate ~ 1 ml/min

10. Wash Ni column with 200~500  ml '''Ni Washing Buffer (Buffer A). '''Use Buffer A as blank, measure A280.

11. Wash''' '''Ni column with 50 ml  20 mM Tris-HCl Buffer, pH 8.0.

12. Elute Taq enzyme with ~200 ml '''Buffer B (low salt, concentration of NaCl is 100mM), '''Collect fractions 5 ml/tube using a fraction collector.

13.Use Buffer B as blank, measure protein concentration in each fraction. Identify protein peak, combine fractions with high A280 value (4-9). (A280>0.30)

{| width="500" border="1" cellspacing="1" cellpadding="1"
|-
| Num 
| A280(mg/mL) 
| Num 
| A280(mg/mL) 
| Num 
| A280(mg/mL) 
|-
| 1 
| 0.19 
| 6 
| 1.58 
| 11 
| 0.22 
|-
| 2 
| 0.16 
| 7 
| 0.97 
| 12 
| 0.18 
|-
| 3 
| 0.22 
| 8 
| 0.66 
| 13 
| 0.17 
|-
| 4 
| 0.60 
| 9 
| 0.42 
| 14 
| 0.16 
|-
| 5 
| 1.68 
| 10 
| 0.26 
| 15 
| 0.15 
|}

14. Add 1 volume of cold '''Dilution Buffer.'''

15.''' '''Load Taq onto a  20~25 ml Q HP column (1.6 X10) using a peristaltic pump. With speed 15.

16. Use Dilution Buffer as blank, measure A280 of sample, A280=0.03.

17. Wash column with 100 ml '''Tris-HCl Buffer, flow rate 2 ml/min'''

18. Elute Taq using 200 ml of linear gradient of  

'''A:'' '''''Q HP Washing Buffer

'''B: Q HP Elution Buffer ''' 

Flow rate 1.5 or 2 ml/min. Use ddH2O as blank, measure A280.

[[File:108-2295 yPolyA QHP 3 22 2017.jpg|RTENOTITLE]]

{| style="width: 500px" border="1" cellspacing="1" cellpadding="1"
|-
| 18 
| 0.01 
|-
| 19 
| 0.01 
|-
| 20 
| 0.07 
|-
| 21 
| 0.10 
|-
| 22 
| 0.26 
|-
| 23 
| 0.30 
|-
| 24 
| 0.37 
|-
| 25 
| 0.44 
|-
| 26 
| 0.52 
|-
| 27 
| 0.52 
|-
| 28 
| 0.40 
|-
| 29 
| 0.51 
|-
| 30 
| 0.29 
|-
| 31 
| 0.18 
|-
| 32 
| 0.08 
|-
| 33 
| 0.06 
|-
| 34 
| 0.06 
|-
| 35 
| 0.04 
|-
| 42 
| 0.03 
|-
| 44 
| 0.05 
|}

19. Run SDS-PAGE gel on the first peak. If there is anothe peak at ~600~800 mM KCl, it could be DNA/RNA peak, discard the DNA/RNA peak.

{| style="width: 500px" border="1" cellspacing="1" cellpadding="1"
|-
| Number 
| 1 
| 2 
| 3 
| 4 
| 5 
| 6 
| 7 
| 8 
| 9 
| 10 
| 11 
| 12 
|-
| sample 
| 21 
| 22 
| 23 
| 24 
| 25 
| 26 
| MW 
| 27 
| 28 
| 29 
| 30 
| 31 
|-
| uL 
| 10 
| 4 
| 4 
| 3 
| 3 
| 2 
| 10 
| 2 
| 3 
| 2 
| 4 
| 6 
|-
| ddH2O uL 
| \ 
| 10 
| 10 
| 10 
| 10 
| 10 
| \ 
| 10 
| 10 
| 10 
| 10 
| 10 
|-
| buffer uL 
| 4 
| 4 
| 4 
| 4 
| 4 
| 4 
| \ 
| 4 
| 4 
| 4 
| 4 
| 4 
|}

[[File:Protein Expression SDS-PAGE 108-2295 yPolyA QHP 3 22 2017 1.jpg|RTENOTITLE]]

[[File:2292 2295 2296 2044 2312 2301.jpg]]

2295 2014/9/5 lane2 [http://excellgen.net/eln/index.php/2292_2295_2296_2044_2312_2301 http://excellgen.net/eln/index.php/2292_2295_2296_2044_2312_2301]

20. Combine fractions containing Taq.

21. Use ddH2O wash the gel. Boil the gel in microwave for 2 min, two times.

22. Use N3-EF wash Buffer to wash the gel in microwave for 2 min.

23. Use ddH2O wash the gel in microwave for 2 min, two times.

24. Dialysis. Add combined fractions into dialysis bag. Use '''High salt Buffer B '''to wash the tube and add into bag.

25. Collect the liquid in Dialysis bag, use '''Dialysis Buffer''' to wash bag, 1mL per time, twice, add into collect tube.

26. '''Measure A280 1=0.69 mg/ml, total 4.14 mg.'''

     '''Measure A280 2=0.83 mg/ml, total 11.87 mg.'''

27. Use '''Dialysis Buffer''' to wash bag, 1mL per time,

 '''    A280 1 wash #1 0.59mg/ml, wash #2 0.53mg/ml'''

'''     A280 2 wash #1 0.79mg/ml, wash #2 0.60mg/ml'''

28. Sterilization. Use 0.22 um filter.

29. Store at -80 oC.

== Regeneration of Ni column ==

1. Wash Ni column with 5 CV of water.

2. Wash column with 3 CV of 100 mM EDTA, 500 mM NaCl.

3. Wash the column with 5 CV of H2O.

4. Recharge the column with 1 column volume of 100 mM NiSO4, or NiCl2.

5. Wash the column with 10 column volumes of H2O.

6. Equilibrate with 3 volumes of a suitable buffer (''e.g., ''Buffer A).

'''Buffer A'''

20 mM Tris-HCl, pH 7.9~8.0

500 mM NaCl

0.1 mM EDTA

0.1% Tween 20

0.1% Nonident-P40

 '''Ni Washing Buffer'''

20 mM Tris-HCl, pH 7.9~8.0

800 mM NaCl

10 mM imidazole 

'''Tris-HCl Buffer '''

20 mM Tris-HCl, pH 7.9~8.0

'''Buffer B (Low Salt)'''

20 mM Tris-HCl, pH 7.9~8.0

100 mM NaCl

150 mM imidazole 

5% glycerol

'''Dilution Buffer'''

20 mM Tris-HCl, pH 7.9~8.0, optional: 5% glycerol

'''Q HP Washing Buffer'''

20 mM Tris-HCl, pH 7.9~8.0

50 mM KCl

5% Trehalose

'''Q HP Elution Buffer'''

20 mM Tris-HCl, pH 7.9~8.0

1 M KCl

5% Trehalose

'''Buffer E'''

20 mM HEPES (pH 8.3) or Tris-HCl, pH 8.0 

'''Buffer F'''

20 mM HEPES (pH 8.0) or Tris-HCl, pH 7.9 

50 mM KCl. 

'''Buffer G'''

20 mM HEPES or Tris-HCl, pH 7.9 or pH 8.0

500 mM KCl.

'''Storage Buffer'''

20 mM HEPES, pH 7.5

100 mM KCl

0.1 mM EDTA

1 mM dithiothreitol

50% glycerol.
[[Category:My ELN]] [[Category:Protein Expression]] [[Category:QC]]

MS 3 20 2017 - Revision history

Crm at 18:12, 3 May 2017