Editing MS 3 1 2017 (section)

= <span dir="auto">MS 3 1 2017</span> =
<div id="bodyContent" class="mw-body-content"><div id="contentSub"></div></div>
1. Grow 1995 in LB/K/C

== <span class="mw-headline" id="Expression"><span class="mw-headline" id="Expression">Expression</span></span> ==

1. Take out 1995 protein expression stock and inoculate&nbsp;200 ml LB//Kan/Chl.

2. Grow at 37&nbsp;ºC o/n.

3. Split the overnight culture &nbsp;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 &nbsp;2~4 hours.

5. When cell density OD<sub>600</sub> 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.&nbsp;

6. In the morning, spin down cells, combine. Weigh cell pellet, record weight is 40.67g. Store cell pellet at -20 ºC.



== <span class="mw-headline" id="Purification"><span class="mw-headline" id="Purification">Purification</span></span> ==

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

2. Homogenize the cell suspension with a homogenizer.

3. Incubate the homogenate&nbsp;at 75&nbsp;<sup style="line-height: 1">o</sup>C for 10 min, mix ocasionally, cool on ice for 15 min.

4. Spin at 6000 rpm for 15&nbsp;min at 4&nbsp;<sup style="line-height: 1">o</sup>C using high speed centrifuge, collect supernatant.&nbsp;

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

6. Spin at 6000 rpm for 15&nbsp;min at 4&nbsp;<sup style="line-height: 1">o</sup>C using high speed centrifuge, collect supernatant.&nbsp;

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

8. Spin at 25,000 rpm for 30 min at 4&nbsp;<sup style="line-height: 1">o</sup>C 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&nbsp;Ni column with 200~500 &nbsp;ml&nbsp;'''Ni Washing Buffer (Buffer A). '''Use Buffer A as blank, measure A<sub>280</sub>.

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

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

13.Use Buffer B as blank, measure protein concentration in each fraction. Identify protein peak, combine fractions&nbsp;with high A<sub>280</sub> value (18-42). (A<sub>280</sub>>0.05)

{| style="width: 500px" cellspacing="1" cellpadding="1" border="1"
|-
| Num<br/>
| A<sub>280</sub>(mg/mL)<br/>
| Num<br/>
| A<sub>280</sub>(mg/mL)<br/>
| Num<br/>
| A<sub>280</sub>(mg/mL)<br/>
| Num<br/>
| A<sub>280</sub>(mg/mL)<br/>
|-
| 1<br/>
| 0.08<br/>
| 17<br/>
| 0.22<br/>
| 33<br/>
| 0.58<br/>
| 49<br/>
| 0.37<br/>
|-
| 2<br/>
| 0.08<br/>
| 18<br/>
| 0.40<br/>
| 34<br/>
| 0.63<br/>
| 50<br/>
| 0.34<br/>
|-
| 3<br/>
| 0.06<br/>
| 19<br/>
| 1.07<br/>
| 35<br/>
| 0.62<br/>
| 51<br/>
| 0.31<br/>
|-
| 4<br/>
| 0.05<br/>
| 20<br/>
| 1.72<br/>
| 36<br/>
| 0.66<br/>
| 52<br/>
| 0.30<br/>
|-
| 5<br/>
| 0.04<br/>
| 21<br/>
| 2.08<br/>
| 37<br/>
| 0.52<br/>
| 53<br/>
| 0.29<br/>
|-
| 6<br/>
| 0.12<br/>
| 22<br/>
| 1.13<br/>
| 38<br/>
| 0.64<br/>
| 54<br/>
| 0.27<br/>
|-
| 7<br/>
| 0.06<br/>
| 23<br/>
| 0.93<br/>
| 39<br/>
| 0.62<br/>
| 55<br/>
| 0.23<br/>
|-
| 8<br/>
| 0.05<br/>
| 24<br/>
| 0.97<br/>
| 40<br/>
| 0.57<br/>
| 56<br/>
| 0.24<br/>
|-
| 9<br/>
| 0.05<br/>
| 25<br/>
| 0.87<br/>
| 41<br/>
| 0.57<br/>
| 57<br/>
| 0.23<br/>
|-
| 10<br/>
| 0.06<br/>
| 26<br/>
| 0.79<br/>
| 42<br/>
| 0.53<br/>
| 58<br/>
| 0.22<br/>
|-
| 11<br/>
| 0.06<br/>
| 27<br/>
| 0.74<br/>
| 43<br/>
| 0.45<br/>
| 59<br/>
| 0.20<br/>
|-
| 12<br/>
| 0.07<br/>
| 28<br/>
| 0.51<br/>
| 44<br/>
| 0.47<br/>
| 60<br/>
| 0.20<br/>
|-
| 13<br/>
| 0.05<br/>
| 29<br/>
| 0.42<br/>
| 45<br/>
| 0.46<br/>
| 61<br/>
| 0.19<br/>
|-
| 14<br/>
| 0.06<br/>
| 30<br/>
| 0.58<br/>
| 46<br/>
| 0.42<br/>
| 62<br/>
| 0.20<br/>
|-
| 15<br/>
| 0.09<br/>
| 31<br/>
| 0.53<br/>
| 47<br/>
| 0.40<br/>
| 63<br/>
| 0.17<br/>
|-
| 16<br/>
| 0.13<br/>
| 32<br/>
| 0.59<br/>
| 48<br/>
| 0.39<br/>
| 64<br/>
| 0.15<br/>
|}

14. Add 1 volume of&nbsp;cold '''Dilution Buffer.'''

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

16. Use Dilution Buffer as blank, measure A<sub>280</sub> of sample, A<sub>280</sub>=0.05.

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

18. Elute Taq using 200 ml of linear gradient of &nbsp;

'''A:''&nbsp;'''''<b>Q HP Washing Buffer</b>

'''B: Q HP Elution&nbsp;Buffer&nbsp;'''&nbsp;

Flow rate 1.5 or 2 ml/min. Use ddH<sub>2</sub>O as blank, measure A<sub>280</sub>.

[[File:107-1995 Kfu QHP 3 9 2017.jpg|RTENOTITLE]]

{| style="width: 500px" cellspacing="1" cellpadding="1" border="1"
|-
| 19<br/>
| 0.06<br/>
|-
| 20<br/>
| 0.04<br/>
|-
| 21<br/>
| 1.68<br/>
|-
| 22<br/>
| 0.77<br/>
|-
| 23<br/>
| 0.38<br/>
|-
| 24<br/>
| 0.31<br/>
|-
| 25<br/>
| 0.26<br/>
|-
| 26<br/>
| 0.26<br/>
|-
| 27<br/>
| 0.31<br/>
|-
| 28<br/>
| 0.15<br/>
|-
| 29<br/>
| 0.12<br/>
|-
| 30<br/>
| 0.13<br/>
|}

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" cellspacing="1" cellpadding="1" border="1"
|-
| Number<br/>
| 1<br/>
| 2<br/>
| 3<br/>
| 4<br/>
| 5<br/>
| 6<br/>
| 7<br/>
| 8<br/>
| 9<br/>
| 10<br/>
| 11<br/>
| 12<br/>
|-
| sample<br/>
| MW<br/>
| 20<br/>
| 21<br/>
| 22<br/>
| 23<br/>
| 24<br/>
| 25<br/>
| 26<br/>
| 27<br/>
| 28<br/>
| 29<br/>
| 30<br/>
|-
| uL<br/>
| 10<br/>
| 25<br/>
| 2<br/>
| 2<br/>
| 3<br/>
| 4<br/>
| 4<br/>
| 4<br/>
| 4<br/>
| 7<br/>
| 9<br/>
| 8<br/>
|-
| ddH<sub>2</sub>O uL<br/>
| \<br/>
| \<br/>
| 10<br/>
| 10<br/>
| 10<br/>
| 10<br/>
| 10<br/>
| 10<br/>
| 10<br/>
| 10<br/>
| 10<br/>
| 10<br/>
|-
| buffer uL<br/>
| \<br/>
| 5<br/>
| 3<br/>
| 3<br/>
| 3<br/>
| 3<br/>
| 3<br/>
| 3<br/>
| 3<br/>
| 4<br/>
| 4<br/>
| 4<br/>
|}

[[File:Protein Expression SDS-PAGE 107-1995 Kfu QHP 3 9 2017.jpg|RTENOTITLE]]

[[File:1 24 14 protein exp screening 1.jpg]]

2014/1/24 lane9&nbsp;[http://excellgen.net/eln/index.php/1631_1693_1898_1934_1924_1926_1942_1995_2001 http://excellgen.net/eln/index.php/1631_1693_1898_1934_1924_1926_1942_1995_2001]





20. Combine fractions containing Taq (21-22).

21. Use ddH<sub>2</sub>O 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 ddH<sub>2</sub>O wash the gel in microwave for 2 min, two times.

24. Dialysis. Add combined fractions into dialysis bag. Use '''Dialysis Buffer (Storage Buffer) '''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 A<sub>280</sub>=3.27 mg/ml, total 5.3 ml.

27. Use '''Dialysis Buffer''' to wash bag, 1mL per time, until A<sub>280</sub> is low. #1 0.78, #2 0.72, #3 0.65.

28. Store at -80 <sup style="line-height: 1">o</sup>C.





== <span class="mw-headline" id="Regeneration_of_Ni_column"><span class="mw-headline" id="Regeneration_of_Ni_column">Regeneration of Ni column</span></span> ==

1. Wash Ni&nbsp;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.,&nbsp;''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

<br/>'''Ni Washing Buffer'''

20 mM Tris-HCl, pH 7.9~8.0

800 mM NaCl

10 mM imidazole&nbsp;



'''Tris-HCl Buffer&nbsp;'''

20 mM Tris-HCl, pH 7.9~8.0



'''Buffer B&nbsp;(Low Salt)'''

20 mM Tris-HCl, pH 7.9~8.0

'''100''' mM NaCl

150&nbsp;mM imidazole&nbsp;

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&nbsp;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&nbsp;



'''Buffer F'''

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

50 mM KCl.&nbsp;



'''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:Protein Expression]] [[Category:QC]] [[Category:My ELN]]