[molecular biology work] ◈_gel_filtration_protocol-1

[Seung Jae Jeong]

◈ Gel filtration

 

Kim Jinyoung

 

1.     p43,p38,wild type의 MEF cell을 100π dish 20장에 culture(37℃ CO2 5%).

2.     TE처리후 cold 1XPBS로 spindown 하고, 다시 한번 cold 1XPBS로 washing한다.

3.     hypotonic solution(10mM HEPES pH7.6, 10mM KCL, 1.5mMMgCl2, 0.5mM EGTA, 10mM NaF, 1mM PMSF, Protease  inhibitor cocktail )과 syringe(needle size 23G)로 lysis 시킴.

4.     4℃, 14000 rpm에서 15min간 centrifuge.

5.     transfer sup. into new tube, 4℃, 14000 rpm에서 15min간 centrifuge

6.     sup과 동일 volume의 solution(10mM HEPES pH7.6, 150mM KCL, 1.5mMMgCl2, 0.5mM EGTA, 10mM NaF, 1mM PMSF, Protease  inhibitor cocktail )을 첨가해서 80mM KCl solution으로 만든다.

7.     8mg/ml로 vivaspin(VIVASCIENCE)을 이용하여 농축 시킨다(4℃, maximum 4000 g Tommy High speed). 0.22um filter로 filtering을 한 후 단백질 농도를 잰다.

8.     (10mM HEPES pH7.6, 80mM KCL, 1.5mMMgCl2, 0.5mM EGTA, 10mM NaF, 1mM PMSF, Protease  inhibitor cocktail ) buffer로 FPLC pump와 column(Pharmacia, Sephacryl S-300 High resolution, separate range 10Kda-1500KDa )을 equilibrium 시킨다.

 

l  FPLC를 사용하기 전에는 꼭 pump calibration과 pump washing을 꼭 해준다.

Pump calibration은 FPLC protocol 107page, pump washing은 97page에 잘 설명되어 있음.

–          protocol 마지막 부분 참고!!

 

 

9.     sample 500ul(4mg/500ul) injection 후 flow rate 1.0ml/min, fraction size 1ml로 gel filtration program을 4℃에서  돌린다.

–          Program

–          Set pressure limit : 0.35MPa

–          Set flow rate : 1.0 ml/min

–          Set fraction size : 1.0ml

–          Set equilibration volume : 1.0ml

–          Set sample injection volume : 0.5ml

–          Set elution volume : 180ml

–          Set wash volume : 0ml

10.  일부 fraction을 취해서 western blotting을 한다.

–         
70ul loading

8.12 Removing trapped air bubbles

Remove trapped air bubbles in the flow path by purging the pump with liquids. Use the liquids in the following order: 1. deionized water, 2. 20% ethanol, 3. deionized water and 4. buffer solution.

Note: All liquids used must be degassed.

Note: When using degassed ethanol, make sure that the concentration dose not fall below the required value.

Puring can be done manually through inlet A1, while carefully immersing the tubing in the respective liquid. Set the infection valve to position WASTE. Run 30 ml of each liquid at 50 ml/min. press the pause/cont button to start and stop the pump when changing liquid.

An automatic purging procedure that uses additional inlet tubings is decribed below:

1  Connect at inlet tybing to buffer valve port 4. Immerse the tubing in a vessel filled with deionized water.

2  Connect an inlet tubing to buffer valve port 3. Immerse the tubing in a vessel filled with 20% ethanol.

3  Connect an inlet tubing to buffer valve port 2. Immerse the tubing in a vessel filled with deionized water.

4  Connect an inlet tubing to buffer valve port 1 is immersed in a buffer solution.

5  Fill the empty inlet tubing manually.

6  In Templates, select the System Wash Method under Application template.

7  Select ports 2, 3 and 4 to be washed. Port 1 is pre-selected. De-select port B.

8  Press OK to start the run.

   The pump first draws the liquids in the following order: deionized water, ethanol, deionized water, and finally the buffer solution .
   Consequently, when the run is finished, the system is filled with the buffer solution connected to buffer valve port 1.

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