[molecular biology work] Bio Rad IEF cell protocol

[Seung Jae Jeong]

Bio Rad IEF cell(Model #PROTEAN IEF Cell) protocol

                                                                                                                                                         Arthur

 

 

 

1. spin down the cell at 1,500Xg for 5 min

2. wash the cell with ice cold PBS and lyse the cells with lysis buffer(1X PBS containing 1% triton X-100, 1mM DTT, 1mM PMSF, 5ug/ml aprotinin)

3. centrifuge at 26,000 Xg for 15min at 4 degree

4. take supernatant and quantify the protein concentration

5. precipitate the whole protein with 10% TCA with slow addition.

6. centrifuge at 10,000 Xg for 10 min at 4 degree and take the pellet

7. resuspend the pellet with rehydration buffer(9M urea, 1% CHAPS, 10mM DTT, 0.2% Bio-Lytes, 0.001% bromophenol Blue)

8. load protein lysates 500ug to the gel-strip and cover with mineral oil to inhibit evaporation.

9. perform the rehydration for 12 hrs with 50 voltage

10. after rehydration, run with 250V for 15 min to remove salt ions and charged contaminants

11. Focus the strip with 4,000V for 48hrs

12. After focusing, remove the strip from the focusing tray.

13. equilibrate the strips with equilibration buffer for 2^nd dimension gel electrophoresis(6M urea, 2% SDS,         0.4M Tris-HCl(pH 8.8), 20% glycerol, 130mM DTT) for 15 min

14. equilibrate the strips again with equilibration buffer II(6M urea, 2% SDS, 0.4m Tris-HCl(pH 8.8), 20% glycerol, 135mM iodoacetamide) for 15 min

15. place the strips on the second dimension gel and run.

16. you can choose -commassie or silver staining, – western blot

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