Jan. 05. 2000

Purufication of aaRS complex from Bovine Liver

(Final Version)

 

 

1. Chop the 400g liver into 4-5 g pieces with clear knife(immediately after sacrifice)

2. Gentle grinding of fresh tissue 400g with homogenizer for 10 sec 2 times in 1L buffer A and Centrifuge at 26,000Xg for 45min è harvest supernatant

3. Ultracentrifuge at 100,000Xg for 90min to get the post ribosomal proteinè harvest sup

4. Slowly add 50% PEG stock sol (1ml/min) with gentle stirring to final 2% and contimuous stirring for 30min.

5. Centrifuge at 15,000Xg for 20min. Do NOT discard the pellet(resuspend the pellet in buffer A) and take sup.

6. Slowly add 50% PEG stock sol (1ml/min) with gentle stirring to final 8.5% and stirring for 30min

7. Centrifuge at 15,000Xg for 20min. take the pellet and sup. Resuspend the 2-8.5 % pellet in 500ml buffer A and centrifuge at 26,000Xg for 30 min to remove the un dissolved particle.

=è Check the IRS activity(crude, 2% PEG pellet, 8.5% pellet and sup)

8. You can see the IRS activity in the fraction between 2% and 8.5%.

9. Load the lysate to 40ml Heparin Column (0.4ml/min) equilibrated with Buffer C.

10. Wash the column until O.D run on the baseline with buffer C.

11. Make KCl gradient(35-400mM)with 10 bed volume and take 8ml per fraction.

12. Calculate the protein conc. and check the IRS activity.

13. collect fractions(30-44) showing the IRS activity and immediately dilute by slow addition, with stirring, of 2.5volumes of buffer E.

14. Diluted Solution was applied onto a 10ml yeast tRNA conjugated sepharose column equilibrated in buffer F with 0.8ml/min.

15. Wash the column until O.D run on the baseline with buffer F.

16. Make phosphate gradient(25-400mM) with 10 bed volume and take 4ml per fraction

17. repeat step 12, 13

18. Diluted Solution was applied to 5ml hydroxyapatite column equilibrated in buffer H with 0.25ml/min.

19. wash the column until O.D run on the baseline with buffer H.

20. Make phosphate gradient(100-400mM) with 10 bed volume and take 2.5ml per fraction.

21. repeat step 12 or check the complex through immunoblot.

22.Harvest the fraction showing the IRS activity and concentrate to 10mg/ml

23. run the sample to the superdex 200HR equilibrated in buffer A with 0.2ml/min and take 400ul per fraction.

 

***** Unless described, All procedures are performed at 4 degree.

 

 

 

 

 

 

 

 

 

 

 

aaRS Complex purification buffer

 

                                     Jan. 05. 2000      Sang Gyu Park

 

Buffer A(Homogenization Buffer)

 

50mM Tris-HCl, 100mM KCl, 5mM MgCl2, 0.2mM EDTA, 5mM DTT, 15% Glycerol

pH 7.5

 

Buffer B(50% PEG 6000 Stock Sol)

 

50mM Tris-HCl, 5mM MgCl2, 0.1mM EDTA, 1mM DTT, pH 7.5.

 

Buffer C(Heparin equilibration buffer)

 

50mM Tris-HCl, 35mM KCl, 10mM 2-mercaptoethanol, 5mM MgCl2,

0.1mM EDTA, 10% glycerol, pH 7.5

 

Buffer D(Heparin gradient buffer)

 

50mM Tris-HCl, 400mM KCl, 10mM 2-mercaptoethanol, 5mM MgCl2,

0.1mM EDTA, 10% glycerol, pH 7.5

 

Buffer E(2.5 X Dilution Buffer)

 

10mM KPO4, 10mM 2-marcaptoethanol, 0.1mM EDTA, 10% glycerol, pH 7.5

 

 

Buffer F(tRNA column equilibration buffer)

 

25mM KPO4, 10mM 2-mercaptoethanol, 0.1mM EDTA, 10% glycerol, pH 7.5

 

Buffer G(tRNA column gradient buffer)

 

400mM KPO4, 10mM 2-mercaptoethanol, 0.1mM EDTA, 10% glycerol, pH 7.5

 

 

Buffer H(Hydroxyapatite equilibration buffer)

 

100mM KPO4, 10mM 2-marcaptoethanol, 0.1mM EDTA, 10% glycerol, pH 7.5

 

#### Must be added ####

 

1mM PMSF(from 200mM Stock), 10mM b-glycerophosphate, 10ug/ml TPCK, 10ug/ml TLCK,

5ug/ml elastidinol, 10mM DIFP(DiIsopropyl Fluro-Phosphate), 1X protease inhibitor cocktail(from 1000X)

 

Protease Inhibitor cocktail(1000X)

5mg/ml Pepstatin A, 5mg/ml Leupeptin, 5mg/ml Antipain, 5mg/ml Chymostatin

=> must be melted at DMSO=>store at -20 degree

=> use 1X