Paul.xu at 20:10, 14 January 2013

2013-01-14T20:10:20Z

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'''Chemicals''' 

Bromoacetic acid, Ne-benzyloxycarbonyl-L-lysine, palladium on charcoal, epibromohydrin and Sepharose CL-6B can be obtained from Sigma. 

'''Method''' 

1. Dissolve '''bromoacetic acid '''(41.7 g) in 150 ml 2 N NaOH and cool to 0 ºC.

 

2. Prepare a solution of (CBZ-epsilon-lysine) '''N-benzyloxycarbonyl-L-lysine '''(42 g) in 2 M NaOH.

3. Add the protected amino acid solution to the bromoacetic solution slowly dropwise at 0 ºC with stirring.

4. After 2 hours remove the cooling and continue stirring the mixture overnight.

5. Heat the mixture to 50 ºC for 2 hours.

6. Add 450 ml 1 M HCl and cool the mixture in ice.

7. Filter off the crystals produced, dissolve them in a minimum volume of 1 M NaOH and reprecipitate with 1 M HCl.

8. Filter off the crystals and allow to dry (NTA). 

9. Dissolve a 7.9 g portion of the above product (NTA) in 49 ml 1M NaOH , add a small portion of '''5%Pd''' on charcoal and hydrogenate at room temperature and pressure.

10. When the hydrogenation has gone to completion filter off the catalyst and concentrate the filtrate by rotary evaporation. (drying is not necessary) 

11. Wash a 100 ml volume of '''Sepharose CL-6B''' twice on a glass suction filter with 500 ml water.

12. To activate the Sepharose heat it to 30 ºC for 4 hours with 16 ml 4 M NaOH and 8.22 ml '''epibromohydrin.'''

13. Filter off the Sepharose and wash to neutrality with water.

14. Dissolve 6 g of the hydrogenated reaction product in 50 ml water and add to the activated Sepharose together with 10.6 g solid sodium carbonate. Stir the reaction mixture slowly at 60 ºC overnight.

15. Filter off the Sepharose and wash with water.

16. To charge the resin with nickel wash through slowly with 100 ml aqueous NiSO4, 200 ml water, 200 ml 0.2 M acetic acid containing 0.2 M sodium chloride and 0.1% Tween-20 and finally 200 ml water.

The resin should bind up to 10 mg/ml of tagged protein.

 

NTA can be coupled to sepharose using epoxy activation with 1,4-butanediol diglycidyl ether according to Porath and Axen, Meth. Enz. 44: 19-45. 

 

'''Preparation of Epoxy Sepharose''' EPS was prepared by a standard procedure (Axen et al., 1967).

Wash 40 g of Sepharose 4B well with distilled water

Suspend in 2 Μ sodium hydroxide (40 ml).

Add Butanedioldiglycidyl ether (10 ml)  to the suspendsion.

Stir overnight at room temperature.

Filter the suspension on a sintered funnel.

Wash extensively with water until the filtrate is neutral (2 litres of water), then with acetone (200 ml) and finally with 500 ml of water.

Store the gel as aqueous suspension at 4°C for further use. 

'''Ref''' 

Hochuli E, Dobeli H and Schacher A, 1987, J Chromatography, 411, 177-184.

 

 

[[Category:Protocols]]

Ni-NTA Beads Preparation - Revision history

Paul.xu at 20:10, 14 January 2013