| Sugden lab,McArdle Laboratory for Cancer Research, University of Wisconsin-Madison Medical School
http://mcardle.oncology.wisc.edu/sugden/Protocols/html files/Purification of 6xHis-tagged proteins by Ni-NTA-Agarose ver1 (6 x his tagged protein) ver.1.htm
Buffers:
Lysis Buffer (1 liter)
50 mM NaH2PO4 6.90g NaH2PO4.H2O ( MW 137.99g/mol )
300 mM NaCl( up to 2M) 17.54gNaCl(MW 58.44) or 60ml 5M
10 mM Imidazole ( up to 100mM) 0.68g ( MW 68.08 )
10 mM BME ( up to 20 mM) 0.69 ml stock ( 14.4M )
Adust pH to 8.0 using NaOH
Washing Buffer (1 liter)
50 mM NaH2PO4 6.90g NaH2PO4.H2O( MW 137.99 )
300 mM NaCl( up to 2M) 17.54g NaCl ( MW 58.44 ) or 60ml 5 M
20 mM Imidazole( up to 100mM) 1.36g Imidazole ( MW 68.08 )
10% Glycerol 100ml 100% stock
10 mM BME 0.69ml stock (14.4M )
Adjust pH to 8.0 by NaOH
Elution Buffer (1 liter)
50 mM NaH2PO4 6.90g NaH2PO4.H2O( MW 137.99 )
300 mM NaCl 17.54g NaCl ( MW 58.44 ) or 60ml 5 M
250 mM Imidazole 17.00g Imidazole ( MW 68.08 )
Adjust pH to 8.0 by NaOH
Preparing cleared lysates under Native Conditions
1.Thaw the cell pellet for 15’ on ice and resuspend the cells in lysis buffer at 2-5 ml per gram weight ( I use 10ml for 500ml culture)
2. Add lysozyme to 1mg/ml and 1xprotease inhibitors and incubate on ice for 30’
3.Sonicate on ice (use 6x10” bursts with a 10” cooling period)
Centrifuge lysate at 10,000xg for 20’ at 40C to pellet the cellular debris and save supernatant.
Add 5ul 2xSB to 5ul Supernatant and store at -200C for SDS-PAGE analysis
Batch purification under Native Conditions
Add 2.5 ml of the 50% Ni-NTA slurry to 10 ml cleared lysate and mix gently by shaking at 40C for 60’
Load the lysate-Ni-NTA mixture into a column with the bottom outlet capped ( 5ml per column)
Remove bottom cap and collect the column flow through
Wash twice with 4ml washing buffer every time for each column and Save Wash
Elute the protein 4 times with 0.5 ml elution buffer per column each time, collect in 4 tubes and analyze by SDS-PAGE ( run 5ul, 0.05%)
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