This protocol has been modified and adapted in the Espenshade Lab, Johns Hopkins School of Medicine
<Buffers> add BME when your protein has cysteines
Denaturing/Lysis buffer (1 L)
100 mM NaH2PO4 [13.8 g NaH2PO4·H2O (MW 137.99 g/mol)]
10 mM Tris·HCl [1.2 g Tris base (MW 121.1 g/mol)]
8 M urea 480.5 g (MW 60.06 g/mol)
(optional) 5 mM BME
Adjust pH to 8.0 using NaOH
Denaturing Washing buffer (1 L)
100 mM NaH2PO4 [13.8 g NaH2PO4·H2O (MW 137.99 g/mol)]
10 mM Tris·HCl [1.2 g Tris base (MW 121.1 g/mol)]
8 M urea 480.5 g (MW 60.06 g/mol)
(optional) 5 mM BME
Adjust pH to 6.3 using HCl.
Denaturing Elution buffer I (1 L)
100 mM NaH2PO4 [13.8 g NaH2PO4·H2O (MW 137.99 g/mol)]
10 mM Tris·HCl [1.2 g Tris base (MW 121.1 g/mol)]
8 M urea 480.5 g (MW 60.06 g/mol)
(optional) 5 mM BME
Adjust pH to 5.9 using HCl.
Denaturing Elution buffer II (1 L)
100 mM NaH2PO4 [13.8 g NaH2PO4·H2O (MW 137.99 g/mol)]
10 mM Tris·HCl [1.2 g Tris base (MW 121.1 g/mol)]
8 M urea 480.5 g (MW 60.06 g/mol)
(optional) 5 mM BME
Adjust pH to 4.5 using HCl.
Note: Due to the dissociation of urea, the pH of Buffers B, C, D, and E should be adjusted immediately before use. Do not autoclave
1. Add 1 ml of the pre-washed Ni-NTA slurry to 20 ml of the Inclusion body solution (~2 mg/ml) and mix gently by rotating for 30 min at RT.
2. Load lysate-resin mixture carefully into an empty column and collect the flow through (20 μl) for SDS-PAGE analysis.
4. Wash once with 5 ml Denaturing/Lysis buffer. Keep wash fractions (20 μl) for SDS-PAGE analysis.
5. Wash twice with 5 ml Denaturing Wash buffer. Keep wash fractions (20 μl) for SDS-PAGE analysis.
6. Elute the recombinant protein 4 times with 1 ml Denaturing Elution buffer I, followed by 4 times with 1 ml Denaturing Elution buffer II. Collect fractions and analyze by SDS-PAGE. Monomers generally elute in Denaturing Elution buffer I, while multimers, aggregates, and proteins with two 6x His tags will generally elute in Denaturing Elution buffer II.