Editing Generic Protein Purification Protocol (section)

=== FPLC purification ===

Load to 10 ml Ni column using peristaltic pump, wash with 200 ml buffer A, then run FPLC program: <u>'''method 64'''</u>



for 2 L culture, a 5 ml HisTrap cartridge can be used. The cartridge should be equilibrated with at least 5 column volumes (25 ml) of buffer A.&nbsp; Load supernatant using peristaltic pump. Then wash the catridges with 100 ml Washing Buffer AKTA Prime FPLC program: <u>'''method 65'''</u> for step gradient & elution; or method <u>'''method 66''' </u>for step gradient wash and linear gradient elution<br/>

For 8 L culture, use 20 ml Ni column and method <u>'''method 63'''</u> (high expression)



19. Run SDS-PAGE to check purity, Optionally: Western blot.

20. Combine fractions containing desired protein. Concentrate using spin column.

21. If the target protein is fused to a tag, such as MBP, GST, TF, TRX, and has a 3C protease site,&nbsp; digest the protein with 3C protease&nbsp; (1: 50 to 1:100) o/n at 4 oC: Transfer protein to a float dializer, put the dializer in a beaker, add at least 20 X volume of dialysis buffer,&nbsp; dialysis o/n at 4 oC.

'''&nbsp;'''22. Next day, load digested protein solution to a 5 ml&nbsp; HiTrap Ni column, collect FT. Wash column with 2 CV of Dialysis Buffer or PBS buffer containing 5% glycerol, 1 mM beta-ME.

23. Concentrate protein using spin column(s).

24. Load protein solution&nbsp; to a Tosho gel filtration column, such as BioSil TSK250. Elute protein using 20 mM Tris-HCl, pH 8.0 or 7.5, 0.1 mM EDTA, 5 mM beta-ME, 150 mM NaCl.

25. Combine fractions containing desired protein.

Optional:&nbsp; Load to SP column (basic protein), or Q column (acidic protein). Elute with NaCl gradient.

26. Concentrate protein using Pierce spin concentators (10 KDa) or by dialysis vs 50% glycerol in a storage buffer.

27. Measure protein concentration using nanodrop.

28. Aliquot 1 ml/tube, freeze in dry ice and store them at -80 oC

<br/><u>'''Buffers'''</u>

For basic proteins (pI >8.0), use pH 7.4 buffers.

For acidic proteins, use pH 7.4 or 8.0 buffers.

'''Buffer A''': 10 mM imidazole, 20 mM Tris-HCl pH 8.0 (or 7.4), 500 mM NaCl, 10% Glycerol (optional), 0.05 mM EDTA, 1 mM beta-ME



'''Buffer B''': <u>'''500'''</u> mM imidazole, 20 mM Tris-HCl pH 8.0 (or 7.4), 300 mM NaCl, 0.05 mM EDTA, 10% Glycerol (optional), 1 mM beta-ME

'''Washing Buffer:''' 35 mM imidazole, 20 mM Tris-HCl pH 8.0 (or 7.4), 500 mM NaCl,&nbsp;0.05 mM EDTA, 0.051% Triton X-100, 1 mM beta-ME

'''Dialysis Buffer''' (example): 20 mM Tris-HCl pH 8.0 or 7.4, 150 mM NaCl, 10% Glycerol (optional), 0.05 mM EDTA, 1 mM beta-ME, some proteins will aggregate at low salt concentration, for these proteins >200 mM NaCl should be used.



For proteins with lot of cysteine: add beta-ME and 0.05 mM EDTA.

For unstable proteins, add 5~10% glycerol and/or 50 mM arginine.