Editing Generic Protein Purification Protocol

1. Grow up cells in 100 ~150 ml of LB medium supplemented with proper antibiotics at 37 oC o/n.

2. Next morning, add 20 ml of culture to 1 L LB medium supplemented with proper antibiotics, grow at 37 oC.

For high-throughput protein purification: 2 L per protein.

3. Check OD600, add 0.1 to 1 mM (final concentration) of IPTG or 0.02% to 0.2% arabinose when OD600 ~=0.6 to 1.5 (about 2 to 3 hours after culturing in 1 L flask). Induce at 37 oC or 30 oC or 25 oC for 4 hours or 6 hours or overnight.

Usually these combinations:

37 oC for 2 hours

37 oC for 4 hours

30 oC for 3 ~ 4 hours

25 oC for 4 ~ 6 hours

30 oC o/n

25 oC overnight.



5. Spin down cells, 6000 rpm for 5 min.

6. Transfer to 50 ml tubes using dH2O, spin down cells.

7. Add ~30 ml (per 10 g wet pellet) lysis buffer (Buffer A containing 10% glycerol and 5 mM beta-ME). Freeze cells with dry ice.

8. Thaw cells in 25~37 <sup>o</sup>C water bucket.

9. Usually for BL21 cells with pLysS, pLysN, cells will be lysed upon one or 2 freeze/thaw cycle(s). If Top10 cell is used,&nbsp; 50 ul lysozyme may be required for cell lysis. After adding lysozyme, &nbsp; incubate at RT for 30 min to 2 hours or in the cold room overnight.

10. Use sonicator to lyse cells and break the sticky DNA. For lysed BL21 cells, homogenizer should be used to break sticky DNA.

<br/>11. Spin at 12000 RPM for 20 min.

12. Collect supernatant. Do not discard pellet. Wash pellet with 10 ml buffer A/g wet cells (add buffer, homogenize, spin at 12000 rpm, collect supernatant).

13. Spin supernatant for 20 min at 25000 RPM (ultracentrifuge), 4 oC.



=== Manual Purification ===

14. Load cleared supernatant to 10 ml Ni column, do not throw away flow through (FT).

15. Wash beads with Buffer A until no protein is detectable (using Braford reagent).

16. Wash beads with 100 ml Wash Buffer (Buffer A with 20 mM Imidazole).

'''if protein is not so pure (less than 80% purity) an additional wash step using &nbsp;100 ml Wash Buffer (Buffer A with 35 to 50 mM&nbsp;Imidazole) is required.'''

17. Elute protein with ~25 to 45 ml Buffer B (300 mM imidazole), collect '''2 ml''' fractions.

18. Measure protein concentration using nanodrop

<s>10 ul from each fraction and 75 ul Braford reagent.</s>



=== 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.



== Stripping and recharging ==

<br/>The column does not have to be stripped and recharged between each purification if the same protein is going to be purified; it is sufficient to strip and recharge it after 5–7 purifications, depending on the cell extract, extract volume, target protein, etc.

<br/>Recommended stripping buffer:20 mM sodium phosphate, 0.5 M NaCl, 50 mM EDTA, pH 7.4

<br/>Strip the column by washing with at least 5–10 column volumes of stripping buffer. Wash with 5 CV of 0.5 M NaCl and water, then at least 5–10 column volumes of binding buffer and 5–10 column volumes of distilled water before recharging the column.

<br/>Recharge the water-washed column by loading 2.5 ml of 0.1 M NiSO4 in distilled water on HisTrap HP 5 ml column. Salts of other metals, chlorides, or sulfates, may also be used (see “Optimization”).&nbsp; Wash with 5 column volumes distilled water, and 5 column volumes binding buffer (to adjust pH) before storage in 20% ethanol.



== '''Column Regeneration and Storage''' ==

Use a peristalic pump to pump&nbsp;50~100 ml of '''Column Regeneration Buffer''' to 10 ml column:

20 mM sodium phosphate, pH 7.4, 0.5 M NaCl, 50 mM EDTA.

Wash column with 20 ml water, then pump 5~10 ml 8M Urea, wash with 100 ml water.

Charge column with 5 ml 0.1M NiSO4.

Wash with 100 ml water.

Store column in water or 20% ethanol (long term storage)

Before use, equilibrate column with Buffer A.



The Ni2+-stripped column can be cleaned by the following methods;<br/>• Remove ionically bound proteins by washing the column with several column volumes of 1.5 M NaCl; then wash the column with approximately 10 column volumes of distilled water.

<br/>• Remove precipitated proteins, hydrophobically bound proteins, and lipoproteins by washing the column with 1 M NaOH, contact time usually 1–2 hours (12 hours or more for endotoxin removal). Then wash the column with approximately 10 column volumes of binding buffer, followed by 5–10 column volumes of distilled water.

<br/>• Remove hydrophobically bound proteins, lipoproteins, and lipids by washing the column with 5–10 column volumes 30% isopropanol for about 15–20 minutes. Then wash the column with approximately 10 column volumes of distilled water.

<br/>Alternatively, wash the column with 2 column volumes of detergent in a basic or acidic solution. Use, for example, 0.1–0.5% nonionic detergent in 0.1 M acetic acid, contact time 1–2 hours. After treatment, always remove residual detergent by washing with at least 5 column volumes of 70% ethanol.



Then wash the column with approximately 10 column volumes of distilled water.<br/>

For 5 ml HisTrap HP column, use half volumes.



== Trouble shooting ==

1. Protein is difficult to dissolve or precipitates during purification.<br/>

The following additives may be used: 2% Triton X-100, 2% Tween 20, 2% NP-40, 2% cholate, 1% CHAPS, 1.5 M NaCl, 50% glycerol, 20 mM ß-mercaptoethanol, 1–3 mM DTT or DTE (up to 5 mM is possible but depends on the sample and the sample volume), 5 mM TCEP, 10 mM reduced glutathione, 8 M urea, or 6 M Gua-HCl. Mix gently for 30 min to aid solubilization of the tagged protein (inclusion bodies may require longer mixing). Note that Triton X-100 and NP-40 (but not Tween) have a high absorbance at 280 nm. Furthermore, detergents cannot be easily removed by buffer exchange.<br/>

If protein has arelady precipitated onto the column, use 4–8 M urea or 4–6 M Gua-HCl to clean the column<br/>
[[Category:Protocols]]