Protein PurificationExtraction and Clarification
Choice of lysis buffer and additives
The first choice we have to make is that of the nature and the pH of the buffer system we want to use. This depends on:
- the stability of the target protein with respect to pH and the bufferring compound.
- the purification procedure. To avoid time and protein loss caused by an additional buffer exchange step, it is advisable to choose a buffer that is compatible with the first chromatography step (see chromatography).
Buffers and their pH ranges are listed in the table below. The most used buffers are indicated in red. They are normally used at concentrations of 20-50 mM.
|Citric acid - NaOH||2.2 - 6.5|
|Sodium citrate - citric acid||3.0 - 6.2|
|Sodium acetate - acetic acid||3.6 - 5.6|
|Cacodylic acid sodium salt - HCl||5.0 - 7.4|
|MES - NaOH||5.6 - 6.8|
|Sodium dihydrogen phosphate - disodium hydrogen phosphate||5.8 - 8.0|
|Imidazole - HCl||6.2 - 7.8|
|MOPS - KOH||6.6 - 7.8|
|Triethanolamine hydrochloride - NaOH||6.8 - 8.8|
|Tris - HCl||7.0 - 9.0|
|HEPES - NaOH||7.2 - 8.2|
|Tricine - NaOH||7.6 - 8.6|
|Sodium tetraborate - boric acid||7.6 - 9.2|
|Bicine - NaOH||7.7 - 8.9|
|Glycine - NaOH||8.6 - 10.6|
- Most buffers show a pH-dependence with temperature. This is especially true for Tris buffers. The pKa changes from 8.06 at 25°C to 8.85 at 0°C.
- HEPES interferes with the Lowry protein assay (not the Bradford assay). It can form radicals under various conditions and should not be used in systems where radicals are being studied.
- Tris possesses a potentially reactive amine and participates in various enzymatic reactions. The pH of a Tris buffer is affected by the temperature (see above) and the concentration. The pH decreases 0.1 unit upon a tenfold dilution.
- Phosphates buffers are incompatible with the use of divalent cations (e.g. Mg2+ ions).
Depending on the target protein, it may be necessary to add compounds to the lysis buffer:
- to improve the stability of the target protein.
- to keep the protein in solution.
The most used additives, their effective concentrations, and their general purpose are listed in the table below.
Only use additives when it is really necessary!
|Class of additive||example||concentration||purpose|
|Salts||NaCl, KCl, (NH4)2SO4||50-150 mM||maintain ionic strength of medium|
|0.1-1%||solubilization of poorly soluble proteins|
|Glucose or sucrose||25 mM||Stabilize lysosymal membranes, reduce protease release|
|Metal chelators||EDTA, EGTA||1 mM||reduce oxidation damage, chelate metal ions|
|Reducing agents||DTT, DTE
|reduce oxidation damage|
|Ligands, metal ions||Mg2+, ATP, GTP||1-10 mM||stabilization|
An important class of additives are the protease inhibitors. In general cell disruption leads to the release of proteolytic enzymes which could lower the overall yield. To control this undesirable proteolysis it may be necessary to add a cocktail of protease inhibitors to the cell suspension. Since many of these compounds are not very stable in aqueous solutions it is important that they are added to the lysis buffer from a stock solution in an organic solvent (methanol, ethanol, isopropanol, or DMSO) immediately before use.
|Protease inhibitor||concentration||stock conc.||solvent||inhibition of|
|Aprotinin||1-2 µg/ml||10 mg/ml||water||serine proteases|
|Benzamidine||15 µg/ml||10 mg/ml||water||serine proteases|
|EDTA, EGTA||1-10 mM||0.5 M (pH 8)||water||metallo proteases|
|Leupeptin||1-2 µg/ml||10 mg/ml||water||cysteine and serine proteases|
|PMSF||0.1-1.0 mM||100 mM||isopropanol||serine proteases|
|Pepstatin A||1µg/ml||1 mg/ml||methanol||aspartic proteases|
- PMSF (phenylmethylsulfonyl fluoride) has a short half-life time in aqueous solutions. A stock solution of 100 mM in isopropanol should be made and diluted into buffer immediately before use. Pefablock is a less toxic and more soluble and stable alternative for PMSF.
- The use of EDTA and EGTA is not compatible with the presence of Mg2+ ions (required for nucleotide binding) in the buffer or with affinity chromatography on Ni2+-columns.
Although it is impossible to give a general lysis buffer, a good starting buffer would be:
|50 mM Tris-HCl pH 7.5|
|100 mM NaCl|
|1 mM DTT (for intracellular proteins)|
|5% glycerol (possibly)|
The addition of protease inhibitors depends strongly on the host used and the suggested compounds are listed in the table below. If results are not satisfactory changes or further addition to the lysis buffer should be made.
|E. coli||Yeast||Insect cells|
|1 mM EDTA||1 mM EDTA||1 mM EDTA|
|1 mM PMSF||1 mM PMSF||1 mM PMSF|
|1 µg/ml Leupeptin||1 µg/ml Leupeptin|
|1 µg/ml Pepstatin A||1 µg/ml Pepstatin A|