Additive | Concentration | Usage |
ethanol | 5-20% | solubilization (steroids) additive for crystallization |
methanol | 5-15% | solubilization (phospholipids combined with MPD) |
hexafluoropropanol | 1-5% | solubilization (very versatile, peptides and mimetics, steroids, etc.) |
2-propanol | 5-20% | solubilization(steroids), (cryoprotectant at >70% best in combination with others), additive in crystallization |
Glycerol | 15-45% | cryoprotectant and additive for crystallization |
DMSO | 2-20% | solubilization of ligands and cryoprotectant |
Ethylene Glycol | 15-45% | cryoprotectant |
PEG 200-600 | 35-50% | cryoprotectant, precipitant |
Sucrose | >50%w/v | cryoprotectant |
MPD | 0.5-50% | solubilization (phospholipids combined with methanol) Additive for crystallization, cryoprotectant(25-40%), precipitant |
1,6 Hexane diol | 0.5-10% | Used instead of MPD as a crystallization additive |
1,2,3 Heptane Triol | 0.5-15% | Membrane protein crystallization (Amphiphile) |
Benzamidine | 0.5-15% | Membrane protein crystallization (Amphiphile) |
Erythritol | >50%w/v | potential cryoprotectant |
Xylitol | 20-50% | cryoprotectant when combined with others / compatible with crystallization |
Inositol | 20-50% | cryoprotectant similar to xylitol |
Raffinose | >50%w/v | potential cryoprotectant |
Trehalose | 20-50% | cryoprotectant when combined with others |
Glucose | - | (usually together with other cryoprotectants) |
l-2,3-Butanediol | - | cryoprotectant (levo isomer should be best, but racemic mixture also works |
Propylene glycol | - | similar to ethylene glycol |
HES hydroxyethyl starch | - | used in cryobiology |
PVP polyvinyl pyrolidone | - | used in cryobiology, potential uses as precipitant |
Divalent metals are used for the crystallization of various enzymes, which require such metals for activity. It is important to chose a buffer that will not chelate the metals. Cacodylate, acetate are good candidates. Since metals like zinc, copper cobalt have a tendency to bind to non-specific sites, imidazole chelation may become useful. In the table below are suggested concentrations for use of these additives:
Additive | Concentration | Usage |
Calcium chloride or acetate | 1-20mM | Serine proteases, Avoid phospahte buffers |
Magnesium chloride or sulfate | 5-100mM | Can also be used as a precipitant. Avoid Phosphate in the buffer |
Zinc acetate or sulfate | 0.2-5mM | It inevitably reduces protein solubility. It can act as an inhibitor. Essential for activity of various enzymes |
Cadmium chloride | 0.02-0.2% | Check at concentrations (0.02,0.06,0.12,0.2%). It is very concentration dependent |
Barium chloride or acetate | 1-20mM | Same as calcium Heavy Atom |
Copper sulfate | 0.05-2mM | Similar to zinc |
Manganse chloride | 1-100mM | Calcium or magnesium substitute |
Cobalt chloride | 1-100mM | Some enzymes depend on it. |
Erbium or Samarium acetate | 0.1-10mM | use to replace other divalent metals. Heavy Atom |
Salts are used to precipitate as well as solubilize proteins. At low concentartions sodium chloride will tend to help increase solubilty while at higher concentration will tend to act as a co-precipitant
Additive | Concentration | Usage |
Sodium chloride | 50-500mM | Additive to PEG and MPD. May help solubility |
Sodium chloride | 0.15-2 M | Additive to PEG. Co-precipitant, used to speed up equilibration and gain better control on crystallization with PEG. |
Potassium chloride | 0.05-2 M | alternative to NaCl. NaCl is generally better |
Lithium chloride | 0.05-2 M | alternative to NaCl. The differences are worth checking out |
Sodium Fluoride | 20-300mM | Inhibitor. Use for Divalent metal binding proteins. |
Ammonium sulfate (A.S.) | 20-300mM | alternative to NaCl as an additive to PEG. Popular in Hampton screens |
Lithium sulfate | 0.05-2 M | alternative to A.S. |
Sodium or Ammonium Thiocyanate | 50-500mM | Additive to PEG and MPD and A.S. Acts as a co-precipitant. Of particular interest its use with basic proteins. See references: (1, 2, 3) |