Organic Solvents, Additives and Cryoprotectants.
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:

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)

Additives for Crystallization from Hampton Research:

Additive Screen 1

Additive Screen 2

Updated/Created on Sep-23-97 by EAS, TSRI