Additives


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:

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