The Low Ionic Strength Screen is effective in determining the preliminary crystallization conditions of intact monoclonal antibodies.1 However, this screen is not just an intact antibody screen. The screen has effectively determined the preliminary crystallization conditions for numerous monoclonal antibody fragments as well as other soluble proteins.
The screen should be utilized as a low ionic strength crystallization screen for proteins where this strategy could be effective in determining preliminary crystallization screens.
In this screen, the concentration of a high purity, monodisperse PEG 3,350 is varied from 4 to 24% w/v (4, 8, 12, 16, 20 and 24% w/v) versus a pH range of 2 to 12 (2, 3, 3.5, 4, 4.5, 5, 5.5, 6.0, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12). Stock buffer concentrations are 50 mM. Final buffer concentration in the drop is typically 10 mM.
Unique features of this screen: (1) Low ionic strength. Buffer concentration is supplied as 50 mM, resulting in an initial drop concentration of 10 mM (Drop = 4 µl sample, 2 µl buffer, 5 µl precipitant). (2) The Polyethylene glycol (PEG) 3,350 is a special, high-purity, monodisperse preparation with an Mr of 3,300-3,400. Most PEGs of this molecular weight have an Mr of plus or minus 500 rather than 50. (3) An extremely broad range of pH 2 to 12 is sampled. At low ionic strength, the effects of pH and temperature upon sample solubility are amplified. Hence this screen allows one to critically evaluate the effects of temperature and pH upon sample solubility and crystallization. It is recommended the screen be repeated at several temperatures between 4°C and 37°C to take advantage of the low ionic strength feature.
The format of this screen is different than other screens offered by Hampton Research. The screen is supplied as a set of eighteen 50 mM buffers, six PEG 3,350 concentrations, and a single dehydrant, PEG 3,350. This formulation allows one to screen up to 108 conditions if desired. The formulation is designed to offer options. Coarse sample of pH and/or precipitant concentration can be performed with 24 or even 12 drops. This will eliminate large regions of sampling space and conserve sample. Subsequent screens and perhaps even optimization can encompass an increasingly finer grid matrix of pH, precipitant concentration as well as other variables significant for sample crystallization. The protocol requires the following pipetting steps for a typical vapor diffusion experiment: (1) Pipet dehydrant to reservoir. (2) Pipet drop. (3) Pipet buffer to drop. (4) Pipet precipitant to drop.
Each kit contains 24 unique reagents of varying pH and PEG 3,350 concentration. Buffers of 50 mM are supplied for pH 2, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, and 12 in 1 ml volumes. The precipitant Polyethylene glycol 3350 is supplied as 4, 8, 12, 16, 20, and 24% w/v in 1 ml volumes. Dehydrant is available separately as 200 ml of 24% w/v PEG 3,350 (catalog number HR2-519). Combining each pH and precipitant supplied can generate 108 crystallization screening conditions. All solutions are formulated using ultra-pure water and are sterile filtered.
Note: Crystallization kit (HR2-120) and Dehydrant/reservoir solution (HR2-519) are sold separately. Both items are needed to perform the screen.
1. Harris, et al., Crystallization of intact monoclonal antibodies, Proteins: Structure, Function, and Genetics (1995) 23:285-289.