Difference: MicroDialysisPlatesTool (10 vs. 11)

Micro-dialysis plates

Overview (Martin's archive - July 2010)

Design

An advantage of 2D crystallization relative to 3D crystallization is the use of relatively low protein concentrations, thus potentially minimizing the amount of proteins required for screening. Currently 2.5mg of protein is required to set up a 96-well crystallization trial using our crystallization block. Although this amount of protein is not prohibitive, the 50μl sample size could easily be reduced and still provide sufficient material for evaluation of the screen.

Our automated staining protocol requires 2μl of each dialyzed sample, but it is desirable to have additional sample available if backup grids are required for thorough evaluation of the outcome. Therefore, an amount of approximately 10μl per crystallization condition is necessary to meet all potential needs. By using 10μl of sample per condition instead of 50μl, the total amount of protein needed for a 96-well screen would be reduced to around 0.5mg.

We have collaborated with GN Biosystems Inc, (Hayward, CA) to develop an alternative minidialysis system for low-volume, high-throughput crystallization trials.

Microdialysis plate overview

The new minidialysis plate has, just as our crystallization block, the capacity to dialyze 96 different samples against the same number of unique buffer conditions. The volume loaded into each sample well is flexible, and ranges between 10-35μl. Each buffer chamber holds 500μl and thus, each buffer exchange has the potential to dilute the sample up to 50-fold.

The sample wells are loaded from the top and due to their open design, the contents remain accessible also when the plate is in use. This design is advantageous for screening 2D-crystal formation, since it allows for sampling of each condition throughout the dialysis regimen, and thus allows for monitoring 2D crystal formation in real time. To prevent loss of sample by evaporation, the entry to the wells is covered with an adhesive plastic film during dialysis.

Schematic top view

Initially, the sample wells were designed as small vertical cylinders with the dialysis membranes glued on the bottom of the tubes. However, since frequently 2D crystallization trials result in large particles (aggregates, proteoliposomes, and crystals) that may precipitate and restrict the flow through the dialysis membrane, a new L-shaped (or J-shaped) design for the well was developed. In the new design, the dialysis membrane is attached at the end of the L-shaped appendage, and any precipitate formed will instead accumulate in the bend of the well. The modified design also makes the device more robust, since the risk of puncturing the dialysis membrane due to improper handling during addition or removal of sample into/from the well is reduced.

Schematic cross-section view

Procedures for using XZ-96-D plates:

1. Pippette 0.5ml of reagent into the reagent wells.
2. Pippette 4~10ml of protein sample into the protein wells.
3. Centrifuge the plate at 1000rpm for 1min to get rid of any trapped bubbles.
4. Seal the plate.

Setup

A prototype 96-well microfluidic dialysis plate was obtained from GN Biosystems Inc. (Hayward, CA), which accommodates sample and buffer volumes of 4 and 160μl, respectively. To load each row of 12 wells, 48μl of protein sample was pipetted into the corresponding reservoir on the side of the tray. A rotary vacuum pump (RV3, BOC Edwards, Crawley UK) was employed to suck in (<2Torr) the protein solution across the plate and fill each of the 12 sample wells. The buffer chambers were filled individually with 160μl of buffer, and the plate was incubated at the desired dialysis temperature. Buffer was exchanged twice a day for a period of two weeks. Samples were recovered through slits at the bottom of each dialysis chamber after removing the sealing film. The rate of detergent removal obtained with the microfluidic dialysis plate was quantified essentially as described in Vink et al. (Vink et al., 2007). In brief, 5mg/ml of either OG or DDM were loaded into 24 sample wells and each well was dialyzed against 160μl of buffer (10mM Tris-Cl pH 7.5, 5mM NaN3^?). Buffer was replaced twice daily and at the end of the experiment, the pooled buffers were lyophilized and the detergent content quantified by colorimetric (Urbani and Warne, 2005).