Crystallization by dialysis is an underutilized method. This protocol presents a Novo approach to this, which will increase the range of crystallization strategies currently available for structured determination. The main advantages of this technique are that it is high throughput, simple to set up with no specialist equipment required, and allows easy monitoring of crystal growth.
The individual performing this technique must be comfortable with low-volume pipetting and using multichannel pipettes. The drops must be pipetted as sufficiently as possible to prevent partial dehydration. To set up the microdialysis experiment, place a commercially-available 96-well microdialysis plate in the protein side up orientation and remove the adhesive cover tape by peeling off the 200-micron pressure adhesive spacer.
After peeling off the adhesive cover tape, note the position of well A1.Next, using a multi-channel pipette, load a maximum of 3.2 microliters of properly prepared protein sample into each well. Properly position the 200-micron UV cover film over the 96-well plate with the film facing up. Then, to activate and seal the pressure adhesive, press down on the UV cover film using a sealing paddle and check its integrity.
Now, invert the plate to bring it to the buffer side up orientation and take note of the mirrored position of the marked well A1.Using a multi-channel pipette, load a maximum of 350 microliters of the dialysis solution into each well of the plate and carefully seal the wells with the reservoir cover film. Then, place the plate in the buffer side up orientation inside a suitable temperature-controlled incubator at 20 degrees Celsius to allow crystal growth. To examine crystal formation under the microscope, remove the protective cover film from the 200-micron UV cover film and place the plate below the eyepiece with the protein side facing upwards.
To set up a large scale crystallization experiment, adopt the conditions in which micro crystal growth occurred in the microdialysis plate and replicate the same conditions in large containers. Select the container size depending on the volume of the dialysis solution. Pipette a maximum volume of 500 microliters of protein sample into the dialyzer tube prior to sealing the tube using the red plastic cap.
Place the sealed 500-microliter dialyzer tube in the floating rack inside the container filled with dialysis solution. Next, place the container undisturbed inside a suitable temperature-controlled incubator at 20 degrees Celsius to achieve crystal growth. To check for the formation of crystals, pipette one to two microliters of the solution from the dialyzer onto a glass cover slide.
Cover the solution on the slide with another glass slide and view it under a microscope. Images captured using a stereo high-magnification microscope showed successful microcrystal formation of four proteins using the microdialysis plates with 10-kilodalton molecular weight cutoff membranes. Lysozyme was found to form crystals when dialyzed against 0.1 molar sodium acetate at pH 4 containing appropriate additives.
Domatin formed crystals when dialyzed against 0.1 molar bis-tris propane at pH 6.6 containing appropriate additives. Optimized dialysis conditions led to microcrystal formation by the membrane proteins, namely the E.coli multidrug efflux pump protein AcrB and the E.coli lactose transporter LacY. Large-scale crystallization in the dialyzer tubes employing the optimized dialysis conditions obtained from the microdialysis experiments led to the formation of thousands of microcrystals for each of the proteins.
For crystallization of fomitin, 250 microliters of protein was dialyzed against 50 milliliters of the dialysis solution. For AcrB, 250 microliters of the protein was dialyzed against 25 microliters of dialysis solution. The LacY crystallization was also set up at the same ratio, with 100 microliters of protein to 10 milliliters of dialysis solution.
When inverting the plate to the buffer side up, it is important to note the position of A1, so that solutions from the crystallization screen can be dispensed accordingly. The crystals obtained from this method can be used for downstream applications, such as serial crystallography and micro ED.
