Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

The overall goal of the following experiment is to visualize the planes of ice bound by fluorescently labeled antifreeze proteins. This is achieved by growing single ice crystals to which the antifreeze proteins can bind as a second step. Each crystal is viewed through crossed polarizers to establish its singularity and orientation.

Next, an ice crystal is mounted onto a temperature controlled cold finger and formed into a hemisphere. It is then submerged into a solution of fluorescently labeled antifreeze proteins in order to absorb the antifreeze protein onto specific binding planes of the growing hemispherical. Ice results are obtained by visualizing and imaging ice bound by fluorescently labeled antifreeze protein, using wavelength specific light and filters in a darkened cold room.

The main advantage of this technique over existing methods like ice etching, is that the antifreeze proteins are fluorescently labeled To allow immediate visualization of the protein bound ice planes Start crystal growing by preparing a temperature controlled ethylene glycol bath at minus 0.5 degrees celsius and finding a clean metal pan that fits into and can float on it. The crystals are formed in molds. Use cylindrical molds three to four centimeters high cut from a polyvinyl chloride pipe.

Each mold should have a one millimeter wide, two millimeter high notch on one side. Apply a light film of vacuum grease to the side of the ring from which the notch was cut. Seal this greased notched surface onto the metal pan with the notch oriented away from the center of the pan.

Be sure not to fill or obstruct the notches with grease. Prepare as many molds as can fit into the pan. Next, add filtered Degas and deionized water to the center of the pan outside of the molds.

Be careful not to introduce any bubbles. As the water layer is raised to five millimeters, the water should slowly enter the molds through the notches. When done, placed the pan perfectly level into the ethylene glycol bath.

After the pan and water have reached minus 0.5 degrees Celsius, add a small piece of ice to the middle of the pan outside of the molds. Incubate overnight to form a layer of ice over the next three days. Return to the bath to add water to the molds.

Deposit 13 milliliters of four degrees Celsius, Degas and deionized water to each mold. Once a day after adding the water, reduce the temperature of the ethylene glycol bath. By day four, the molds should be completely filled with ice.

Retrieve the molds and prepare a clean surface for the ice. Pull each mold off the pan and push the ice crystal out. Place the clean surface with the molds in a minus 20 degrees Celsius freezer for one hour.

Before handling. When the ice is ready, take it to a freezer room or cold room to determine if it is a single crystal. Place the ice between two crossed polarizers.

If the ice is a single crystal, no cracks or discontinuities should be seen and the light direction should not change within the crystal. With the polarizer still in place, determine the orientation of the C axis by observing the light transmitted when the ice is rotated. To determine the orientation of the A AEs, wrap the ice tightly in aluminum foil.

Orient a needle normal to the sea axis and poke a small hole through the foil and into the ice. Next, place the ice in a 0.5 millibar vacuum for 20 minutes. Once the crystal is retrieved, uncover it in the cold room.

Observe the hexagonal etch on the basal plane. The A AEs run through the vertices of the six pointed star. This crystal will be cut in half along a line parallel to opposite points on the star.

To expose a primary prism plane, hold the crystal securely on a bench. Use a hacksaw to cut the crystal in half. Collect the pieces for mounting on a cold finger.

The ice crystal must be further prepared for mounting on a cold finger, find two aluminum rods with slightly different diameters, but comparable in size to the cold finger. Alternate using the rods to melt a cavity into the top of the crystal stop when there is a cavity into which the cold finger can fit. Cool the cold finger to minus 0.5 degrees celsius and place it in the ice cavity.

Hold the crystal in place until it freezes to the metal. Next, fill a hemispherical cup with approximately twice the diameter of the ice crystal with filtered deionized water cooled to four degrees Celsius. Submerged the cold finger bound ice crystal in the cup, remove excess water so the top of the ice crystal is approximately level with the liquid and the ice is not touching.

The cup walls cover the cup with insulation and lower the temperature to minus five degrees Celsius. Allow the ice to form a hemisphere over about an hour. Prepare to add the fluorescent protein.

When the hemisphere is ready. Remove the ice crystal from the cup, remove water from the cup. Then add 25 to 30 milliliters of pre cooled fluorescently labeled protein solution at the desired concentration.

While keeping the total liquid volume unchanged res, submerge the ice crystal into the cup.Again. Make sure the top of the crystal is level with the liquid and is not touching the cup walls. Drop the cold finger temperature to minus eight degrees Celsius and let the protein solution freeze into the crystal for two to three hours.

Stop the ice growth when at least five millimeters of ice has formed from the protein solution. With the cold finger still attached, remove the ice crystal from the cup. Detach the cold finger by warming the coolant to just above zero degrees Celsius.

Wait until the ice crystal melts off. When the crystal melts off the cold finger, place it flat side down onto a clean surface. Be careful not to touch the newly formed ice.

Store the crystal at minus 20 degrees Celsius for at least 20 minutes Before proceeding to visualize the fluorescence work in a cold room that can be darkened. Prepare lamps with wavelength specific excitation filters to excite the fluorescent label and camera emission filters. To block out non-specific light, place the ice flat side down under the lamps, darken the room and observe the patterns in the illuminated ice.

To estimate the ice planes that are bound by the antifreeze proteins, compare a traditional ice etch image with that of the corresponding fluorescence based ice plane Affinity analysis using trixy both show type one antifreeze proteins produced by the winter flounder pseudo pectus Americana. The technique can be used to simultaneously compare ice binding patterns of different antifreeze proteins. In this case, Pacific Blue labeled type three NFEA FP eight and trixy labeled type one antifreeze protein.

This is the result of visualizing only type three N-F-E-A-F-P eight here only the type one antifreeze protein is visualized in this image. The two are visualized together. Note the C axis is the same for each image.

After watching this video, you should have a good understanding of how to grow and orient single ice crystals and use the fluorescence based ice plain affinity analysis to assess ice plain binding patterns of fluorescently labeled antifreeze proteins.