The overall goal of this protocol is to identify novel ice-binding proteins or IBPs from plants by testing for ice-binding activity and isolating native peptides using ice-affinity purification. This method can help answer key questions in the field of plant freeze tolerance, specifically ice-binding proteins. The main advantage of this technique is that it allows you isolate ice-binding proteins from native extracts without the use of complicated purification methods.
Visual demonstration of this method is critical, since the apparatus set-up can be difficult to conceptualize. Begin this protocol with splat apparatus setup as described in the text protocol. To induce the expression of ice-binding proteins, or IBPs, in freeze-tolerant grasses or other freeze-tolerant plant species, cold-acclimate plants for up to one week at four degrees Celsius under low light conditions.
Obtain approximately 0.1 gram of leaf tissue by cutting at the base of the stem and then placing in a 1.5 milliliter tube. Immediately flash-freeze the sample in liquid nitrogen and store at minus 80 degrees Celsius until use. To lyse the cells, grind the tissue sample into a fine powder using a mortar and pestle under liquid nitrogen.
Ensure that the liquid nitrogen does not evaporate during the homogenization process. Immediately place the ground samples on ice and allow the liquid nitrogen to completely evaporate. Then add one milliliter of a native protein extraction buffer and pipette to mix.
Gently shake the samples overnight at four degrees Celsius and conduct the remaining steps at the same temperature. The next day, remove cellular debris by centrifuging the samples at approximately 1, 600 times G for five minutes. Retain the soluble fraction and centrifuge for an additional five minutes if cellular debris persists.
Clarified samples can be stored at minus 20 degrees Celsius until use. Pour 100 milliliters of hexane into the external chamber set up with the polarized film and add five to six desiccation beads to prevent moisture buildup on the slides. Then add a small amount of vacuum grease to the plate covering the bath and twist to ensure a tight seal and prevent evaporation of the hexane.
40 minutes prior to starting the experiment, place the cooling block in a polystyrene box directly below the plastic tube and cover it completely with dry ice. Before beginning the assay, ensure that the tube is level. Then remove the dry ice from the top of the cold block.
First, test this procedure with water to determine where on the cooling block the sample will drop. Dip the disposable tip affixed to an automatic pipette in immersion oil, which will allow the sample to fall rather than adhere to the outside of the pipette. Use a paper towel to remove any excess oil.
Then aspirate 10 microliters of water. Place the pipette directly over the plastic tubing before releasing the water. A distinct splat sound should be heard when the water hits the cooling block, creating a monolayer of ice crystals.
Next, place a glass microscope cover slide on the drop mark that has been determined with water. Immediately before starting the assay, turn on the light source and scrape any ice that has formed on the bottom of the glass chamber. Then perform the splat assay with the sample, as done for the water, releasing 10 microliters of the sample onto the glass cover slide to create a monolayer of ice crystals.
Quickly and carefully remove the microscope slide from the cold block using tweezers and place in the hexane bath on top of the polarizing film. Looking under the microscope, put the microscope slide into the field of view and adjust the objective lens so that the cross-polarization allows for the visualization of high-contrast ice crystals. Attach the camera to the microscope and adjust the settings to macro to allow clear visualization of ice crystals and capture the image.
Repeat these steps for all samples. Typically place up to six slides with a different sample in the hexane chamber. Once finished, turn off the light source.
Then place a plastic plate over the hexane bath, ensuring a tight seal, and allow ice crystals to anneal overnight. Following incubation, capture images of the ice films as before. After cold-acclimating the plant tissue as before, collect approximately 100 to 150 grams of the ground biomass in 20-milliliter tubes.
Immediately flash-freeze the samples, and keep them at minus 80 degrees Celsius before use. Prepare the sample as before, using a one-to-one ratio for re-suspension of the leaf tissue. Use additional protease inhibitor tablets to avoid the degradation of IBPs by endogenous proteases.
To remove cellular debris, sift the lysate through two layers of cheesecloth three times. Pellet the debris by centrifugation at 30, 000 times G for 40 minutes at four degrees Celsius. Then increase the volume of sample to 120 milliliters using native protein extraction buffer and use the lysade immediately for ice-affinity purification to avoid any loss of ice-binding activity.
After cooling the ice finger to minus 0.5 degrees Celsius, subsequently submerse it into a 50-milliliter tube containing cold distilled water. Add a few ice chips to facilitate ice nucleation and allow a thin layer of ice to form on the finger. This process typically takes between 20 and 30 minutes.
Place the sample in a 150-milliliter glass beaker containing a small magnetic stir bar, which is placed in a polystyrene container on top of the magnetic stirrer. Make sure that the ice finger is centered and lowered at least halfway into the liquid sample. Then seal the container.
Allow the program to run for approximately two days, checking every 24 hours. Once 50%of the sample is frozen, stop the program. Incorporation of IBPs into the ice hemisphere will result in ice etching.
To remove the ice hemisphere, warm the probe to four degrees Celsius. Then rinse the hemisphere with distilled water to remove unbound protein and thaw at four degrees Celsius. If the ice hemisphere still contains pigment, or to increase the sample purification, repeat the purification steps two to three times.
Shown here are extracts collected from a buffer control lacking IBPs and extracts from wild-type Arabidopsis thaliana that do not contain IBPs. After 18 hours, the buffer control and A.thaliana extracts are unable to restrict ice crystal growth. In contrast, the small ice crystals seen with extracts of transgenic A.thaliana expressing IBPs from cold-acclimated Lolium perenne restrict ice crystal growth substantially.
After the first round of purification, the high degree of ice etching on the hemisphere surface indicates that IBPs have adsorped to the ice and modified growth of the ice crystals. To remove additional solutes, pigments, and contaminating proteins, a second round of ice-affinity was used, resulting in a clearer ice hemisphere. Following this procedure, ice shaping and thermal hysteresis can be evaluated using a nanoliter osmometer in order to further characterize ice-binding proteins.
Additionally, isolated fractions can be sent for mass spectometry in order to identify the peptide sequences.
