This work was funded by an NSERC (Canada) grant to VKW.

1. Splat Apparatus Setup
<ol>
	<li>Fill a temperature-programmable circulating water bath with ethylene glycol (50% v/v in water). 
	NOTE: Green automotive ethylene glycol can be used.</li>
	<li>To assemble the external chamber, use insulated plastic tubing to attach the water bath to a double-walled glass bowl. Insulate the glass bowl with polystyrene foam and cover with a plastic petri dish lid. Cut a 3-4 inch hole in the bottom of the polystyrene chamber so that the light source can be seen through the glass chamber.</li>
	<li>Mount the external chamber on a dissection microscope, fitted with a camera port and a 4X polarized objective lens with a 10x ocular lens. Place an additional piece of polarized film in the bottom of the external chamber.</li>
	<li>Clamp a 1-1.5 m tube (~5 cm diameter) onto a retort stand and place inside a large polystyrene container, along with a cooling block positioned underneath the tube at the base of the retort stand.</li>
</ol>
2. Preparation of Native Protein Extracts for IRI Analysis
<ol>
	<li>To induce the expression of IBPs in freeze-tolerant grasses or other freeze-tolerant plant species, cold acclimate plants for up to 1 week at 4 &#176;C, under low light conditions (6 h light/ 18 h dark). This does not apply to samples collected from the field that have already been exposed to low temperature conditions and acclimation periods.</li>
	<li>Obtain approximately 0.1 g of leaf tissue by cutting at the base of the stem and then place in a 1.5 mL tube. Immediately flash freeze the sample in liquid nitrogen and store at -80 &#176;C until use.</li>
	<li>To lyse cells, grind 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.</li>
	<li>Immediately place ground samples on ice and allow the liquid nitrogen to completely evaporate. Add 1 mL of a native protein extraction buffer containing 10 mM Tris-HCl, 25 mM NaCl (pH 7.5), with two EDTA-free protease inhibitor tablets added immediately before use. Pipette to mix; do not vortex. 
	NOTE: Additional additives may need to be used if the lysate contains secondary metabolites (see Discussion).</li>
	<li>Gently shake samples overnight (18-24 h) at 4 &#176;C. Conduct the remaining steps at 4 &#176;C.</li>
	<li>Remove cellular debris by centrifuging samples at ~16,000 x g for 5 min. Retain the soluble fraction and centrifuge for an additional 5 min if cellular debris persists. Clarified samples can be stored at -20 &#176;C until use.</li>
</ol>
3. Splat Assay Pre-experiment Set up
<ol>
	<li>Pour 100 mL of hexane into external chamber set up with the polarized film and add 5-6 desiccation beads to prevent moisture build up on the slides. 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.</li>
	<li>Cool the hexane bath between -4 &#176;C and -6 &#176;C using the programmable water bath approximately 2-3 h before starting the experiment. Make sure to check the temperature of the hexane with a thermometer prior to beginning the experiment.</li>
	<li>Place the cooling block in a polystyrene box directly below the plastic tube and cover completely with dry ice, 40 min prior to starting the experiment.</li>
	<li>Before beginning the assay, ensure that the tube is level. To determine where on the cooling block the sample will drop, first test the procedure with water and indicate where the sample drops with a marker (as described below).</li>
	<li>Remove dry ice from the top of the cold block and place a glass microscope cover slide on the drop mark that has been determined with water.</li>
	<li>Immediately before starting assay, turn on the light source and scrape any ice that has formed on the bottom of the glass chamber. Avoid keeping the light source on unless ice crystals are being visualized to prevent heating the hexane.</li>
</ol>
4. Conducting the Splat Assay
<ol>
	<li>Dip the disposable tip affixed to an automatic pipette (1-20 &#181;L) in immersion oil, using a paper towel remove any excess oil. This step will allow the sample to fall rather than adhere to the outside of the pipette. 
	NOTE: Removing excess oil from the pipette is critical to ensure that oil droplets do not form on the ice crystals, making visualization difficult.</li>
	<li>Pipette 10 &#181;L of sample and place pipette directly over plastic tubing before releasing the sample. A distinct &#39;splat&#39; sound should be heard when the sample hits the glass cover slide, creating a monolayer of ice crystals.</li>
	<li>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.</li>
	<li>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. Adjust magnification to 40x.</li>
	<li>Attach the camera to the microscope and adjust the settings to &#34;macro&#34; to allow clear visualization of ice crystals and capture the image. 
	NOTE: Waiting up to 1 h to allow ice crystals to grow can give a clearer picture.</li>
	<li>Repeat steps 4.1-4.5 for all samples. Typically, place up to 6 slides with a different sample in the hexane chamber.</li>
	<li>Turn off the light source and place a plastic plate over the hexane bath, ensuring a tight seal. Allow ice crystals to anneal overnight (12-24 h, keeping a consistent annealing period within a particular assay). Following incubation, capture images of the ice films as described above. 
	NOTE: Some samples may recrystallize more quickly than others. If no recrystallization is apparent after 12 h, allow crystals to anneal for up to 24 h to ensure that no recrystallization will occur.</li>
</ol>
5. Splat Assay Data Analysis
<ol>
	<li>Upload the photos to a computer and directly compare the size of ice crystals before and after annealing. Samples with ice recrystallization activity will not grow, whereas samples without no IRI activity will recrystallize forming noticeably larger ice crystals. Light interference will make large ice crystals appear in slightly different hues and thus are easy to see.</li>
</ol>
6. Ice-affinity Purification Equipment Setup
<ol>
	<li>Fill a temperature-programmable circulating water bath with ethylene glycol. Green automotive ethylene glycol can be used.</li>
	<li>Connect the bath to a hollow, brass probe using insulated plastic tubing15.</li>
	<li>Construct a polystyrene container into which a 150 mL beaker can fit snuggly. Create a lid for the container with a hole in the center for the brass cold finger.</li>
</ol>
7. Preparation of Samples for Ice-affinity Purification
<ol>
	<li>Cold acclimate plant tissue as described in section 2.1.</li>
	<li>Collect ~100-150 g of the ground biomass (step 2.3) in 20 mL tubes and immediately flash freeze. Samples can be kept at -80 &#176;C before use.</li>
	<li>Prepare sample as described in sections 2.3-2.5. Use a 1:1 ratio (mg of tissue: mL of native protein extraction buffer) for resuspension of leaf tissue. Use additional protease inhibitor tablets to avoid the degradation of IBPs by endogenous proteases. 
	NOTE: If the tissue sample is too concentrated, adjust the ratio to 1:2 or 1:3 (mg of tissue: mL of native protein extraction buffer).</li>
	<li>To remove cellular debris, sieve lysate through 2 layers of cheesecloth, 3 times. Pellet the debris by centrifugation at 30,000 x g for 40 min at 4 &#176;C.</li>
	<li>Increase the volume of sample to 120 mL using native protein extraction buffer. Use the lysate immediately for ice-affinity purification to avoid any loss of ice-binding activity.</li>
</ol>
8. Conducting Ice-affinity Purification
<ol>
	<li>Prior to purification, set the programmable water bath to cool from -0.5 &#176;C to -3 &#176;C at a rate of -0.04 &#176;C/h. 
	NOTE: The cooling rate can be adjusted depending on the initial volume of sample.</li>
	<li>Cool the ice finger to -0.5 &#176;C and subsequently submerse it into a 50 mL 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 min. 
	NOTE: Any lumps on the ice-coated finger should be smoothed with a gloved hand prior to placing in sample. If an ice layer does not form at -0.5 &#176;C, lower the temperature to -0.75 &#176;C.</li>
	<li>Place sample in a 150 mL glass beaker containing a small magnetic stir bar into a polystyrene container on top of the magnetic stirrer. Make sure that the ice finger is centered and lowered at least half way into the liquid sample. Seal the container.</li>
	<li>Allow the program to run for approximately two days, checking every 24 h. Once 50% of the sample is frozen, stop the program. Incorporation of IBPs into the ice hemisphere will result in &#34;ice-etching&#34;. 
	NOTE: Purification experiments can be done using larger sample volumes with the length of time at which the program runs adjusted accordingly.</li>
	<li>In order to remove the ice hemisphere, warm the probe to 4 &#176;C, rinse the hemisphere with distilled water to remove unbound protein, and then thaw at 4 &#176;C. 
	NOTE: Most IBPs are not stable in water and an appropriate buffer (i.e. 50 mM Tris-HCl, pH 8 or 50 mM ammonium bicarbonate, pH 8) will need to be added periodically during the thawing process (approximately 1 mL/2 h). Ice hemispheres can be wrapped in aluminum foil and stored at -20 &#176;C prior to thawing.</li>
	<li>If the ice-hemisphere still contains pigment, or to increase the sample purification, repeat steps 8.1-8.5 2-3 times. 
	NOTE: Although the concentration of IBP may be lower following multiple rounds of purification, purity is more valuable than yield if the purified samples will be analyzed using MS. If sufficient material is available, three rounds of ice-affinity purification is recommended.</li>
</ol>
9. Identification of IBPs
<ol>
	<li>Since IBPs are contained in a large volume following thawing of the ice-hemisphere, concentrate the samples. Concentrate samples containing ~100 g of tissue to ~1-3 mL using centrifugal concentration tubes with a 3,000 Da molecular weight cut-off to avoid the loss of small proteins.</li>
	<li>Prior to sending samples for MS, determine the protein concentration using a protein quantification assay such as a BCA or Bradford assay. Estimate the purity of samples by gel electrophoresis such as SDS-PAGE. Test the purified proteins for IRI activity prior to sending for MS to ensure that active protein has been retained through the purification and concentration steps.</li>
	<li>Use concentrated samples directly for mass spectrometry analysis10.</li>
</ol>

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E., Laybourn-Parry, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+of+antifreeze+protein+activity+in+Antarctic+lake+bacteria.">Demonstration of antifreeze protein activity in Antarctic lake bacteria.</a> Microbiology. 150 (Pt 1), 171-180 (2004).</li><li>Knight, C. A., Cheng, C. C., DeVries, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adsorption+of+alpha-helical+antifreeze+peptides+on+specific+ice+crystal+surface+planes.">Adsorption of alpha-helical antifreeze peptides on specific ice crystal surface planes.</a> Biophys. L. 59 (20), 409-418 (1991).</li><li>Yeh, Y., Feeney, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifreeze+proteins:+structures+and+mechanisms+of+function.">Antifreeze proteins: structures and mechanisms of function.</a> Chem. Rev. 96 (2), 601-618 (1996).</li><li>Smolin, N., Daggett, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+ice-like+water+structure+on+the+surface+of+an+antifreeze+protein.">Formation of ice-like water structure on the surface of an antifreeze protein.</a> J. Phys. Chem. B. 112 (19), 6193-6202 (2008).</li><li>Graham, L. A., Davies, P. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperactive+antifreeze+protein+in+a+fish.">Hyperactive antifreeze protein in a fish.</a> Nature. 429 (6988), 153 (2004).</li><li>Zhang, D. Q., Liu, B., Feng, D. R., He, Y. M., Wang, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression,+purification+and+antifreeze+activity+of+carrot+antifreeze+protein+and+its+mutants.">Expression, purification and antifreeze activity of carrot antifreeze protein and its mutants.</a> Protein Expr. Purif. 35 (2), 257-263 (2007).</li><li>Gupta, R., Dreswal, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+temperature+stress+modulated+secretome+analysis+and+purification+of+antifreeze+protein+from+Hippophae+rhamnoides,+a+Himalayan+wonder+plant.">Low temperature stress modulated secretome analysis and purification of antifreeze protein from Hippophae rhamnoides, a Himalayan wonder plant.</a> Proteome Res. 11 (5), 2684-2696 (2012).</li><li>Kuiper, M. J., Lankin, C., Gauthier, S. Y., Walker, V. K., Davies, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+antifreeze+proteins+by+adsorption+to+ice.">Purification of antifreeze proteins by adsorption to ice.</a> Biochem. Biphys. Res. Commun. 300 (3), 645-648 (2003).</li><li>Middleton, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+ice-binding+proteins+from+higher+plants.">Isolation and characterization of ice-binding proteins from higher plants.</a> Methods Mol. Biol. 1166, 255-277 (2014).</li><li>Bredow, M., Vanderbeld, B., Walker, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ice-binding+proteins+confer+freezing+tolerance+in+transgenic+Arabidopsis+thaliana.">Ice-binding proteins confer freezing tolerance in transgenic Arabidopsis thaliana.</a> Plant Biotechnol. J. , (2016).</li><li>Olijve, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blocking+rapid+ice+crystal+growth+through+nonbasal+plane+adsorption+of+antifreeze+proteins.">Blocking rapid ice crystal growth through nonbasal plane adsorption of antifreeze proteins.</a> Proc. Natl. Acad. Sci. U.S.A. 113 (14), 3740-3745 (2016).</li><li>Zakharov, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ice+Recrystallization+in+a+Solution+of+a+Cryoprotector+and+Its+inhibition+by+a+Protein:+Synchrotron+X-Ray+Diffraction+Study.">Ice Recrystallization in a Solution of a Cryoprotector and Its inhibition by a Protein: Synchrotron X-Ray Diffraction Study.</a> J. Pharm. Sci. 105 (7), 2129-2138 (2016).</li><li>Capicciotti, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+ice+recrystallization+inhibitors+enable+freezing+of+human+red+blood+cells+with+reduced+glycerol+concentrations.">Small molecule ice recrystallization inhibitors enable freezing of human red blood cells with reduced glycerol concentrations.</a> Sci. Rep. 5, 9692 (2015).</li><li>Knight, C. A., Cheng, C. C., DeVries, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adsorption+of+alpha-helical+antifreeze+peptides+on+specific+ice+crystal+surface+planes.">Adsorption of alpha-helical antifreeze peptides on specific ice crystal surface planes.</a> Biophys. J. 59 (2), 409-418 (1991).</li><li>Van Driessche, E., Beeckmans, S., Dejaegere, R., Kanarek, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiourea:+the+antioxidant+of+choice+for+the+purification+of+proteins+from+phenol+rich+plant+tissues.">Thiourea: the antioxidant of choice for the purification of proteins from phenol rich plant tissues.</a> Anal. Biochem. 141 (1), 184-188 (1984).</li><li>Marshall, C. J., Basu, K., Davies, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ice-shell+purification+of+ice-binding+proteins.">Ice-shell purification of ice-binding proteins.</a> Cryobiology. 72 (3), 258-263 (2016).</li><li>Basu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+the+ice-binding+planes+of+antifreeze+proteins+by+fluorescence-based+ice+plane+affinity.">Determining the ice-binding planes of antifreeze proteins by fluorescence-based ice plane affinity.</a> J. Vis. Exp. (83), e51185 (2014).</li><li>Sandve, S. R., Rudi, H., Asp, T., Rognli, O. 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The authors have nothing to disclose.

For the successful analysis and purification of IBPs, it is important to understand the temperature-sensitive nature of some of these proteins. Certain plant IBPs become unstable at temperatures above 0 &#176;C, resulting in unfolding, precipitation and inactivity. In order to obtain active IBPs, it is often critical that plants are processed in a cold room (~4 &#176;C) and samples are kept on ice during experimentation. Another factor to consider when using whole-cell crude lysates is the degradation of proteins by endogenous proteases. In plants, the expression of IBPs is generally low, and thus any loss in protein yield could result in insufficient material for future experimentation or analysis. Working quickly during protein extraction, with the addition of a reliable protease inhibitor cocktail can reduce such protein loss. Additionally, it is important that the cold-acclimation period be optimized for the greatest accumulation of IBPs in laboratory-grown plants.
Another common problem when working with IBPs is that many of the assays are sensitive to temperature and humidity. In regards to IRI analysis, moisture buildup on the microscope cover slide can result in a layer of ice that makes visualization of ice crystals difficult. Using a dehumidifier and extra desiccation beads can resolve this difficulty; however, it is recommended that this assay is not conducted when laboratory relative humidity levels are above 70%.
The splat assay, as presented, is qualitative. By performing a dilution series prior to analysis, the IRI endpoint can be established to determine the concentration range in which the IBPs can no longer restrict ice crystal growth. Various labs have also used computer software to measure the average size of ice crystal grains18. As previously indicated, while testing for IRI activity is an efficient initial screen, confirmation of ice-binding activity should be established by determining the level of TH or by directly visualizing the adsorption of IBPs to ice crystals16. A notable limitation of IRI analysis is that several non-IBP molecules have been identified that mimic IRI activity. These molecules can include bulky proteins, phenolic glycosides, and synthetic polymers such as polyvinyl alcohol19,20,21. As a result, a buffer control should always be run with samples to ensure that any contaminants or additives do not result in the observed IRI activity.
While ice-affinity purification is easily performed, when not done correctly, other proteins, solutes and metabolites can predominant. An important consideration is the rate of ice crystal growth; by lowering the cooling rate (i.e. to -0.02 &#176;C/h), the ice hemisphere grows more slowly and any non-ice-binding molecules are less likely to become incorporated in the ice crystal lattice. The presence of contaminating molecules can also be avoided by ensuring that only 50% of the lysate is incorporated into the hemisphere (i.e. 50:50, liquid to ice ratio). As previously stated, multiple rounds of ice affinity can also clarify the ice-fraction and result in a sample of higher purity. Notably, secondary metabolites are often overproduced under biotic and abiotic stress conditions in plants. During the purification of phenol-rich plant tissues, if dark brown pigments persist, 1.5% (v/v) polyvinylpyrrolidone (PVP) and 1.5% polyvinylpolypyrrolidone (w/v) (PVPP) can be added to the extraction buffer, to neutralize secondary metabolites. Antioxidant additives such as thiourea (5-10 mM) can also be used19,21.
If information about the IBP of interest is known, additional purification steps, such as high-pressure liquid chromatography (HPLC) can also be used. Modifications to ice-affinity purification can also be considered and have been reported to optimize protein yield and gain additional insight into IBP characteristics including the faster ice shell purification23 and fluorescence-based ice plane affinity (FIPA)24.
When optimized, the techniques described above can yield a purified protein pool for the identification of IBPs; however, unspecific incorporation of other highly abundant proteins may be inevitable. While IBPs from the Pooideae family share sequence homology25, due to the high diversity between IBPs from different organisms, it is typically not possible to identify an IBP by sequence homology alone. A common feature of IBPs is the presence of amino acids known to bind to ice, including threonine, serine and valine26; identifying peptides sequences rich in these residues may be useful when mining sequence data.

Ice-binding proteins (IBPs) are a diverse family of protective proteins that have been discovered in a number of organisms including plants1, insects2, fish3, and microbes4. The key feature of these proteins is their unique ability to specifically and efficiently adsorb to ice crystals, modifying their growth. IBPs have several documented properties, with the two most well characterized being thermal hysteresis (TH) and ice-recrystallization inhibition (IRI). TH activity is more readily observed in IBPs produced in freeze-intolerant animals. This activity results in lowering of the freezing point of organisms&#39; circulatory or interstitial fluids to prevent freezing. In contrast, in freeze-tolerant organisms, which will inevitably freeze at subzero temperatures, IBPs appear to have low TH activity. Despite the low TH activity, a high IRI activity to restrict ice crystal growth is often observed with these proteins. For the freeze-tolerant organism, this IRI activity presumably helps protect cells from the uncontrolled growth of ice in extracellular compartments.
The &#34;mattress button&#34; model can be used to describe the mechanism by which IBPs prevent the growth of ice crystals5. Under this model, IBPs specifically adsorb to the ice crystal surface at intervals, such that water molecules can only incorporate with the growing ice crystal lattice in the space between bound IBPs. This creates a curvature that makes the incorporation of additional water molecules unfavorable, an event that can be described by the Gibbs-Thomson effect6. The anchored clathrate waters hypothesis provides a mechanism for the specific binding of IBPs to the ice crystal surface whereby the presence of charged residues, specifically positioned on the protein ice binding site, results in the reorganization of water molecules so they match one or more planes of the ice crystal lattice7.
TH activity can be quantified by measuring the difference between the melting and freezing temperatures of a single ice crystal in the presence of an IBP. While TH activity is a widely accepted method of evaluating the activity of IBPs, the low TH gap produced by plant IBPs (typically only a fraction of a degree) normally requires a high protein concentration, specialized equipment and operator experience. Although non-IBPs may restrict ice crystal growth, it is a property shared by all IBPs and thus testing for IRI activity is an effective initial screen for the presence of IBPs, especially for those with low TH activity. The methodology used to test this activity is known as a &#39;splat assay&#39;, whereby a protein sample is flash frozen to produce a monolayer of small ice crystals, which are observed over a period of time to determine if ice crystal growth is restricted. Unlike other methods used to screen a source tissue sample for the presence of IBPs, this technique is applicable to low protein concentrations in the range of 10-100 ng, utilizes easily-fabricated equipment, and generates data that is quickly and easily interpreted. However, it is important to stress that this assay provides an initial screen for IBPs that should be followed by the determination of TH and ice crystal shaping.
The isolation of native proteins is challenging, often requiring information regarding the structural and biochemical properties of a protein of interest. The affinity of IBPs for ice allows for the isolation of these proteins using ice as a substrate for purification purposes. Since the majority of molecules are pushed ahead of the ice-water boundary during ice crystal growth, slow growth of an ice-hemisphere in the presence of an IBP sample results in a highly-purified sample, devoid of large quantities of contaminating proteins and solutes. This method has been used to identify IBPs from insects8,9,10, bacteria11, fish12 and plants13,14. Additionally, the IBP-enriched fractions achieved through this method can also be used for downstream biochemical analysis. This paper outlines the identification of IBPs in plants through the induction and extraction of IBPs, analysis of IRI activity to confirm the presence of proteins, and the isolation of proteins using ice affinity purification.

<table><tbody><tr><td>1.5 mL microcentrifugetubes</td><td>Fisher</td><td>05-408-129</td><td></td></tr><tr><td>Adjustable lab jack</td><td>Fisher</td><td>S63080</td><td></td></tr><tr><td>Benchtop centrifuge</td><td>Desaga MC2</td><td></td><td></td></tr><tr><td>Brass probe</td><td>Custom built</td><td></td><td></td></tr><tr><td>Camera/camera port</td><td>Canon</td><td></td><td>Canon Power Shot SX110 digitial camera; custom built microscope port</td></tr><tr><td>Cheesecloth</td><td>Purewipe/Fisher Scientific</td><td>06-665-25A</td><td></td></tr><tr><td>Concentration tubes (0.5 mL)</td><td>EMD Millipore</td><td>UFC501008</td><td></td></tr><tr><td>Concentration tubes (15 mL)</td><td>EMD Millipore</td><td>UFC900308</td><td></td></tr><tr><td>Conical tubes (50 mL)</td><td>Thermo Fisher</td><td>AM12502</td><td></td></tr><tr><td>Cooling block</td><td>VWR</td><td>13259</td><td>Use a metal heating block</td></tr><tr><td>Dehumidifier</td><td>Whirlpool</td><td></td><td>50 pint Energy Star dehumidifier; purchase from local supplier</td></tr><tr><td>Dessciation beads</td><td>t.h.e. Dessicant/VWR</td><td>EM-DX0017-2</td><td>6-8 mesh size; 100% indicating</td></tr><tr><td>Dissection microscope</td><td>Olympus Tokyo</td><td></td><td></td></tr><tr><td>Double walled glass bowl</td><td>Generic</td><td></td><td>Purchase from local lab glassware supplier</td></tr><tr><td>Dry ice</td><td>Generic</td><td></td><td>Use local supplier; hazardous&amp;nbsp;</td></tr><tr><td>EDTA-protease inhibitor tablets</td><td>Sigma Aldrich</td><td>11836170001</td><td>Roche cOmplete mini</td></tr><tr><td>Ethylene glycol</td><td>Generic</td><td></td><td>Green automotive ethylene glycol can be purchased from any local hardware store (&lt;em&gt;i.e.&lt;/em&gt; Home Depot)</td></tr><tr><td>Hexane</td><td>Sigma Aldrich</td><td>296090</td><td>Anhydrous, 95%; hazardous</td></tr><tr><td>Immersion oil</td><td>Sigma Aldrich</td><td>56822</td><td></td></tr><tr><td>JA10/20 centrifuge</td><td>Beckman</td><td></td><td></td></tr><tr><td>Large plastic Petri dish</td><td>Generic</td><td></td><td></td></tr><tr><td>Liquid nitrogen</td><td>Generic</td><td></td><td>Use local supplier; hazardous&amp;nbsp;</td></tr><tr><td>Magnetic stir plate</td><td>Hanna Instruments</td><td>HI190M</td><td></td></tr><tr><td>Microscope cover slides</td><td>Fisher</td><td>12-542A</td><td></td></tr><tr><td>Plastic tube</td><td>Generic</td><td></td><td>Purchase PVC pipe from local hardware store</td></tr><tr><td>Polarized film</td><td>Edmund Optics</td><td>43-781</td><td></td></tr><tr><td>Polystyrene foam</td><td>Generic</td><td></td><td>Can be constructed from polystyrene shipping boxes</td></tr><tr><td>Poreclain mortar and pestle</td><td>Fisher</td><td>FB961</td><td></td></tr><tr><td>PVPP</td><td>Sigma Aldrich</td><td>77627</td><td>110 &amp;micro;m particle size</td></tr><tr><td>Retort Stand</td><td>Fisher</td><td>12-000</td><td></td></tr><tr><td>Small stir bar</td><td>Fisher</td><td>14-513-51</td><td></td></tr><tr><td>Temperature-programmable water bath</td><td>VWR</td><td>13271-118</td><td></td></tr><tr><td>Vacuum grease</td><td>Dow Corning/Sigma Aldrich</td><td>Z273554</td><td></td></tr><tr><td>Vinyl tubing</td><td>Generic</td><td></td><td></td></tr></tbody></table>

identification of plant ice-binding proteins through assessment of ice-recrystallization inhibition and isolation using ice-affinity purification

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In plants, freeze damage occurs when extracellular ice crystals grow, resulting in the rupture of plasma membranes and possible cell death. Adsorption of IBPs to ice crystals restricts further growth by a process known as ice-recrystallization inhibition (IRI), thereby reducing cellular damage. IBPs also demonstrate the ability to depress the freezing point of a solution below the equilibrium melting point, a property known as thermal hysteresis (TH) activity. These protective properties have raised interest in the identification of novel IBPs due to their potential use in industrial, medical and agricultural applications. This paper describes the identification of plant IBPs through 1) the induction and extraction of IBPs in plant tissue, 2) the screening of extracts for IRI activity, and 3) the isolation and purification of IBPs. Following the induction of IBPs by low temperature exposure, extracts are tested for IRI activity using a &#39;splat assay&#39;, which allows the observation of ice crystal growth using a standard light microscope. This assay requires a low protein concentration and generates results that are quickly obtained and easily interpreted, providing an initial screen for ice binding activity. IBPs can then be isolated from contaminating proteins by utilizing the property of IBPs to adsorb to ice, through a technique called &#39;ice-affinity purification&#39;. Using cell lysates collected from plant extracts, an ice hemisphere can be slowly grown on a brass probe. This incorporates IBPs into the crystalline structure of the polycrystalline ice. Requiring no a priori biochemical or structural knowledge of the IBP, this method allows for recovery of active protein. Ice-purified protein fractions can be used for downstream applications including the identification of peptide sequences by mass spectrometry and the biochemical analysis of native proteins.

For ease of set-up, Figure 1 and Figure 2 are included as visual representations of the equipment used for IRI analysis and ice-affinity purification, respectively. Results of IRI analysis using extracts collected from mustard weed with and without IBPs are depicted in Figure 3. These results show that extracts collected from mustard weed, which is not freeze-tolerant, were unable to restrict ice crystal growth due to the lack of IBPs present. In contrast, the small ice crystals seen with transgenic plants containing IBPs from perennial ryegrass restrict ice crystal growth substantially.&#160; Figure 4 shows ice hemispheres grown using perennial ryegrass extracts expressing IBPs after two rounds of purification. After the first round of purification the high degree of ice-etching on the hemisphere surface indicates that IBPs have adsorbed to the ice and modified growth of the ice crystals (Figure 4A). The second round of purification shown in Figure 4B has clearer ice, indicating that there was some exclusion of contaminating molecules. Ice-etching on the hemisphere surface indicates that IBPs are still present. Figure 5 shows an SDS-PAGE following ice-affinity purification, of proteins collected from perennial ryegrass, and subsequently stained with a silver stain. A number of proteins were identified with seven distinct bands, corresponding to the most abundant proteins that adsorb to ice.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55302/55302fig1.jpg" /> 
Figure 1: Diagrammatic representation of the splat apparatus16. Equipment used for the generation of a monolayer of ice crystals (A) and for the visualization of ice crystal growth (B). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55302/55302fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55302/55302fig2.jpg" /> 
Figure 2: Apparatus used for ice-affinity purification of IBPs16. A brass &#34;ice finger&#34; attached to a programmable ethylene glycol bath is used to slowly grow an ice-hemisphere. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55302/55302fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55302/55302fig3.jpg" /> 
Figure 3: Representative data obtained using IRI analysis17. A monolayer of ice crystals formed using plant extracts is visualized at 40x magnification. Extracts from wild-type Arabidopsis thaliana that do not contain IBPs (&#8722;IBPs) were compared to extracts of transgenic A. thaliana expressing IBPs from Lolium perenne (+IBPs). Following an 18 h incubation period at -4 &#176;C, ice crystals had recrystallized in the buffer and wild-type A. thaliana samples. In contrast, ice crystal growth was restricted in the A. thaliana extracts expressing an IBP. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55302/55302fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55302/55302fig4.jpg" /> 
Figure 4: Ice-hemispheres grown using IBP extracts collected from the freeze-tolerant grass, L. perenne. After grass extracts were subjected to one round of ice-affinity purification, ice-etching of the surface of the ice hemisphere indicates the successful incorporation of IBPs (A). To remove additional solutes, pigments, and contaminating proteins, a second round of ice-affinity was used, resulting in a clearer ice-hemisphere (B). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55302/55302fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55302/55302fig5.jpg" /> 
Figure 5: Monitoring the purification of native IBPs from perennial ryegrass. Following two rounds of ice-affinity purification and centrifugal concentration, native grass extracts were electrophoresed on a denaturing polyacrylamide gel (SDS-PAGE) and then visualized using silver stain. Lane 1: molecular weight protein ladder; Lane 2: undiluted extract; Lane 3-5: samples diluted in water prior to gel loading with a 1:2, 1:5, and 1:10 (sample:water) ratio. Since this particular plant species contains a number of IBPs, multiple bands are observed which may correspond to different IBP isoforms. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55302/55302fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification at JoVE.com

Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification

This paper outlines the identification of ice-binding proteins from freeze-tolerant plants through the assessment of ice-recrystallization inhibition activity and subsequent isolation of native IBPs using ice-affinity purification.

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In ...

identification-plant-ice-binding-proteins-through-assessment-ice

Nanyang Technological University

Research

JoVE Journal

Biochemistry

8.6K Views.  Queen's University.  Dit document beschrijft de identificatie van ijs bindende eiwitten uit freeze-tolerante planten door de beoordeling van ijs herkristallisatie inhibitie activiteit en daaropvolgende isolatie van natieve IBP gebruik ice-affiniteitzuivering. 

Video: Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification

LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition proteins, are found in cold-adapted organisms and protect them from freeze injuries by interacting with ice crystals. IBPs are found in a variety of organism, including fish1, plants2, 3, arthropods4, 5, fungi6, and bacteria7. IBPs adsorb to the surfaces of ice crystals and prevent water molecules from joining the ice lattice at the IBP adsorption location. Ice that grows on the crystal surface between the adsorbed IBPs develops a high curvature that lowers the temperature at which the ice crystals grow, a phenomenon referred to as the Gibbs-Thomson effect. This depression creates a gap (thermal hysteresis, TH) between the melting point and the nonequilibrium freezing point, within which ice growth is arrested8-10, see Figure 1. One of the main tools used in IBP research is the nanoliter osmometer, which facilitates measurements of the TH activities of IBP solutions. Nanoliter osmometers, such as the Clifton instrument (Clifton Technical Physics, Hartford, NY,) and Otago instrument (Otago Osmometers, Dunedin, New Zealand), were designed to measure the osmolarity of a solution by measuring the melting point depression of droplets with nanoliter volumes. These devices were used to measure the osmolarities of biological samples, such as tears11, and were found to be useful in IBP research. Manual control over these nanoliter osmometers limited the experimental possibilities. Temperature rate changes could not be controlled reliably, the temperature range of the Clifton instrument was limited to 4,000 mOsmol (about -7.5 &deg;C), and temperature recordings as a function of time were not an available option for these instruments.
We designed a custom-made computer-controlled nanoliter osmometer system using a LabVIEW platform (National Instruments). The cold stage, described previously9, 10, contains a metal block through which water circulates, thereby functioning as a heat sink, see Figure 2. Attached to this block are thermoelectric coolers that may be driven using a commercial temperature controller that can be controlled via LabVIEW modules, see Figure 3. Further details are provided below. The major advantage of this system is its sensitive temperature control, see Figure 4. Automated temperature control permits the coordination of a fixed temperature ramp with a video microscopy output containing additional experimental details.
To study the time dependence of the TH activity, we tested a 58 kDa hyperactive IBP from the Antarctic bacterium Marinomonas primoryensis (MpIBP)12. This protein was tagged with enhanced green fluorescence proteins (eGFP) in a construct developed by Peter Davies' group (Queens University)10. We showed that the temperature change profile affected the TH activity. Excellent control over the temperature profile in these experiments significantly improved the TH measurements. The nanoliter osmometer additionally allowed us to test the recrystallization inhibition of IBPs5, 13. In general, recrystallization is a phenomenon in which large crystals grow larger at the expense of small crystals. IBPs efficiently inhibit recrystallization, even at low concentrations14, 15. We used our LabVIEW-controlled osmometer to quantitatively follow the recrystallization of ice and to enforce a constant ice fraction using simultaneous real-time video analysis of the images and temperature feedback from the sample chamber13. The real-time calculations offer additional control options during an experimental procedure. A stage for an inverted microscope was developed to accommodate temperature-controlled microfluidic devices, which will be described elsewhere16.
The Cold Stage System
The cold stage assembly (Figure 2) consists of a set of thermoelectric coolers that cool a copper plate. Heat is removed from the stage by flowing cold water through a closed compartment under the thermoelectric coolers. A 4 mm diameter hole in the middle of the copper plate serves as a viewing window. A 1 mm diameter in-plane hole was drilled to fit the thermistor. A custom-made copper disc (7 mm in diameter) with several holes (500 &mu;m in diameter) was placed on the copper plate and aligned with the viewing window. Air was pumped at a flow rate of 35 ml/sec and dried using Drierite (W.A. Hammond). The dry air was used to ensure a dry environment at the cooling stage. The stage was connected via a 9 pin connection outlet to a temperature controller (Model 3040 or 3150, Newport Corporation, Irvine, California, US). The temperature controller was connected via a cable to a computer GPIB-PCI card (National instruments, Austin, Texas, USA).

Ice binding proteins (IBPs), also known as antifreeze proteins, inhibit ice growth and are a promising additive for use in the cryopreservation of tissues. The main tool used to investigate IBPs is the nanoliter osmometer. We developed a home-designed cooling stage mounted on an optical microscope and controlled using a custom-built LabVIEW routine. The nanoliter osmometer described here manipulated the sample temperature in an ultra-sensitive manner.

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition ...

Chemistry

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

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

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice ...

Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve classes of intrinsically disordered proteins (IDPs) to reduce oxidative and osmotic stress. This article uses a combination of capillary gel electrophoresis (CGE) and mobility shift affinity electrophoresis (ACE) in order to describe the binding behavior of different conformers of the IDP AtHIRD11 from Arabidopsis thaliana. CGE is used to confirm the purity of AtHIRD11 and to exclude fragments, posttranslational modifications, and other impurities as reasons for complex peak patterns. In this part of the experiment, the different sample components are separated by a viscous gel inside a capillary by their different masses and detected with a diode array detector. Afterward, the binding behavior of the sample towards various metal ions is investigated by ACE. In this case, the ligand is added to the buffer solution and the shift in migration time is measured in order to determine whether a binding event has occurred or not. One of the advantages of using the combination of CGE and ACE to determine the binding behavior of an IDP is the possibility to automate the gel electrophoresis and the binding assay. Furthermore, CGE shows a lower limit of detection than the classical gel electrophoresis and ACE is able to determine the manner of binding a ligand in a fast manner. In addition, ACE can also be applied to other charged species than metal ions. However, the use of this method for binding experiments is limited in its ability to determine the number of binding sites. Nevertheless, the combination of CGE and ACE can be adapted for characterizing the binding behavior of any protein sample towards numerous charged ligands.

This protocol combines the characterization of a protein sample by capillary gel electrophoresis and a fast-binding screening for charged ligands by affinity capillary electrophoresis. It is recommended for proteins with a flexible structure, such as intrinsically disordered proteins, to determine any differences in binding for different conformers.

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve ...

A Microfluidic Approach for the Study of Ice and Clathrate Hydrate Crystallization

An accurate mechanistic description of water crystallization is challenging and requires a few key elements: superb temperature control to allow the formation of single microscopic crystals and a suitable microscopy system coupled to the cold stage. The method described herein adds another important feature that includes exchanging solutions around ice and clathrate hydrate crystals. The described system comprises a combination of unique and home-developed instruments, including microfluidics, high-resolution cold stages, and fluorescence microscopy. The cold stage was designed for microfluidic devices and allows for the formation of micron-sized ice/hydrate crystals inside microfluidic channels and the exchange of solutions around them. The temperature resolution and stability of the cold stage is one millikelvin, which is crucial for controlling the growth of these small crystals. This diverse system is used to study the different processes of ice and hydrate crystallization and the mechanism by which the growth of these crystals is inhibited. The protocol describes how to prepare microfluidic devices, how to grow and control microscopic crystals in the microfluidic channels, and how the utilization of the flow of liquids around ice/hydrate crystals affords new insights into the crystallization of water.

The present protocol describes the crystallization of microscopic ice crystals and clathrate hydrates in microfluidic devices, enabling liquid exchange around the formed crystals. This provides unparalleled possibilities to examine the crystallization process and binding mechanisms of the inhibitors.

An accurate mechanistic description of water crystallization is challenging and requires a few key elements: superb temperature control to allow the ...

TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been successfully used for identification of protein-protein interactions (PPIs) in mammalian cells. However, requirements of toxic hydrogen peroxide (H2O2) in APEX-based PL, longer incubation time with biotin (16&#8211;24 h), and higher incubation temperature (37 &#176;C) in BioID-based PL severely limit their applications in plants. The recently described TurboID-based PL addresses many limitations of BioID and APEX. TurboID allows rapid proximity labeling of proteins in just 10&#8201;min under room temperature (RT) conditions. Although the utility of TurboID has been demonstrated in animal models, we recently showed that TurboID-based PL performs better in plants compared to BioID for labeling of proteins that are proximal to a protein of interest. Provided here is a step-by-step protocol for the identification of protein interaction partners using the N-terminal Toll/interleukin-1 receptor (TIR) domain of the nucleotide-binding leucine-rich repeat (NLR) protein family as a model. The method describes vector construction, agroinfiltration of protein expression constructs, biotin treatment, protein extraction and desalting, quantification, and enrichment of the biotinylated proteins by affinity purification. The protocol described here can be easily adapted to study other proteins of interest in Nicotiana and other plant species.

Described here is a proximity labeling method for identification of interaction partners of the TIR domain of the NLR immune receptor in Nicotiana benthamiana leaf tissue. Also provided is a detailed protocol for the identification of interactions between other proteins of interest using this technique in Nicotiana and other plant species.

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been ...

Immunology and Infection

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Biology

Assaying Proteasomal Degradation in a Cell-free System in Plants

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions.
The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.

Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular &#8220;tags&#8221; for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

AirID-Based Proximity Labeling for Protein-Protein Interaction in Plants

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA (known as BioID) have proven successful in identifying protein-protein interactions (PPIs). APEX, BioID, and TurboID, a revised version of BioID have some restrictions in addition to being valuable technologies. The recently developed AirID, a novel version of BioID for proximity identification in protein-protein interactions, overcame these restrictions. Previously, AirID has been used in animal models, while the current study demonstrates the use of AirID in plants, and the results confirmed that AirID performs better in plant systems as compared to other PL enzymes such as BioID and TurboID for protein labeling that are proximal to the target proteins. Here is a step-by-step protocol for identifying protein interaction partners using AT4G18020 (APRR2) protein as a model. The methods describe the construction of vector, the transformation of construct through agroinfiltration, biotin transformation, extraction of proteins, and enrichment of biotin-labeled proteins through affinity purification technique. The results conclude that AirID is a novel and ideal enzyme for analyzing PPIs in plants. The method can be applied to study other proteins in plants.

Here, we present a step-by-step protocol for performing the proximity labeling (PL) experiment in cucumber (Cucumis sativus L.) using AT4G18020 (APRR2)-AirID protein as a model. The method describes the construction of a vector, the transformation of a construct through agroinfiltration, biotin infiltration, protein extraction, and purification of biotin-labeled proteins through affinity purification technique.

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA ...

Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification

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