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 border="0" cellpadding="0" cellspacing="0" width="1117">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.5 mL microcentrifugetubes</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:119px;">05-408-129</td>
			<td style="width:631px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Adjustable lab jack</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:119px;">S63080</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Benchtop centrifuge</td>
			<td style="width:152px;">Desaga MC2</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Brass probe</td>
			<td style="width:152px;">Custom built</td>
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			<td></td>
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			<td height="21" style="height:21px;width:216px;">Camera/ camera port</td>
			<td style="width:152px;">Canon</td>
			<td style="width:119px;"></td>
			<td>Canon Power Shot SX110 digitial camera; custom built microscope port</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cheesecloth</td>
			<td style="width:152px;">Purewipe/Fisher Scientific</td>
			<td style="width:119px;">06-665-25A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Concentration tubes (0.5 mL)</td>
			<td style="width:152px;">EMD Millipore</td>
			<td style="width:119px;">UFC501008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Concentration tubes (15 mL)</td>
			<td style="width:152px;">EMD Millipore</td>
			<td style="width:119px;">UFC900308</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Conical tubes (50 mL)</td>
			<td style="width:152px;">Thermo Fisher</td>
			<td style="width:119px;">AM12502</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cooling block</td>
			<td style="width:152px;">VWR</td>
			<td style="width:119px;">13259</td>
			<td>Use a metal heating block</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dehumidifier</td>
			<td style="width:152px;">Whirlpool</td>
			<td style="width:119px;"></td>
			<td>50 pint Energy Star dehumidifier; purchase from local supplier</td>
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			<td height="21" style="height:21px;width:216px;">Dessciation beads</td>
			<td style="width:152px;">t.h.e. Dessicant/VWR</td>
			<td style="width:119px;">EM-DX0017-2</td>
			<td>6-8 mesh size; 100% indicating</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dissection microscope</td>
			<td style="width:152px;">Olympus Tokyo</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Double walled glass bowl</td>
			<td style="width:152px;">Generic</td>
			<td style="width:119px;"></td>
			<td>Purchase from local lab glassware supplier</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dry ice</td>
			<td style="width:152px;">Generic</td>
			<td style="width:119px;"></td>
			<td>Use local supplier; hazardous&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA-protease inhibitor tablets</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td>11836170001</td>
			<td>Roche cOmplete mini</td>
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			<td height="21" style="height:21px;width:216px;">Ethylene glycol</td>
			<td style="width:152px;">Generic</td>
			<td style="width:119px;"></td>
			<td>Green automotive ethylene glycol can be purchased from any local hardware store (i.e. Home Depot)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hexane</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:119px;">296090</td>
			<td>Anhydrous, 95%; hazardous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Immersion oil</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:119px;">56822</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">JA10/20 centrifuge</td>
			<td style="width:152px;">Beckman</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Large plastic petri dish</td>
			<td style="width:152px;">Generic</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Liquid nitrogen</td>
			<td style="width:152px;">Generic</td>
			<td style="width:119px;"></td>
			<td>Use local supplier; hazardous&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnetic stir plate</td>
			<td style="width:152px;">Hanna Instruments</td>
			<td style="width:119px;">HI190M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microscope cover slides</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:119px;">12-542A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plastic tube</td>
			<td style="width:152px;">Generic</td>
			<td style="width:119px;"></td>
			<td>Purchase PVC pipe from local hardware store</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polarized film</td>
			<td style="width:152px;">Edmund Optics</td>
			<td style="width:119px;">43-781</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polystyrene foam</td>
			<td style="width:152px;">Generic</td>
			<td style="width:119px;"></td>
			<td>Can be constructed from polystyrene shipping boxes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Poreclain mortar and pestle</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:119px;">FB961</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PVPP</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:119px;">77627</td>
			<td>110 &#181;m particle size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Retort Stand</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:119px;">12-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Small stir bar</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:119px;">14-513-51</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Temperature-programmable water bath</td>
			<td style="width:152px;">VWR</td>
			<td style="width:119px;">13271-118</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vacuum grease</td>
			<td style="width:152px;">Dow Corning/Sigma Aldrich</td>
			<td style="width:119px;">Z273554</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vinyl tubing</td>
			<td style="width:152px;">Generic</td>
			<td style="width:119px;"></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. 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.

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

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic purification methods. This manuscript illustrates the technical details involved in the anaerobic purification process, including the preparation of buffers and reagents, the methods for column chromatography in a glove box, and the desalting of the protein prior to kinetics. Also described are the methods for preparing and using an oxygen electrode to perform kinetic characterization of an oxygen-utilizing enzyme. These methods are illustrated using the dioxygenase enzyme DesB, a gallate dioxygenase from the bacterium Sphingobium sp. strain SYK-6.

Here we present a protocol for anaerobic protein purification, anaerobic protein concentration, and subsequent kinetic characterization using an oxygen electrode system. The method is illustrated using the enzyme DesB, a dioxygenase enzyme which is more stable and active when purified and stored in an anaerobic environment.

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic ...

Isolation of Physiologically Active Thylakoids and Their Use in Energy-Dependent Protein Transport Assays

Chloroplasts are the organelles in green plants responsible for carrying out numerous essential metabolic pathways, most notably photosynthesis. Within the chloroplasts, the thylakoid membrane system houses all the photosynthetic pigments, reaction center complexes, and most of the electron carriers, and is responsible for light-dependent ATP synthesis. Over 90% of chloroplast proteins are encoded in the nucleus, translated in the cytosol, and subsequently imported into the chloroplast. Further protein transport into or across the thylakoid membrane utilizes one of four translocation pathways. Here, we describe a high-yield method for isolation of transport-competent thylakoids from peas (Pisum sativum), along with transport assays through the three energy-dependent cpTat, cpSec1, and cpSRP-mediated pathways. These methods enable experiments relating to thylakoid protein localization, transport energetics, and the mechanisms of protein translocation across biological membranes.

We present protocols herein for high-yield isolation of physiologically active thylakoids and protein transport assays for the chloroplast twin arginine translocation (cpTat), secretory (cpSec1), and signal recognition particle (cpSRP) pathways.

Chloroplasts are the organelles in green plants responsible for carrying out numerous essential metabolic pathways, most notably photosynthesis. Within ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

We here present a proven and tested protocol for the purification of chloroplast protein import complexes (TOC-TIC complex) using the TAP-tag. The one-step affinity-isolation protocol can potentially be applied to any protein and be used to identify new interaction partners by mass spectrometry.

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used strategy for RBP capture exploits the polyadenylation [poly(A)] of target RNAs, which mostly occurs on eukaryotic mature mRNAs, leaving most binding proteins of non-poly(A) RNAs unidentified. Here we describe the detailed procedures of a recently reported method termed click chemistry-assisted RNA-interactome capture (CARIC), which enables the transcriptome-wide capture of both poly(A) and non-poly(A) RBPs by combining the metabolic labeling of RNAs, in vivo UV cross-linking, and bioorthogonal tagging.

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy

A detailed protocol for applying the click chemistry-assisted RNA-interactome capture (CARIC) strategy to identify proteins binding to both coding and noncoding RNAs is presented.

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

Identification of Functional Protein Regions Through Chimeric Protein Construction

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences in a structurally similar protein, in order to determine the functional importance of these regions. Such chimeras are generated by means of a nested PCR protocol using overlapping DNA fragments and adequately designed primers, followed by their expression within a mammalian system to ensure native secondary structure and post-translational modifications. 
The functional role of a distinct region is then indicated by a loss of activity of the chimera in an appropriate readout assay. In consequence, regions harboring a set of critical amino acids are identified, which can be further screened by complementary techniques (e.g. site-directed mutagenesis) to increase molecular resolution. Although limited to cases in which a structurally related protein with differing functions can be found, chimeric proteins have been successfully employed to identify critical binding regions in proteins such as cytokines and cytokine receptors. This method is particularly suitable in cases in which the protein's functional regions are not well defined, and constitutes a valuable first step in directed evolution approaches to narrow down the regions of interest and reduce the screening effort involved.

Structurally related proteins frequently exert distinct biological functions. The exchange of equivalent regions of these proteins in order to create chimeric proteins constitutes an innovative approach to identify critical protein regions that are responsible for their functional divergence.

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences ...

Looking Outwards: Isolation of Cyanobacterial Released Carbohydrate Polymers and Proteins

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The identification and characterization of these biomolecules can improve knowledge about their secretion pathways and help to manipulate them. Furthermore, some of these biomolecules are also interesting in terms of biotechnological applications. Described here are two protocols for easy and rapid isolation of cyanobacterial released carbohydrate polymers and proteins. The method for isolation of released carbohydrate polymers is based on conventional precipitation techniques of polysaccharides in aqueous solutions using organic solvents. This method preserves the characteristics of the polymer and simultaneously avoids the presence of contaminants from cell debris and culture medium. At the end of the process, the lyophilized polymer is ready to be used or characterized or can be subjected to further rounds of purification, depending on the final intended use. Regarding the isolation of the cyanobacterial exoproteome, the technique is based on the concentration of the cell-free medium after removal of the major contaminants by centrifugation and filtration. This strategy allows for reliable isolation of proteins that reach the extracellular milieu via membrane transporters or outer membrane vesicles. These proteins can be subsequently identified using standard mass spectrometry techniques. The protocols presented here can be applied not only to a wide range of cyanobacteria, but also to other bacterial strains. Furthermore, these procedures can be easily tailored according to the final use of the products, purity degree required, and bacterial strain.

Here, protocols for the isolation of cyanobacterial released carbohydrate polymers and isolation of their exoproteomes are described. Both procedures embody key steps to obtain polymers or proteins with high purity degrees that can be used for further analysis or applications. They can also be easily adapted according to specific user needs.

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological functions in developmental processes, interorganismal interactions, and environmental adaptation. Due to these various bioactivities, many diterpenoids are also of economic importance as pharmaceuticals, food additives, biofuels, and other bioproducts. Advanced genomics and biochemical approaches have enabled a rapid increase in the knowledge of diterpenoid-metabolic genes, enzymes, and pathways. However, the structural complexity of diterpenoids and the narrow taxonomic distribution of individual compounds in often only a single species remain constraining factors for their efficient production. Availability of a broader range of metabolic enzymes now provide resources for producing diterpenoids in sufficient titers and purity to facilitate a deeper investigation of this important metabolite group. Drawing on established tools for microbial and plant-based enzyme co-expression, we present an easily operated and customizable protocol for the enzymatic production of diterpenoids in either Escherichia coli or Nicotiana benthamiana, and the purification of desired products via silica chromatography and semi-preparative HPLC. Using the group of maize (Zea mays) dolabralexin diterpenoids as an example, we highlight how modular combinations of diterpene synthase (diTPS) and cytochrome P450 monooxygenase (P450) enzymes can be used to generate different diterpenoid scaffolds. Purified compounds can be used in various downstream applications, such as metabolite structural analyses, enzyme structure-function studies, and in vitro and in planta bioactivity experiments.

Here we present easy to use protocols for producing and purifying diterpenoid metabolites through the combinatorial expression of biosynthetic enzymes in Escherichia coli or Nicotiana benthamiana, followed by chromatographic product purification. The resulting metabolites are suitable for various studies including molecular structure characterization, enzyme functional studies, and bioactivity assays.

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological ...