Name: Reconstitution Of β-catenin Degradation In Xenopus Egg Extract (Video) | JoVE Journal
Uploaded: 2014-06-17T20:30:00.000+00:00
Duration: 9 min 41 s
Description: Learn to analyze beta-catenin degradation using Xenopus egg extract. This cell-free biochemical method enables identification of protein turnover modulators through radiolabeling and luminescence detection.

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5 minutes

We thank Laurie Lee for critical reading of the manuscript. T.W.C is supported by an American Heart Association Predoctoral Fellowship (12PRE6590007). M.R.B. is supported by a National Cancer Institute training grant (T32 CA119925). S.S.H is supported by National Institutes of Health (R01DK078640). E.L. is supported by the National Institutes of Health (R01GM081635 and R01GM103926).

1. Preparation of Xenopus&#160;Egg Extract
NOTE: Each frog yields approximately 1 ml&#160;of usable egg extract. Extracts from 10 frogs are typically prepared at one time, and the volume of buffer described below is for performing a 10 frog Xenopus egg extract prep. The buffer volume can be adjusted accordingly for larger or smaller preparations of egg extract. Preps generated in this manner consistently yield protein concentrations &#8805; 50 mg/ml. The process of collecting eggs and processing them into extract is most efficient when conducted by two people. (For basic frog husbandry techniques, see Sive&#160;et al.33).
<ol>
	<li>Egg Collection
	<ol>
		<li>To prime the frogs, inject each female frog with 100 U of Pregnant Mare Serum Gonadotropin (PMSG) from a freshly made 250 U/ml&#160;stock. Use a 3 ml&#160;tuberculin syringe with a 27 G&#160;needle to inject subcutaneously, with the bevel of the needle up, into the dorsal lymph sac, which is located approximately 1 cm away from the midline from the notched discolorations along the length of the legs of the frog.</li>
		<li>Store primed frogs in water (plus 20 mM NaCl, see&#160;Sive&#160;et al.33) at 18 &#176;C for 5-10 days. For standing water tank systems, the animal density is approximately 4 L of water per female frog. NOTE: The minimum time required for priming to take effect is 5 days, and the effects of priming wear off after 10 days.</li>
		<li>Prepare 0.5x Marc&#8217;s Modified Ringers (MMR) solution from a 20x MMR stock. 20x MMR consists of 2 M sodium chloride, 40 mM potassium chloride, 40 mM calcium chloride, 20 mM magnesium chloride, and 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4.</li>
		<li>Set up buckets for all injected frogs (one frog per 4 L bucket). NOTE: Although more than one frog can be placed in the same bucket for egg collection, if one of the frogs lays predominantly poor quality eggs, a substantial amount of effort will be required to separate the poor quality eggs from those that are suitable for making extract. Thus, maximizing the number of frogs in the same tank to minimize the amount of buffer used for egg collect is not worth the risk.</li>
		<li>Inject 750 U Human Chorionic Gonadotropin (HCG) into the dorsal lymph sac of each frog using a 27 G needle as described in 1.1.1.</li>
		<li>Place each of the HCG-injected frogs into individual 4 L buckets containing 0.5x MMR cooled to 16 &#176;C.</li>
		<li>Place the containers with the frogs in a 16 &#176;C incubator to collect eggs O/N (15-16 hr). NOTE: Maintaining the proper temperature is critical for the entire procedure, from collecting eggs to preparing egg extract.</li>
	</ol>
	</li>
	<li>Dejellying Eggs 
	NOTE: Eggs are covered with a jelly coat that must be removed prior to making extract. The likelihood of spontaneous lysis of the eggs increases as the time between egg laying and extract preparation increases. Thus, it is important to proceed through the following steps as rapidly as possible.
	<ol>
		<li>Prepare 4 L of 1x MMR, 50 ml&#160;of 0.1x MMR, and 400 ml&#160;of 2% cysteine, pH 7.7, made in distilled water. Maintain all solutions at 16 &#176;C.</li>
		<li>To expel additional eggs, gently squeeze the lower back and abdomen of the frog.</li>
		<li>Remove the frogs and the bulk of the MMR, to leave the eggs in approximately 1-200 ml of MMR in each bucket.</li>
		<li>Remove debris with a transfer pipette, and assess the quality of the eggs: high quality eggs are generally marked by a clear separation between the darkly pigmented animal hemisphere and the lightly colored vegetal hemisphere and have the highest dark-to-light contrast. Discard with a transfer pipette any eggs that appear stringy, mottled, or lysed (white and puffy) as they will decrease the overall quality of the extract. If &#62;10% of the eggs are of poor quality, the entire batch should be discarded.</li>
		<li>Combine eggs into a 500 ml glass beaker and pour out as much MMR as possible while keeping eggs submerged.</li>
		<li>Rinse eggs by gentle swirling with twice the egg volume of MMR. Repeat twice, and remove any debris or obviously poor quality eggs.</li>
		<li>Add approximately 100 ml of 2% cysteine to the glass beaker, swirl gently to mix, and allow eggs to settle for 5 min&#160;at 16 &#176;C. Pour off the cysteine. NOTE: Dejellying is marked by the gradual appearance of jelly coats floating above the eggs and the more compact packing of the eggs as they now occupy a smaller volume without the jelly coat.</li>
		<li>Add another 100 ml of 2% cysteine, gently swirl, wait 5 min, and then slowly pour off the cysteine. Repeat until eggs have become tightly compacted (usually by the third cysteine treatment). Note: If eggs are left too long in cysteine, they are prone to lysis. Similarly, dejellied eggs are fragile and prone to mechanical lysis if they are swirled too vigorously or if they are exposed to air. Once the eggs have been dejellied, it is important to rapidly proceed to the centrifugation steps.</li>
		<li>Pour off cysteine, and rinse away the jelly coat and other debris by gently washing eggs in 1x MMR. Pour off buffer carefully along the side of the beaker. Repeat twice&#160;or until MMR solution is no longer cloudy. While rinsing with 1x MMR continue to remove the bad eggs using a transfer pipette.</li>
		<li>Perform a final gentle rinse with 30 ml 0.1x MMR, and gently pour off as much of the buffer as possible. Again, remove any obviously bad eggs.</li>
	</ol>
	</li>
	<li>Packing and Crushing Eggs by Centrifugation 
	NOTE: The extract described below that is used for &#946;-catenin degradation is a variant of the cytostatic factor extract (metaphase II-arrested). In contrast to the low-speed and high-speed extract used for cell cycle studies, intermediate speed extract works best for &#946;-catenin degradation. Interphase extract similarly promotes robust &#946;-catenin degradation although is more labor intensive to prepare.
	<ol>
		<li>Add Leupeptin, Pepstatin, Aprotinin mixture (LPA, a protease inhibitor) at 10 &#181;g/ml (diluted from a 10 mg/ml stock solution in DMSO) and Cytochalasin D at 20 &#181;g/ml (diluted from a 10 mg/ml stock solution in DMSO) into the remaining 20 ml of 0.1x MMR.</li>
		<li>Add 0.&#160;x MMR containing LPA and Cytochalasin D to the washed eggs, swirl gently, and incubate for 5 min&#160;at 16 &#176;C.</li>
		<li>Transfer eggs into 16 &#176;C pre-chilled 50 ml centrifuge tubes, allow the eggs to settle, and remove residual buffer from the top. To prevent exposure to air, withdraw a small amount of buffer into the transfer pipette prior to withdrawing eggs for transfer. Continue to transfer additional eggs into the centrifuge tubes and remove residual buffer from the top of the tube until the eggs fill the centrifuge tube to the top, which will maximize the yield of extract.</li>
		<li>To pack the eggs, spin centrifuge tubes at 400 x g for 60 sec&#160;at 4 &#176;C using a fixed angle rotor. Remove residual buffer from the top of the centrifuge tubes.</li>
		<li>For the crushing spin, spin tubes at 15,000 x g for 5 min&#160;at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Collecting Cytoplasmic Layer of Extract 
	NOTE: The method described below differs from the classic method of puncturing the side of the centrifuge tube in order to collect the cytoplasmic layer. This protocol has been adapted for simplification and for use with particular centrifuge tubes, which are reusable. No noticeable differences in the kinetics of &#946;-catenin degradation are found with either method. At this point extract should be kept cold during throughout the process, and all steps should be performed at 4 &#176;C.
	<ol>
		<li>Clear a hole in the lipid layer using P1000 pipette&#160;tip.</li>
		<li>Collect the cytoplasmic layer (between the dark pigmented layer and the light lipid layer) using a new P1000 pipette tip&#160;into clean pre-chilled centrifuge tubes (Figure 1). For high-quality extract that robustly degrades &#946;-catenin, minimize the amount of pigmented and lipid layer that is withdrawn with the cytoplasmic layer.</li>
		<li>Spin extracted cytoplasmic layer at 15,000 x g for 10 min&#160;at 4 &#176;C and again collect the cytoplasmic layer. Repeat the spin and extraction 1X. NOTE: The extract should be &#34;straw colored.&#34; If there is substantial contamination with the pigmented and lipid layers at this point, one can repeat the spin one more time, although excessive spins will decrease the capacity of the extract to degrade &#946;-catenin.</li>
		<li>Add LPA and Cytochalasin D to the extract at final concentrations of 10 &#181;g/ml each. NOTE: Using the method described yields a fairly consistent total protein concentration of approximately 50 mg/ml (52.41 &#177; 5.40 mg/ml, n=3 separate preps) in Xenopus egg extracts.</li>
		<li>(Optional) For translation assays, add capped mRNA (0.1 mg/ml), RNAsin (1.5 U/ml), and energy regeneration mix (2.2.1) and incubate the reaction at RT for 2 hr (for further information see Salic&#160;et al.2 and Sive&#160;et al.33). Use translated extract immediately for &#946;-catenin degradation assays or snap-freeze in liquid nitrogen for later use. NOTE: Freshly prepared extract has a high capacity to translate exogenously added mRNA, but unfortunately, this capacity is lost once the extract is frozen. Capped mRNA can be readily prepared using commercially available kits.</li>
		<li>Snap-freeze extract in liquid nitrogen. NOTE: Extracts are stored in small (200 &#181;l) aliquots for single use because they rapidly lose their capacity to degrade &#946;-catenin if refrozen. For long-term storage, extract can be stored in liquid nitrogen. For short-term storage, extract can be stored at -80 &#176;C, although the capacity of extract to degrade &#946;-catenin can be dramatically reduced with extended storage at -80 &#176;C (longer than 2 months).</li>
	</ol>
	</li>
</ol>
2. Preparing Extract for &#946;-catenin Degradation Assay
<ol>
	<li>Depletion from Xenopus&#160;Extract 
	NOTE: A major advantage of Xenopus extract is the capacity to readily deplete components of a pathway and precisely add back a defined amount of a protein in order to determine its dose-dependent effects.
	<ol>
		<li>Use freshly prepared Xenopus egg extract or quickly thaw frozen extract and place on ice. Perform all manipulations in the cold.</li>
		<li>Add extract to 1/10 the volume of pelleted antibody or affinity beads (e.g., 20 ml pelleted beads to 200 ml extract). In order to minimize dilution of the extract, withdraw as much liquid from the beads as possible before addition of the extract using gel loading tips with long, tapered tips.</li>
		<li>Rotate extract-bead mix at 4 &#176;C for 1 hr.</li>
		<li>Spin extract-bead mix at 12,600 x g in microfuge at 4 &#176;C for 30 sec. Alternatively, if magnetic beads are used, apply magnetic field to collect beads.</li>
		<li>Transfer depleted extract to a fresh microfuge tube on ice. Be careful not to transfer any beads with the extract.</li>
		<li>Confirm efficiency of depletion by immunoblotting both depleted extract and beads.</li>
		<li>Prepare extract for &#946;-catenin degradation assay as described in 2.2.</li>
	</ol>
	</li>
	<li>Optimizing Xenopus&#160;Extract for &#946;-catenin Degradation 
	NOTE: &#946;-catenin degradation in Xenopus egg extract is an energy-dependent process that quickly depletes the endogenous ATP stores. Consequently, an energy regeneration system is required to maintain robust &#946;-catenin degradation.
	<ol>
		<li>Prepare a 20x energy regeneration (ER) mix consisting of 150 mM creatine phosphate, 20 mM ATP, 600 &#181;g/ml creatine phosphokinase, and 20 mM MgCl2. ER should be aliquoted and stored at -80 &#176;C. Avoid repeated freeze/thaw cycles by using small frozen aliquots.</li>
		<li>Quickly thaw Xenopus egg extract by rubbing the frozen tube between hands. Place the tube on ice just before all of the extract has melted.</li>
		<li>Add 10 &#181;l&#160;of energy regeneration mix (20x ER) into an aliquot (200 &#956;l) of Xenopus egg extract. Mix thoroughly by quickly flicking the tube and pulse vortexing. Pulse-spin and immediately place on ice.</li>
		<li>(optional) The turnover of &#946;-catenin can be slightly enhanced in Xenopus egg extract by addition of ubiquitin (1.25 mg/ml final). Cycloheximide (0.1 mg/ml final) can also be added to minimize translation of endogenous transcripts.</li>
		<li>Aliquot the appropriate volumes for degradation assay into pre-chilled microfuge tubes on ice. For radiolabeled &#946;-catenin degradation assays, withdraw 2-5 ml extract for each time point.</li>
	</ol>
	</li>
</ol>
3. Radiolabeled &#946;-catenin degradation assay in Xenopus egg extract
NOTE: All steps should be performed on ice unless otherwise indicated.
<ol>
	<li>Preparing Radiolabeled &#946;-catenin
	<ol>
		<li>Prepare freshly in vitro-synthesized [35S]methionine-radiolabeled protein using commercially available kits. NOTE: Generating 35S-labeled (half-life 87 days) proteins are easily and efficiently produced using commercially available in vitro-coupled transcription-translation kits. It is important that the translated protein is sufficiently labeled such that changes in protein turnover can be readily visualized.</li>
		<li>To confirm successful radiolabeling, perform SDS-PAGE/autoradiography with 0.5 &#181;l&#160;of the translated protein. Quantify the intensity of the radiolabeled &#946;-catenin band using ImageJ, ImageQuant, or an alternative quantitative software program. NOTE: The radiolabeled &#946;-catenin protein band should be clearly visible on film within a few hours (4-6 hr).</li>
		<li>Snap-freeze the radiolabeled protein in liquid nitrogen for storage until use. Note: Prolonged storage (&#62;2 months) and multiple freeze/thaw (greater than 2) can severely impact the capacity of the radiolabeled &#946;-catenin to degrade robustly in Xenopus egg extract. Typically, the stability of radiolabeled proteins declines significantly after 1&#160;month of storage at -80 &#176;C; thus, relatively freshly prepared radiolabeled &#946;-catenin gives best results in these degradation assays.</li>
	</ol>
	</li>
	<li>Performing &#946;-catenin Degradation Assay
	<ol>
		<li>Add 1-3 &#181;l&#160;(depending on the strength of the radiolabeled band signal) of in vitro-translated &#946;-catenin (and other proteins, small molecules, etc. that are being tested) into 20 &#181;l&#160;of Xenopus reaction mix on ice. Mix thoroughly by quickly flicking the tube and a short pulse of vortexing; this is an important step as Xenopus egg extract is very viscous, and incomplete mixing will affect the consistency of the results. Pulse spin and place on ice.</li>
		<li>Start the &#946;-catenin degradation reaction by shifting the tubes to RT.</li>
		<li>At the designated time point, remove 1-5 ml of the sample and mix immediately with SDS sample buffer (5x volume) to stop the reaction. To make sure the degradation reaction is completely terminated, flick tube several times and vortex vigorously.</li>
		<li>Perform SDS-PAGE/autoradiography. Run 1 ml&#160;equivalents (~50 mg of protein) of the extract for each time point/lane. Degradation of &#946;-catenin in Xenopus egg extract should be evidenced by the time-dependent decrease in intensity of the radiolabeled &#946;-catenin band Figure 2. Quantify results using ImageJ, ImageQuant, or other preferred imaging software if necessary.</li>
		<li>(optional) Soak SDS-polyacrylamide gel in fixing solution (10% acetic acid and 30% methanol in distilled water) prior to drying to decrease background radioactivity and increase the quality of the image.</li>
	</ol>
	</li>
</ol>
4. &#946;-catenin-luciferase Degradation Assay in Xenopus Egg Extract
Perform all steps on ice unless otherwise indicated.
<ol>
	<li>Preparing &#946;-catenin-luciferase
	<ol>
		<li>Synthesize non-radiolabeled, luciferase-tagged &#946;-catenin using the transcription-translation coupled system with complete amino acid mix.</li>
		<li>Confirm production of the luciferase-tagged &#946;-catenin by measuring luciferase activity from 0.5-1 &#181;l&#160;of the reaction. Assess background luminescence by measuring luminescence from an untranslated reaction mix. NOTE: Multiple commercial kits are available for measuring luciferase activity. Long-lived luminescence, however, works particularly well for the degradation assay.</li>
	</ol>
	</li>
	<li>Performing &#946;-catenin-luciferase Degradation Assay
	<ol>
		<li>Thaw and prepare Xenopus egg extract as in 2.2.</li>
		<li>Add in vitro-translated &#946;-catenin-luciferase fusion (from 4.1) into prepared Xenopus reaction mix (from 2.2) on ice and mix well as in 3.2.1. NOTE: The activity of the &#946;-catenin luciferase that is added to the extract is typically between 20 - 50,000 relative luminescence units (RLU)/ml of extract (based on measurements obtained from 4.1.2). Starting signal should be approximately 100,000 RLU (2-5 ml of the in vitro-translated &#946;-catenin-luciferase fusion).</li>
		<li>Shift the extract to RT to start the degradation reaction.</li>
		<li>Remove an aliquot of the reaction at the indicated time and snap-freeze in liquid nitrogen. NOTE: Triplicate samples are typically removed for analysis for each time point. Frozen extract can be stored at -80 &#176;C until they are ready to be analyzed.</li>
		<li>Thaw samples ice, transfer samples to standard white 96-well plates on ice, and process for luciferase activity.</li>
	</ol>
	</li>
</ol>

<ol><li>Glotzer, M., Murray, A. W., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cyclin+is+degraded+by+the+ubiquitin+pathway%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Cyclin is degraded by the ubiquitin pathway.</a> Nature. 349, 132-138 (1991).</li>
<li>Salic, A., Lee, E., Mayer, L., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Control+of+beta-catenin+stability%3A+reconstitution+of+the+cytoplasmic+steps+of+the+wnt+pathway+in+Xenopus+egg+extracts%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">Control of beta-catenin stability: reconstitution of the cytoplasmic steps of the wnt pathway in Xenopus egg extracts.</a> Molecular Cell. 5, 523-532 (2000).</li>
<li>Murray, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+cycle+extracts%5BTitle%5D)+AND+%22Methods+in+cell+biology%22%5BJournal%5D)">Cell cycle extracts.</a> Methods in cell biology. 36, 581-605 (1991).</li>
<li>Dabauvalle, M. C., Scheer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assembly+of+nuclear+pore+complexes+in+Xenopus+egg+extract%5BTitle%5D)+AND+%22Biology+of+the+cell+%2F+under+the+auspices+of+the+European+Cell+Biology+Organization%22%5BJournal%5D)">Assembly of nuclear pore complexes in Xenopus egg extract.</a> Biology of the cell / under the auspices of the European Cell Biology Organization. 72, 25-29 (1991).</li>
<li>Tutter, A. V., Walter, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromosomal+DNA+replication+in+a+soluble+cell-free+system+derived+from+Xenopus+eggs%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Chromosomal DNA replication in a soluble cell-free system derived from Xenopus eggs.</a> Methods Mol Biol. 322, 121-137 (2006).</li>
<li>Theriot, J. A., Rosenblatt, J., Portnoy, D. A., Goldschmidt-Clermont, P. J., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Involvement+of+profilin+in+the+actin-based+motility+of+L.+monocytogenes+in+cells+and+in+cell-free+extracts%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Involvement of profilin in the actin-based motility of L. monocytogenes in cells and in cell-free extracts.</a> Cell. 76, 505-517 (1994).</li>
<li>Maresca, T. J., Heald, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+for+studying+spindle+assembly+and+chromosome+condensation+in+Xenopus+egg+extracts%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Methods for studying spindle assembly and chromosome condensation in Xenopus egg extracts.</a> Methods Mol Biol. 322, 459-474 (2006).</li>
<li>Shennan, K. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Xenopus+egg+extracts%3A+a+model+system+to+study+proprotein+convertases%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Xenopus egg extracts: a model system to study proprotein convertases.</a> Methods Mol Biol. 322, 199-212 (2006).</li>
<li>Kornbluth, S., Yang, J., Powers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+the+cell+cycle+using+Xenopus+egg+extracts%5BTitle%5D)+AND+%22Current+protocols+in+cell+biology+%2F+editorial+board%2C+Juan+S.+Bonifacino+...+%5Bet+al.%5D%22%5BJournal%5D)">Analysis of the cell cycle using Xenopus egg extracts.</a> Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]. 11, (2006).</li>
<li>Chan, R. C., Forbes, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+study+of+nuclear+assembly+and+nuclear+import+using+Xenopus+egg+extracts%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">In vitro study of nuclear assembly and nuclear import using Xenopus egg extracts.</a> Methods Mol Biol. 322, 289-300 (2006).</li>
<li>Masui, Y., Markert, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytoplasmic+control+of+nuclear+behavior+during+meiotic+maturation+of+frog+oocytes%5BTitle%5D)+AND+%22The+Journal+of+experimental+zoology%22%5BJournal%5D)">Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes.</a> The Journal of experimental zoology. 177, 129-145 (1971).</li>
<li>Forbes, D. J., Kirschner, M. W., Newport, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spontaneous+formation+of+nucleus-like+structures+around+bacteriophage+DNA+microinjected+into+Xenopus+eggs%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Spontaneous formation of nucleus-like structures around bacteriophage DNA microinjected into Xenopus eggs.</a> Cell. 34, 13-23 (1983).</li>
<li>Lohka, M. J., Masui, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Formation+in+vitro+of+sperm+pronuclei+and+mitotic+chromosomes+induced+by+amphibian+ooplasmic+components%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components.</a> Science. 220, 719-721 (1983).</li>
<li>Newport, J. W., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+the+cell+cycle+during+early+Xenopus+development%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Regulation of the cell cycle during early Xenopus development.</a> Cell. 37, 731-742 (1984).</li>
<li>Mitchison, T., Kirschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+instability+of+microtubule+growth%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Dynamic instability of microtubule growth.</a> Nature. 312, 237-242 (1984).</li>
<li>Blow, J. J., Laskey, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Initiation+of+DNA+replication+in+nuclei+and+purified+DNA+by+a+cell-free+extract+of+Xenopus+eggs%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs.</a> Cell. 47, 577-587 (1986).</li>
<li>Verma, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ubistatins+inhibit+proteasome-dependent+degradation+by+binding+the+ubiquitin+chain%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Ubistatins inhibit proteasome-dependent degradation by binding the ubiquitin chain.</a> Science. 306, 117-120 (2004).</li>
<li>Yu, H., King, R. W., Peters, J. M., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+a+novel+ubiquitin-conjugating+enzyme+involved+in+mitotic+cyclin+degradation%5BTitle%5D)+AND+%22Curr+Biol%22%5BJournal%5D)">Identification of a novel ubiquitin-conjugating enzyme involved in mitotic cyclin degradation.</a> Curr Biol. 6, 455-466 (1996).</li>
<li>Thorne, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Small-molecule+inhibition+of+Wnt+signaling+through+activation+of+casein+kinase+1alpha%5BTitle%5D)+AND+%22Nature+Chemical+Biology%22%5BJournal%5D)">Small-molecule inhibition of Wnt signaling through activation of casein kinase 1alpha.</a> Nature Chemical Biology. 6, 829-836 (2010).</li>
<li>Thorne, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+biochemical+screen+for+identification+of+small-molecule+regulators+of+the+Wnt+pathway+using+Xenopus+egg+extracts%5BTitle%5D)+AND+%22Journal+of+Biomolecular+Screening%22%5BJournal%5D)">A biochemical screen for identification of small-molecule regulators of the Wnt pathway using Xenopus egg extracts.</a> Journal of Biomolecular Screening. 16, 995-1006 (2011).</li>
<li>Hinkson, I. V., Elias, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+dynamic+state+of+protein+turnover%3A+It%27s+about+time%5BTitle%5D)+AND+%22Trends+in+Cell+Biology%22%5BJournal%5D)">The dynamic state of protein turnover: It's about time.</a> Trends in Cell Biology. 21, 293-303 (2011).</li>
<li>Kinzler, K. W., Vogelstein, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lessons+from+Hereditary+Colorectal+Cancer%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Lessons from Hereditary Colorectal Cancer.</a> Cell. 87, 159-170 (1996).</li>
<li>Lee, E., Salic, A., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Physiological+regulation+of+%5Bbeta%5D-catenin+stability+by+Tcf3+and+CK1epsilon%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Physiological regulation of [beta]-catenin stability by Tcf3 and CK1epsilon.</a> J Cell Biol. 154, 983-993 (2001).</li>
<li>Lee, E., Salic, A., Krüger, R., Heinrich, R., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+roles+of+APC+and+Axin+derived+from+experimental+and+theoretical+analysis+of+the+Wnt+pathway%5BTitle%5D)+AND+%22PLoS+Biology%22%5BJournal%5D)">The roles of APC and Axin derived from experimental and theoretical analysis of the Wnt pathway.</a> PLoS Biology. 1, (2003).</li>
<li>Seeling, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+beta-catenin+signaling+by+the+B56+subunit+of+protein+phosphatase+2A%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Regulation of beta-catenin signaling by the B56 subunit of protein phosphatase 2A.</a> Science. 283, 2089-2091 (1999).</li>
<li>Guger, K. A., Gumbiner, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((beta-Catenin+has+Wnt-like+activity+and+mimics+the+Nieuwkoop+signaling+center+in+Xenopus+dorsal-ventral+patterning%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">beta-Catenin has Wnt-like activity and mimics the Nieuwkoop signaling center in Xenopus dorsal-ventral patterning.</a> Dev Biol. 172, 115-125 (1995).</li>
<li>Cselenyi, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((LRP6+transduces+a+canonical+Wnt+signal+independently+of+Axin+degradation+by+inhibiting+GSK3%27s+phosphorylation+of+%CE%B2-catenin%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">LRP6 transduces a canonical Wnt signal independently of Axin degradation by inhibiting GSK3's phosphorylation of β-catenin.</a> Proceedings of the National Academy of Sciences. 105, 8032-8037 (2008).</li>
<li>Jernigan, K. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gbetagamma+activates+GSK3+to+promote+LRP6-mediated+beta-catenin+transcriptional+activity%5BTitle%5D)+AND+%22Science+Signaling%22%5BJournal%5D)">Gbetagamma activates GSK3 to promote LRP6-mediated beta-catenin transcriptional activity.</a> Science Signaling. 3, (2010).</li>
<li>Major, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Wilms+tumor+suppressor+WTX+negatively+regulates+WNT%2Fbeta-catenin+signaling%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Wilms tumor suppressor WTX negatively regulates WNT/beta-catenin signaling.</a> Science. 316, 1043-1046 (2007).</li>
<li>Cross, M. K., Powers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Obtaining+eggs+from+Xenopus+laevis+females%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Obtaining eggs from Xenopus laevis females.</a> J Vis Exp. , (2008).</li>
<li>Cross, M. K., Powers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preparation+and+fractionation+of+Xenopus+laevis+egg+extracts%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Preparation and fractionation of Xenopus laevis egg extracts.</a> J Vis Exp. , (2008).</li>
<li>Willis, J., DeStephanis, D., Patel, Y., Gowda, V., Yan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Study+of+the+DNA+damage+checkpoint+using+Xenopus+egg+extracts%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Study of the DNA damage checkpoint using Xenopus egg extracts.</a> J Vis Exp. , (2012).</li>
<li>Sive, H. L., Grainger, R. M., Harland, R. M. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSive%2C+HL+%22Early+Development+of+Xenopus+Laevis+%3A+A+Laboratory+Manual%22">Early Development of Xenopus Laevis : A Laboratory Manual</a>. , Cold Spring Harbor Laboratory Press. (2000).</li>
<li>Rubinfeld, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Binding+of+GSK3%CE%B2+to+the+APC-%CE%B2-Catenin+Complex+and+Regulation+of+Complex+Assembly%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Binding of GSK3β to the APC-β-Catenin Complex and Regulation of Complex Assembly.</a> Science. 272, 1023-1026 (1996).</li>
<li>Salic, A., King, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identifying+small+molecule+inhibitors+of+the+ubiquitin-proteasome+pathway+in+Xenopus+egg+extracts%5BTitle%5D)+AND+%22Methods+in+Enzymology%22%5BJournal%5D)">Identifying small molecule inhibitors of the ubiquitin-proteasome pathway in Xenopus egg extracts.</a> Methods in Enzymology. 399, 567-585 (2005).</li>
<li>Trinkle-Mulcahy, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identifying+specific+protein+interaction+partners+using+quantitative+mass+spectrometry+and+bead+proteomes%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes.</a> J Cell Biol. 183, 223-239 (2008).</li>
<li>Tan, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Wnt+signalling+pathway+parameters+for+mammalian+cells%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Wnt signalling pathway parameters for mammalian cells.</a> PLoS One. 7, (2012).</li>
</ol>

The authors declare that they have no competing financial interests.

Xenopus egg extract is a robust biochemical system for investigating &#946;-catenin turnover. The concentration of &#946;-catenin in Xenopus egg extract is ~25 nM&#160;2. Under optimal conditions, the egg extract is capable of degrading &#946;-catenin at a rate of 50-100 nM/hr and is half-maximal at 200 nM24. There are several critical steps for successful reconstitution of &#946;-catenin degradation using Xenopus egg extract. These include 1) generating high quality X...

A correction was made to <a href="http://www.jove.com/video/51425">Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract</a>. At the time of publication there were some instances where an incorrect volume notation was used. These instances were corrected from:
2.1.2. Add extract to 1/10 the volume of pelleted antibody or affinity beads (e.g., 20 ml pelleted beads to 200 ml extract). In order to minimize dilution of the extract, withdraw as much liquid from the beads as possible before addition of the extract using gel loading tips with long, tapered tips.
2.2.5. Aliquot the appropriate volumes for degradation assay into pre-chilled microfuge tubes on ice. For radiolabeled &#946;-catenin degradation assays, withdraw 2-5 ml extract for each time point.
3.2.3. At the designated time point, remove 1-5 ml of the sample and mix immediately with SDS sample buffer (5x volume) to stop the reaction. To make sure the degradation reaction is completely terminated, flick tube several times and vortex vigorously.
3.2.4. Perform SDS-PAGE/autoradiography. Run 1 ml equivalents (~50 mg of protein) of the extract for each time point/lane. Degradation of &#946;-catenin in Xenopus egg extract should be evidenced by the time-dependent decrease in intensity of the radiolabeled &#946;-catenin band Figure 2. Quantify results using ImageJ, ImageQuant, or other preferred imaging software if necessary.
4.2.2. Add in vitro-translated &#946;-catenin-luciferase fusion (from 4.1) into prepared Xenopus reaction mix (from 2.2) on ice and mix well as in 3.2.1. NOTE: The activity of the &#946;-catenin luciferase that is added to the extract is typically between 20 - 50,000 relative luminescence units (RLU)/ml of extract (based on measurements obtained from 4.1.2). Starting signal should be approximately 100,000 RLU (2-5 ml of the in vitro-translated &#946;-catenin-luciferase fusion).
to:
2.1.2. Add extract to 1/10 the volume of pelleted antibody or affinity beads (e.g., 20 &#181;l pelleted beads to 200 &#181;l extract). In order to minimize dilution of the extract, withdraw as much liquid from the beads as possible before addition of the extract using gel loading tips with long, tapered tips.
2.2.5. Aliquot the appropriate volumes for degradation assay into pre-chilled microfuge tubes on ice. For radiolabeled &#946;-catenin degradation assays, withdraw 2-5 &#181;l extract for each time point.
3.2.3. At the designated time point, remove 1-5 &#181;l of the sample and mix immediately with SDS sample buffer (5x volume) to stop the reaction. To make sure the degradation reaction is completely terminated, flick tube several times and vortex vigorously.
3.2.4. Perform SDS-PAGE/autoradiography. Run 1 &#181;l equivalents (~50 mg of protein) of the extract for each time point/lane. Degradation of &#946;-catenin in Xenopus egg extract should be evidenced by the time-dependent decrease in intensity of the radiolabeled &#946;-catenin band Figure 2. Quantify results using ImageJ, ImageQuant, or other preferred imaging software if necessary.
4.2.2. Add in vitro-translated &#946;-catenin-luciferase fusion (from 4.1) into prepared Xenopus reaction mix (from 2.2) on ice and mix well as in 3.2.1. NOTE: The activity of the &#946;-catenin luciferase that is added to the extract is typically between 20 - 50,000 relative luminescence units (RLU)/&#181;l of extract (based on measurements obtained from 4.1.2). Starting signal should be approximately 100,000 RLU (2-5 &#181;l of the in vitro-translated &#946;-catenin-luciferase fusion).

Erratum: Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Xenopus laevis egg extract has been used extensively to study many cell biological processes including cytoskeletal dynamics, nuclear assembly and import, apoptosis, ubiquitin metabolism, cell cycle progression, signal transduction, and protein turnover1-17. The Xenopus egg extract system is amenable to the biochemical analysis of a legion of cellular processes because egg extract represents essentially undiluted cytoplasm that contains all the essential cytoplasmic components necessary to execute these processes and enable investigation. Large quantities of egg extract can be prepared at one time for biochemical manipulations that require large amounts of material (e.g., protein purification or high-throughput screening)18-20. Another advantage is that the concentration of specific proteins in Xenopus egg extract can be precisely adjusted by addition of recombinant protein and/or immunodepletion of endogenous proteins in contrast to transfection of plasmid DNA where expression of the protein of interest is difficult to control. In addition, the lack of available recombinant proteins can be overcome by the addition of transcripts encoding the protein of interest, taking advantage of the freshly prepared Xenopus egg extract&#8217;s high capacity to translate exogenously added mRNA.
The regulation of protein degradation is critical for the control of many cellular pathways and processes21. Xenopus egg extract has been used extensively to study protein degradation as the system allows for multiple ways to monitor protein turnover without confounding influences of transcription and translation. The Wnt signaling pathway is a highly conserved signaling pathway that plays critical roles in development and disease. The turnover of &#946;-catenin, the major effector of the Wnt pathway, is highly regulated, and an increased steady-state level of &#946;-catenin is critical for the activation of Wnt target genes. The importance of &#946;-catenin degradation is highlighted by the fact that mutations in the Wnt pathway that inhibit &#946;-catenin degradation found in ~90% of all sporadic cases of colorectal cancer22. &#946;-catenin degradation by components of the Wnt pathway can be faithfully recapitulated in Xenopus egg extract to study the mechanism of its turnover as well as to identify novel small molecule modulators of its degradation2,19,20,23-29 .
Methods for the preparation of Xenopus egg extract for studying the cell cycle have been described in previous JoVE publications30-32. The current protocol describes a modification of these methods and is optimized for the degradation of [35S]-radiolabeled &#946;-catenin and luciferase-tagged &#946;-catenin in Xenopus egg extract. The radiolabeled degradation assay allows for direct visualization of protein levels via autoradiography. [35S]methionine is incorporated into the protein of interest using an in vitro translation reaction that can then be directly added to a degradation reaction. In addition, the radiolabeled protein turnover assay does not require an antibody against the protein of interest or an epitope tag, which can influence protein stability. Because even small changes in protein levels, as reflected in changes in the intensity of the radiolabeled protein band, are readily visualized by autoradiography, the [35S]-radiolabeled degradation assay represents a very useful method for visualization of protein turnover2.
Fusion of &#946;-catenin to firefly luciferase (hereafter referred to as simply &#34;luciferase&#34;) allows for precise and facile quantitative measurements of protein levels, in order to determine the kinetic properties of &#946;-catenin turnover19,20 . A major advantage of the luciferase assay is that it provides a strong quantitative system that is easily scaled up. The following protocol provides simple methods for assaying &#946;-catenin degradation and a robust, efficient, and effective method for high-throughput screening of novel &#946;-catenin modulators.

<table><tbody><tr><td>Pregnant mare serum gonadotropin (PMSG)</td><td>ProSpec</td><td>hor-272-A</td><td>Reconstituted with distilled water before use.&amp;nbsp;</td></tr><tr><td>Human chorionic gonadotropin</td><td>Sigma</td><td>CG10-10VL</td><td></td></tr><tr><td>Potassium chloride</td><td>Fisher</td><td>BP366-1</td><td></td></tr><tr><td>Sodium chloride</td><td>Research Products International</td><td>S23020-5000.0</td><td></td></tr><tr><td>Magnesium chloride</td><td>Fisher</td><td>BP214-500</td><td></td></tr><tr><td>Calcium chloride</td><td>Acros Organics</td><td>AC42352-5000</td><td></td></tr><tr><td>HEPES</td><td>Fisher</td><td>BP310-1</td><td></td></tr><tr><td>Cysteine</td><td>Acros Organics</td><td>AC17360-1000</td><td></td></tr><tr><td>Leupeptin</td><td>Sigma</td><td>L2884-10MG</td><td></td></tr><tr><td>Aprotinin</td><td>Sigma</td><td>A1153-10MG</td><td></td></tr><tr><td>Pepstatin</td><td>Sigma</td><td>P4265-5MG</td><td></td></tr><tr><td>Cytochalasin B</td><td>Sigma</td><td>C8273-10MG</td><td></td></tr><tr><td>3 ml syringe: Luer Lock tip</td><td>Becton Dickinson</td><td>309657</td><td></td></tr><tr><td>27 G needle</td><td>Becton Dickinson</td><td>305109</td><td></td></tr><tr><td>96 well solid white polystyrene microplate, round bottomed</td><td>Corning</td><td>3605</td><td></td></tr><tr><td>Steady-Glo luciferase assay system</td><td>Promega</td><td>E2520</td><td>Store long-term at -80 &amp;deg;C, can store for up to 1 month at -20 &amp;deg;C</td></tr><tr><td>TNT Sp6 coupled reticulocyte lysate system</td><td>Promega</td><td>L4600</td><td></td></tr><tr><td>TNT Sp6 high-yield wheat germ protein expression system</td><td>Promega</td><td>L3260</td><td>Generally higher yield than reticulocyte lysate&amp;nbsp;</td></tr><tr><td>EasyTag Express protein labeling mix [S35]</td><td>Perkin Elmer</td><td>NEG772007MC</td><td></td></tr><tr><td>Creatine phosphate</td><td>Sigma</td><td>27920-5G</td><td></td></tr><tr><td>ATP</td><td>Sigma</td><td>A2383-5G</td><td></td></tr><tr><td>Creatine phosphokinase</td><td>Sigma</td><td>C3755-35KU</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma</td><td>D8418-50ML</td><td></td></tr><tr><td>Dual-Glo luciferase assay system</td><td>Promega</td><td>E2920</td><td>Same storage conditions as Steady-Glo</td></tr><tr><td>50 ml Centrifuge tubes</td><td>Fisher Scientific</td><td>0556214D</td><td></td></tr><tr><td>Sorvall SS-34 fixed angle rotor</td><td>Thermo Scientific</td><td>28020</td><td></td></tr><tr><td>115 V 50/60 Hz Minicentrifuge</td><td>Fisher Scientific</td><td>05-090-128</td><td></td></tr><tr><td>mMessage mMachine Sp6 kit</td><td>Ambion</td><td>AM1340</td><td></td></tr><tr><td>Anti-firefly luciferase antibody</td><td>Abcam</td><td>ab16466</td><td></td></tr><tr><td>Anti-GSK3 antibody</td><td>BD Transduction Laboratories</td><td>610201</td><td></td></tr><tr><td>FLUOStar Optima</td><td>BMG Labtech</td><td></td><td></td></tr><tr><td>Sorvall RC-6 Plus centrifuge</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>16 &amp;deg;C Incubator</td><td>Percival Scientific</td><td></td><td></td></tr></tbody></table>

reconstitution of &#946;-catenin degradation in xenopus egg extract

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3. Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, &beta;-catenin. Two different methods are described to assess &beta;-catenin protein degradation in Xenopus egg extract. One method is visually informative ([35S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess &beta;-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of &beta;-catenin degradation.

A schematic of β-catenin degradation in Xenopus egg extract is shown in Figure 2A. 35S-labeled β-catenin was incubated in Xenopus egg extract, aliquots (1 ml extract equivalent) were removed at the appropriate times, and samples were subjected to SDS-PAGE followed by autoradiography. β-catenin degradation by components of the Wnt pathway is mediated by the ubiquitin-proteasome system2, and degradation of [35S]-radiolabeled β-catenin in Xenopus e...

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...

The overall goal of the following experiment is to use the cell-free biochemical xenopus egg extract system to analyze beta catine in turnover. This is achieved by first adding and or depleting suspected effectors to activated xip egg extract in order to identify modulators of beta-catenin turnover as a second step exogenous in vitro, transcribed and translated beta-catenin, either radio labeled with S 35 or fused with luciferase is added to the XUS egg extract, which is actively degraded under native conditions. Next, collect time points in order to assess changes in beta katine and protein levels over time.Results are obtained that show whether a particular modulation of xenopus egg extract increases or decreases rates of beta catine and degradation based on auto radiography and or luminescence detection. The advantage of this technique over other existing methods, such as pulse chase or cycl heide chase experiments in cultured cells is that xenopus egg extract provides a cell-free biochemical system in which one can analyze protein regulation at the protein level, and it lacks the added complexity of active transcription. This method can help answer the key questions in the signal transduction field by identifying regulators of key signaling proteins such as beta catina.Working with xenopus extract enables the user to readily deplete components of a pathway and add back a defined amount of a protein. To determine its dose dependent effects, use freshly prepared XUS egg extract or quickly thaw, frozen extract and place on ice. Perform all manipulations in the cold, prepare pelleted antibody or affinity beads at one 10th the volume of the extract in order to minimize dilution of the extract, withdraw as much liquid from the beads as possible before addition of the extract.Using gel loading tips with long tapered ends here the extract is added to GST binding resin. Rotate the extract bead mix at four degrees Celsius for one hour. Then spin the extract bead mix at 12, 600 Gs in a micro fuge at four degrees Celsius for 30 seconds.Taking care not to transfer any beads with the extract. Transfer the depleted extract to a fresh micro fuge tube on ice. Confirm the efficiency of depletion by immuno blotting, both depleted extract and beads.Because T and degradation is energy dependent, it quickly depletes the endogenous A TP stores. Consequently, in order to maintain robust T degradation, you must use a energy regeneration mix. Prepare a 20 x energy regeneration or ER mix as detailed in the text protocol.Quickly thaw opus egg extract by rubbing the frozen tube between the hands. Place the tube on ice just before all of the extract has melted. Add 10 microliters of energy regeneration.Mix into an aliquot of xenopus egg extract. Mix thoroughly by quickly flicking the tube and pulse vortexing pulse spin, and immediately place on ice. Aliquot the appropriate volumes for the degradation assay into pre chilled micro fuge tubes on ice.To prepare for radio labeled beta katine and degradation assays, withdraw two to five microliters of extract for each time point to perform the radio labeled beta-Catenin degradation assay in XUS egg extract. First, prepare the radio labeled beta-catenin as described in the text protocol. Then add one to three microliters of in vitro translated beta-catenin to 20 microliters of xip reaction mix on ice mix thoroughly by a quick flicking of the tube and a short pulse of vortexing.This is an important step. A XUS egg extract is very viscous and incomplete mixing will affect the consistency of the results pulse, spin, and place on ice. Start the beta-catenin degradation reaction by shifting the tubes to room temperature at the designated time point.Remove one to five microliters of the sample and mix immediately with a five x volume of SDS sample buffer to stop the reaction to make sure the degradation reaction is completely terminated. Flick the tube several times and vortex vigorously to perform SDS page. Auto radiography run one microliter equivalence of the extract for each time point per lane.Results can be quantitated using image, J image quant, or another preferred imaging software. Degradation of beta-catenin in xap egg extract should be evidenced by the time dependent decrease in intensity of the radio labeled beta kaine in band. To prepare beta-catenin luciferase synthesize non radiolabeled luciferase tagged beta-catenin.Using the transcription translation coupled system with complete amino acid mix. Confirm production of the luciferase tagged beta-catenin by measuring luciferase activity from 0.5 to one microliter of the reaction. Assess the background luminescence by measuring luminescence from an untranslated reaction mix to perform the beta catine luciferase degradation assay First thaw and prepare xenopus egg extract as before.Then add the in vitro translated beta catine and luciferase fusion into the prepared xip reaction. Mix on ice and mix well. As before, shift the extract to room temperature to start the degradation reaction.Remove an aliquot of the reaction at the indicated time point and snap freeze in liquid nitrogen. Remove triplicate samples for analysis at each time point. Frozen extract can be stored at minus 80 degrees Celsius until ready to be analyzed.Thaw the samples on ice and transfer the samples to standard white 96 well plates on ice before processing. For luciferase activity, GS GSK three dependent degradation of beta-catenin can be readily demonstrated in multiple ways using XUS egg extract. Degradation of S 35 radio labeled beta-catenin in XUS extract is inhibited by addition of the proteasome inhibitor MG 1 32.The beta-catenin mutant is stabilized in XUS egg extract relative to wild type betaine and protein inhibitors of GSK three similarly inhibit degradation of beta-catenin. Finally, depletion of GSK three from xip egg extract inhibits degradation of beta-catenin degradation can be rescued by addition of exogenous GSK three. The beta-catenin luciferase degradation assay in xip egg extract was used to assess degradation of beta katine and luciferase.The rate of degradation of the beta katine and luciferase is similar to the untagged beta-catenin protein. The regulation of degradation for the beta katine and luciferase fusion is essentially identical to the non fusion protein. As the addition of lithium chloride resulted in inhibition of beta catine and luciferase turnover changes in the radioactive signal of the beta catina.Luciferase protein parallels the changes in luciferase enzymatic activity over time While attempting this procedure. It's important to keep all reagents on ice and to mix the reactions thoroughly before attempting this assay. After watching this video, you should have a good understanding of how to identify regulators and analyze beta catine and turnover using the cell-free xenopus egg extract system.

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Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Preparation and Fractionation of Xenopus laevis Egg Extracts

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Xenopus egg extract is a useful model system to investigate the DNA damage checkpoint. This protocol is for the preparation of Xenopus egg extracts and DNA damage checkpoint inducing reagents. These techniques are adaptable to a variety of DNA damaging approaches in the study of the DNA damage checkpoint signaling.

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Xenopus laevis as a Model to Identify Translation Impairment

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

Mechanisms of cellular and intra-cellular scaling remain elusive. The use of Xenopus embryo extracts has become increasingly common to elucidate mechanisms of organelle size regulation. This method describes embryo extract preparation and a novel nuclear scaling assay through which mechanisms of nuclear size regulation can be identified.

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

We present a method for the generation of in vitro self-sustained mitotic oscillations at the single-cell level by encapsulating egg extracts of Xenopus laevis in water-in-oil microemulsions.

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

We describe a method for the preparation and live imaging of undiluted cytoplasmic extracts from Xenopus laevis eggs.

Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization

Here, we describe a detailed protocol for isolating active nuclear extract from larval stage 4 C. elegans and visualizing transcription activity in an in vitro system.

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Learn to analyze beta-catenin degradation using Xenopus egg extract. This cell-free biochemical method enables identification of protein turnover modulators through radiolabeling and luminescence detection.

Reconstitution Of β-catenin Degradation In Xenopus Egg Extract

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