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.
