Name: Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase (Video) | JoVE Journal
Uploaded: 2019-02-12T13:30:00.000+00:00
Duration: 8 min 59 s
Description: Learn radioactive pulse-chase methods using 35S-labeled methionine and cysteine to analyze protein folding, transport, and degradation in live cells. This sensitive assay tracks protein biosynthesis kinetics over time.

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5 minutes

The authors thank all members of the Braakman lab, past and present, for their fruitful discussions and help in developing the methods presented in this article. This project has received funding from both the European Research Council under the European Union&#39;s Seventh Framework Programme (FP7/2007-2013) N&#176; 235649 and the Netherlands Organization of Scientific Research (NWO) under the ECHO-program N&#176; 711.012.008.

All radioactive reagents and procedures were handled in accordance with local Utrecht University radiation rules and regulations.
1. Pulse Chase
<ol>
	<li>Pulse Chase for Adherent Cells 
	NOTE: The volumes given here are based on 60 mm cell culture dishes. For 35 mm or 100 mm dishes, multiply the volumes by 1/2 or 2, respectively. This protocol uses a pulse time of 10 min and chase times of 0, 15, 30, 60, 120, and 240 min. These can be varied depending on the specific proteins being studied (discussed below).
	<ol>
		<li>Seed adherent cells (e.g., HEK 293, HeLa, CHO) in six 60&#160;mm cell culture dishes so that they will be subconfluent (80% - 90%) on the day of the pulse chase. Use at least one dish per time point and/or condition.</li>
		<li>If required, transfect the cells with commercially available transfection reagents according to the manufacturer&#39;s instructions; or, virally transduce8 the cells 1 day before the pulse-chase experiment with the appropriate construct for expression.</li>
		<li>Wash the dishes with 2 mL of wash buffer (Hank&#39;s balanced salt solution [HBSS]), add 2 mL of starvation medium (normal culture medium lacking methionine and cysteine and fetal bovine serum [FBS], such as&#160;minimum essential medium [MEM] without methionine and cysteine but with 10 mM HEPES, pH 7.4), and place the dishes in a 37 &#176;C humidified incubator with 5% CO2 for 15 min.</li>
		<li>Transfer the dishes to the racks in a prewarmed 37 &#176;C water bath so that they are in contact with water but do not float. Start a timer.</li>
		<li>At 40 s, aspirate the starvation medium, draw up 600 &#181;L of pulse solution (starvation media + 55 &#181;Ci/35 mm dish label, see the note preceding step 1.1.1) into the pipette, and add it gently to the center of the dish at exactly 1 min. Repeat this step at 1 min intervals for the remaining dishes.&#160;Start with the longest chase sample to save time during your experiment. 
		NOTE: When handling radioactive material, it is essential to follow appropriate precautions and local rules and regulations to prevent accidental exposure and/or contamination.</li>
		<li>At exactly 11 min and for all following dishes, except for the 0 min chase sample, add 2 mL of chase medium directly to the dish, aspirate it, and again, add 2 mL of chase medium. Repeat this step at 1 min intervals for remaining dishes. Transfer all dishes to a 37 &#176;C incubator.</li>
		<li>At exactly 16 min, add 2 mL of chase medium (normal culture medium + 10 mM HEPES [pH 7.4], 5 mM cysteine, and 5 mM methionine) directly to the 0 min sample dish on top of the pulse medium to stop labeling; then, aspirate immediately, transfer the dish to a cooled aluminum plate, and add 2 mL of ice-cold stop buffer (HBSS + 20 mM N-ethylmaleimide [NEM]). 
		NOTE: This is the 0 min chase sample. For this sample, proceed directly to step 1.1.9.</li>
		<li>Transfer each chase dish back to the water bath 2 min before each chase time (e.g., 24 min for a 15 min chase) and, at the exact chase time (e.g., 26 min for a 15 min chase), aspirate the chase media (or transfer it to a microcentrifuge tube if following protein secretion) and transfer the dish to a cooled aluminum plate. Add 2 mL of ice-cold stop buffer.</li>
		<li>Incubate all dishes on ice in the stop buffer for &#8805;5 min; then, aspirate the stop solution and wash it with 2 mL of ice-cold stop solution. Aspirate the wash and lyse dishes with 600 &#181;L of lysis buffer (phosphate-buffered saline [PBS] + nondenaturing detergent [see Table of Materials] + protease inhibitors + 20 mM NEM). Use a cell scraper to ensure that the dishes are lysed quantitatively.</li>
		<li>Transfer the lysate to a 1.5 mL microcentrifuge tube and centrifuge for 10 min at 15,000 - 20,000 x g and 4 &#176;C to pellet the nuclei.</li>
	</ol>
	</li>
	<li>Pulse Chase for Suspension Cells 
	NOTE: To ensure efficient labeling, cells should be pulsed at a concentration of 3 x 106 to 5 x 106 cells/mL, and chase volumes should be 4x the pulse volume. In the following example, we pulsed 5 x 106 cells in a volume of 1 mL for 10 min, to yield five chase time points (0, 15, 30, 60, and 120 min) of 1 mL, containing 1 x 106 cells per time point. All solutions are the same as in section 1.1.
	<ol>
		<li>Culture the suspension cells (e.g., 3T3, Jurkat) according to a previously published protocol9 so that there is a sufficient number of cells at the time of the experiment, at least 1 x 106 cells per time point and/or condition.</li>
		<li>If required, transfect cells with commercially available transfection reagents according to the manufacturer&#39;s instructions, or virally transduce8 the cells 1 day before the pulse-chase experiment with the appropriate construct for expression.</li>
		<li>Pellet 5 x 106 cells per condition in a 50 mL tube for 5 min at 250 x g at room temperature, wash them 1x with 5 mL of starvation media, pellet them again, and resuspend them in 1 mL of starvation media.</li>
		<li>Transfer the cells to a 37 &#176;C water bath and incubate them for 10 - 25 min. Agitate the tubes every 10 - 15 min to prevent the cells from settling at the bottom.</li>
		<li>Start the timer. At exactly 1 min, add 275 &#181;Ci (55 &#181;Ci/1 x 106 cells) of undiluted label directly to the tube containing cells and swirl to mix. 
		NOTE: When handling radioactive material, it is essential to follow appropriate precautions and local rules and regulations to prevent accidental exposure and/or contamination.</li>
		<li>At exactly 11 min, stop labeling by adding 4 mL of chase media. Mix the sample and immediately transfer 1 mL to a 15 mL tube on ice, containing 9 mL of ice-cold stop solution. 
		NOTE: This is the 0 min chase sample.</li>
		<li>Repeat these steps for each successive time point. Once all time points are collected, pellet the cells for 5 min at 250 x g at 4 &#176;C. Aspirate the medium (or transfer it to a new 15 mL tube if following protein secretion). Wash the cells with 5 mL of stop solution and pellet the cells for 5 min at 250 x g at 4 &#176;C.</li>
		<li>Aspirate the stop solution, completely lyse the cells with 300 &#181;L of ice-cold lysis buffer and incubate them for 20 min on ice to ensure a complete lysis. Transfer the lysate to a 1.5 mL microcentrifuge tube and centrifuge for 10 min at 15,000 - 20,000 x g at 4 &#176;C to pellet the nuclei.</li>
	</ol>
	</li>
</ol>
2. Immunoprecipitation
<ol>
	<li>Combine antibody (see the discussion) and 50 &#181;L of immunoprecipitation beads (e.g., protein A-Sepharose) (10% suspension [v/v] in lysis buffer + 0.25% bovine serum albumin [BSA]) in a microcentrifuge tube and incubate them at 4 &#176;C for ~30 min in a shaker.</li>
	<li>Pellet the beads for 1 min at 12,000 x g at room temperature and aspirate the supernatant. Add 200 &#181;L of lysate to the antibody-bead mixture and incubate at 4 &#176;C in a shaker for 1 h or head-over-head if the immunoprecipitation requires &#62;1 h.</li>
	<li>Pellet the beads for 1 min at 12,000 x g at room temperature. Aspirate the supernatant and add 1 mL of immunoprecipitation wash buffer. Place the sample in a shaker at room temperature for 5 min.</li>
	<li>Pellet the beads as described in step 2.3 and repeat the wash 1x. Then, aspirate the supernatant and resuspend the beads in 20 &#181;L of TE buffer, pH 6.8 (10 mM Tris-HCl, 1 mM EDTA). Vortex the sample.</li>
	<li>Add 20 &#181;L of 2x sample buffer without the reducing agent, vortex it, heat it for 5 min at 95 &#176;C, and vortex again. 
	NOTE: If only preparing reducing samples, 2 &#181;L of 500 mM DTT should be added at this point. Then, proceed to step 2.7.</li>
	<li>Pellet the beads as described in step 2.3. Transfer 19 &#181;L of the nonreduced supernatant to a fresh microcentrifuge tube containing 1 &#181;L of 500 mM DTT, centrifuge the sample, and vortex it before heating it for 5 min at 95 &#176;C again.</li>
	<li>Spin down the sample for 1 min at 12,000 x g; this is the reduced sample. Cool it down to room temperature and add 1.1 &#181;L of 1 M NEM to both the reduced and the nonreduced sample. Vortex and spin down the samples.</li>
</ol>
3. SDS-PAGE
<ol>
	<li>First, determine the appropriate SDS-PAGE resolving gel percentage for the protein of interest. For example, HIV-1 gp120, when deglycosylated, runs at ~60 kDa and is analyzed in a 7.5% gel.</li>
	<li>Prepare the resolving gel mixture without TEMED according to the manufacturer&#39;s instructions (x% acrylamide, 375 mM Tris-HCl [pH 8.8,] 0.1% SDS [w/v], and 0.05% ammonium persulfate [APS] [w/v]) and degas under vacuum for &#62;15 min. While the gel mixture is degassing, thoroughly clean the gel glass plates with 70% ethanol and lint-free tissues and place them into a casting apparatus.</li>
	<li>Add TEMED to the resolving gel mixture (at a final concentration of 0.005% [v/v]), mix thoroughly, and pipette between glass plates, leaving ~1.5 cm space for the stacking gel. Carefully overlay the gel with deionized H2O or isopropanol and leave it to polymerize.</li>
	<li>Once the resolving gel has polymerized, prepare the stacking gel mixture (4% acrylamide, 125 mM Tris-HCl [pH 6.8], 0.1% SDS [w/v], and 0.025% APS [w/v]).</li>
	<li>Flush the top of the resolving gel with deionized H2O and, then, remove all water. 
	NOTE: Use filter paper to remove the last drops.</li>
	<li>Add TEMED to the stacking gel mixture (0.005% [v/v]), mix thoroughly, and overlay the resolving gel with stacking gel and insert a 15-well comb. Once the stacking gel has polymerized, transfer it to a running chamber and fill the upper and lower chambers with running buffer (25 mM Tris-HCl, 192 mM glycine [pH 8.3], and 0.1% SDS [w/v]).</li>
	<li>Load 10 &#181;L of sample per lane in a 15-lane minigel. Avoid loading the samples in the first and the last lane on the gel and load the nonreducing sample buffer in all empty lanes to prevent the smiling of bands. Run the gels at constant a 25 mA/gel until the dye front is at the bottom of the gel.</li>
	<li>Remove the gels from the glass plates, stain the gels with protein-staining solution (10% acetic acid and 30% methanol in H2O + 0.25% brilliant blue R250 [w/v]) for 5 min with agitation, and then, destain for 30 min with destaining solution (staining solution without brilliant blue R250).</li>
	<li>Arrange the gels face-down on a plastic wrap and, then, place 0.4 mm chromatography paper on top of them. Place the gel sandwich chromatography paper-side down onto a gel dryer. Following the manufacturer&#39;s instructions, dry the gels for 2 h at 80 &#176;C.</li>
	<li>Transfer the dried gels to a cassette and overlay them with autoradiography film or phosphor screen. If using autoradiography film, this step must be performed in a dark room.</li>
</ol>

<ol><li>Ellgaard, L., McCaul, N., Chatsisvili, A., Braakman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Co-+and+Post-Translational+Protein+Folding+in+the+ER%5BTitle%5D)+AND+%22Traffic%22%5BJournal%5D)">Co- and Post-Translational Protein Folding in the ER.</a> Traffic. 17 (6), 615-638 (2016).</li>
<li>Pisoni, G. B., Molinari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Five+Questions+%28with+their+Answers%29+on+ER-Associated+Degradation%5BTitle%5D)+AND+%22Traffic%22%5BJournal%5D)">Five Questions (with their Answers) on ER-Associated Degradation.</a> Traffic. 17 (4), 341-350 (2016).</li>
<li>Lamriben, L., Graham, J. B., Adams, B. M., Hebert, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((N-Glycan-based+ER+Molecular+Chaperone+and+Protein+Quality+Control+System%3A+The+Calnexin+Binding+Cycle%5BTitle%5D)+AND+%22Traffic%22%5BJournal%5D)">N-Glycan-based ER Molecular Chaperone and Protein Quality Control System: The Calnexin Binding Cycle.</a> Traffic. 17 (4), 308-326 (2016).</li>
<li>Snapp, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure+and+topology+around+the+cleavage+site+regulate+post-translational+cleavage+of+the+HIV-1+gp160+signal+peptide%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">Structure and topology around the cleavage site regulate post-translational cleavage of the HIV-1 gp160 signal peptide.</a> Elife. 6, 26067(2017).</li>
<li>Braakman, I., Hoover-Litty, H., Wagner, K. R., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Folding+of+influenza+hemagglutinin+in+the+endoplasmic+reticulum%5BTitle%5D)+AND+%22Journal+of+Cell+Biology%22%5BJournal%5D)">Folding of influenza hemagglutinin in the endoplasmic reticulum.</a> Journal of Cell Biology. 114 (3), 401-411 (1991).</li>
<li>Jansens, A., van Duijn, E., Braakman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coordinated+nonvectorial+folding+in+a+newly+synthesized+multidomain+protein%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Coordinated nonvectorial folding in a newly synthesized multidomain protein.</a> Science. 298 (5602), 2401-2403 (2002).</li>
<li>Land, A., Braakman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Folding+of+the+human+immunodeficiency+virus+type+1+envelope+glycoprotein+in+the+endoplasmic+reticulum%5BTitle%5D)+AND+%22Biochimie%22%5BJournal%5D)">Folding of the human immunodeficiency virus type 1 envelope glycoprotein in the endoplasmic reticulum.</a> Biochimie. 83 (8), 783-790 (2001).</li>
<li>Singh, Y., Garden, O. A., Lang, F., Cobb, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Retroviral+Transduction+of+Helper+T+Cells+as+a+Genetic+Approach+to+Study+Mechanisms+Controlling+their+Differentiation+and+Function%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function.</a> Journal of Visualized Experiments. (117), e54698(2016).</li>
<li>Das, A. T., Land, A., Braakman, I., Klaver, B., Berkhout, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HIV-1+evolves+into+a+nonsyncytium-inducing+virus+upon+prolonged+culture+in+vitro%5BTitle%5D)+AND+%22Virology%22%5BJournal%5D)">HIV-1 evolves into a nonsyncytium-inducing virus upon prolonged culture in vitro.</a> Virology. 263 (1), 55-69 (1999).</li>
<li>Chen, W., Helenius, J., Braakman, I., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cotranslational+folding+and+calnexin+binding+during+glycoprotein+synthesis%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">Cotranslational folding and calnexin binding during glycoprotein synthesis.</a> Proceedings of the National Academy of Sciences. 92 (14), 6229-6233 (1995).</li>
<li>Daniels, R., Kurowski, B., Johnson, A. E., Hebert, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((N-linked+glycans+direct+the+cotranslational+folding+pathway+of+influenza+hemagglutinin%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">N-linked glycans direct the cotranslational folding pathway of influenza hemagglutinin.</a> Molecular Cell. 11 (1), 79-90 (2003).</li>
<li>Ferreira, L. R., Norris, K., Smith, T., Hebert, C., Sauk, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Association+of+Hsp47%2C+Grp78%2C+and+Grp94+with+procollagen+supports+the+successive+or+coupled+action+of+molecular+chaperones%5BTitle%5D)+AND+%22Journal+of+Cellular+Biochemistry%22%5BJournal%5D)">Association of Hsp47, Grp78, and Grp94 with procollagen supports the successive or coupled action of molecular chaperones.</a> Journal of Cellular Biochemistry. 56 (4), 518-526 (1994).</li>
<li>Hoelen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+primary+folding+defect+and+rescue+of+DeltaF508+CFTR+emerge+during+translation+of+the+mutant+domain%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">The primary folding defect and rescue of DeltaF508 CFTR emerge during translation of the mutant domain.</a> PLoS One. 5 (11), 15458(2010).</li>
<li>Kleizen, B., van Vlijmen, T., de Jonge, H. R., Braakman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Folding+of+CFTR+is+predominantly+cotranslational%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">Folding of CFTR is predominantly cotranslational.</a> Molecular Cell. 20 (2), 277-287 (2005).</li>
<li>Hagiwara, M., Ling, J., Koenig, P. A., Ploegh, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Posttranscriptional+Regulation+of+Glycoprotein+Quality+Control+in+the+Endoplasmic+Reticulum+Is+Controlled+by+the+E2+Ub-Conjugating+Enzyme+UBC6e%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">Posttranscriptional Regulation of Glycoprotein Quality Control in the Endoplasmic Reticulum Is Controlled by the E2 Ub-Conjugating Enzyme UBC6e.</a> Molecular Cell. 63 (5), 753-767 (2016).</li>
<li>Copeland, C. S., Doms, R. W., Bolzau, E. M., Webster, R. G., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assembly+of+influenza+hemagglutinin+trimers+and+its+role+in+intracellular+transport%5BTitle%5D)+AND+%22Journal+of+Cell+Biology%22%5BJournal%5D)">Assembly of influenza hemagglutinin trimers and its role in intracellular transport.</a> Journal of Cell Biology. 103 (4), 1179-1191 (1986).</li>
</ol>

The authors have nothing to disclose.

Pulse-chase methods have been essential for developing scientists&#39; understanding of protein folding in intact cells. While we have attempted to provide a method that is as general as possible, this approach has the potential for almost limitless variations to study various processes that occur during the folding, the transport, and the life of proteins inside the cell.
When performing a pulse chase using adherent cells in dishes, it is essential to treat each dish the same as much as possi...

The folding of even relatively simple proteins involves many different folding enzymes, molecular chaperones, and covalent modifications1. A complete reconstitution of these processes in vitro is practically impossible, given the vast number of different components involved. It is highly desirable, therefore, to study protein folding in vivo, in live cells. Radioactive pulse-chase techniques prove a powerful tool for studying the synthesis, folding, transport, and degradation of proteins in their natural environment.
The metabolic labeling of proteins during a short pulse with 35S-labeled methionine/cysteine, followed by a chase in the absence of a radioactive label, allows specific tracking of a population of newly synthesized proteins in the wider cellular milieu. Then, target proteins can be isolated via immunoprecipitation and analyzed via SDS-PAGE or other techniques. For many proteins, their journey through the cell is marked by modifications that are visible on SDS-PAGE gel. For example, the transport of glycosylated proteins from the endoplasmic reticulum (ER) to the Golgi complex is often accompanied by modifications of N-linked glycans or the addition of O-linked glycans2,3. These modifications cause large increases in the apparent molecular mass, which can be seen by mobility changes in SDS-PAGE. Maturation can also be marked by proteolytic cleavages, such as signal-peptide cleavage or the removal of pro-peptides, resulting in changes in the apparent molecular mass that can be followed easily on SDS-PAGE gel4. Radioactivity has considerable advantages over comparable techniques such as cycloheximide chases, where novel protein synthesis is prevented, as longer treatments are toxic to cells and do not exclude the majority of older, steady-state proteins from the analysis, as some proteins have half-lives of days. The comparison of proteins under both nonreducing and reducing conditions allows the analysis of disulfide bond formation, an important step in the folding of many secretory proteins4,5,6,7.
Here we describe a general method for the analysis of protein folding and transport in intact cells, using a radioactive pulse-chase approach. While we have aimed to provide the method as detailed as possible, the protocol has an almost limitless potential for adaptability and will allow optimization to study each reader&#39;s specific proteins.
Two alternative pulse-chase protocols, one for adherent cells (step 1.1 of the protocol presented here) and one for suspension cells (step 1.2 of the protocol presented here) are provided. The conditions provided here are sufficient to visualize a protein expressed with medium- to high-expression levels. If the reader is working with poorly expressed proteins or various posttreatment conditions, such as multiple immunoprecipitations, it is necessary to increase the dish size or cell number appropriately.
For suspension pulse chase, the chase samples taken at each time point are all taken from a single tube of cells. The wash steps after the pulse are omitted; instead, further incorporation of 35S is prevented by dilution with a high excess of unlabeled methionine and cysteine.
The presented protocols use radioactive 35S-labeled cysteine and methionine to follow cellular protein-folding processes. All operations with radioactive reagents should be performed using appropriate protective measures to minimize any exposure of the operator and the environment to radioactive radiation and be performed in a designated laboratory. As the pulse-chase labeling technique is relatively inefficient at short pulse times (&#60;15 min), less than 1% of the starting amount of radioactivity is incorporated in the newly synthesized proteins. After the enrichment of the target protein via immunoprecipitation, the sample for SDS-PAGE contains less than 0.05% of the starting amount of radioactivity.
Although the 35S methionine and cysteine labeling mix is stabilized, some decomposition, yielding volatile radioactive compounds, will occur. To protect the researcher and the apparatus, some precautions should be taken. The researcher should always obey the local radiation safety rules and may wear a charcoal nursing mask, besides a lab coat and (double) gloves. Stock vials with 35S methionine and cysteine should always be opened in a fume hood, or under a local aspiration point. Known laboratory contamination spots are centrifuges, pipettes, water baths, incubators, and shakers. The contamination of these areas is reduced by using pipette tips with a charcoal filter, positive-seal microcentrifuge tubes (see Table of Materials), aquarium charcoal sponges in water baths, charcoal filter papers glued in the pulse-chase dishes, charcoal guard in the aspiration system, and the placement of dishes containing charcoal grains in incubators and storage containers.

<table><tbody><tr><td>1.5 mL safeseal microcentrifuge tubes</td><td>Sarstedt</td><td>72.706.400</td><td></td></tr><tr><td>Acetic Acid</td><td>Sigma</td><td>A6283</td><td>glacial acetic acid</td></tr><tr><td>BAS Storage phosphor screen 20x25 cm</td><td>GE Life Sciences</td><td>28956475</td><td></td></tr><tr><td>Bromophenol Blue</td><td>Sigma</td><td>B8026</td><td>Molecular biology grade</td></tr><tr><td>Carestream Biomax MR films</td><td>Kodak</td><td>Z350370-50EA</td><td></td></tr><tr><td>Cell-culture media</td><td>Various</td><td>N/A</td><td>Normal cell culture media for specific cell-lines used</td></tr><tr><td>Cell-culture media, no methionine/cysteine</td><td>Various</td><td>N/A</td><td>Same media formulation as normal culture media e.g DMEM/MEM/RPMI, lacking methionine and cysteine</td></tr><tr><td>Charcoal filter paper</td><td>Whatman</td><td>1872047</td><td></td></tr><tr><td>Charcoal filtered pipette tips</td><td>Molecular bioproducts</td><td>5069B</td><td></td></tr><tr><td>Charcoal vacu-guard</td><td>Whatman</td><td>67221001</td><td></td></tr><tr><td>Coomassie Brilliant Blue R250</td><td>Sigma</td><td>112,553</td><td>for electrophoresis</td></tr><tr><td>Cysteine</td><td>Sigma</td><td>C7352</td><td>Molecular biology grade, Make 500 mM stock, store at -20&amp;nbsp;</td></tr><tr><td>Dithiothreitol (DTT)</td><td>Sigma</td><td>10197777001</td><td>Molecular biology grade</td></tr><tr><td>EasyTag Express35S Protein Labeling Mix</td><td>Perkin Elmer</td><td>NEG772014MC</td><td>Other size batches of label are available depending on useage</td></tr><tr><td>EDTA</td><td>Sigma</td><td>E1644</td><td>Molecular biology grade</td></tr><tr><td>Gel-drying equipment</td><td>Various</td><td>N/A</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td>Molecular biology grade</td></tr><tr><td>Grade 3 chromatography paper</td><td>GE Life Sciences</td><td>3003-917</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS)</td><td>Gibco</td><td>24020117</td><td></td></tr><tr><td>Kimwipes delicate task wipes</td><td>VWR</td><td>21905-026</td><td></td></tr><tr><td>MES</td><td>Sigma&amp;nbsp;</td><td>M3671</td><td>Molecular biology grade</td></tr><tr><td>Methanol</td><td>Sigma</td><td>MX0490&amp;nbsp;</td><td></td></tr><tr><td>Methionine</td><td>Sigma</td><td>M5308</td><td>Molecular biology grade, Make 250 mM stock, store at -20</td></tr><tr><td>Minigel casting/running equipment</td><td>Various</td><td>N/A</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S7653</td><td>Molecular biology grade</td></tr><tr><td>N-ethylmaleimide</td><td>Sigma</td><td>E3876</td><td>Molecular biology grade, Make 1M stock in 100% ethanol, store at -20</td></tr><tr><td>PBS</td><td>Sigma</td><td>P5368</td><td>Molecular biology grade</td></tr><tr><td>Protein-A Sepharose fastflow beads</td><td>GE health-care</td><td>17-5280-04</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>Sigma</td><td>L3771</td><td>Molecular biology grade</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td>Molecular biology grade</td></tr><tr><td>Trizma base (Tris)</td><td>Sigma</td><td>T6066</td><td>Molecular biology grade</td></tr><tr><td>Typhoon IP Biomolecular imager</td><td>Amersham</td><td>29187194</td><td></td></tr><tr><td>Unwire Test Tube Rack 20 mm for waterbath</td><td>Nalgene</td><td>5970-0320PK</td><td></td></tr></tbody></table>

analysis of protein folding, transport, and degradation in living cells by radioactive pulse chase

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

The folding and secretion of HIV-1 gp120 from an adherent pulse chase is shown in Figure 2. The nonreducing gel (Cells NR in the figure) shows the oxidative folding of gp120. Immediately after the pulse labeling of 5 min (0 min chase) gp120 appears as a diffuse band higher in the gel, and as the chase progresses, the band migrates down the gel through even more diffused folding intermediates (IT) until it accumulates in the tight band (NT) that represents nat...

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

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Research

JoVE Journal

Biochemistry

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

This video describes a degradation assay for misfolded proteins using cycloheximide treatment along with detergent fractionation. This method aids in studying the dynamics of misfolded proteins and uncovering the in-depth mechanisms of protein turnover.

Misfolding-Prone Protein Degradation Assay: A Technique to Monitor Misfolded Protein Degradation Using Cycloheximide Treatment and Detergent Fractionation

This video describes an in vitro fluorescence-based microplate assay to study the degradation kinetics of a fluorescently-labeled misfolded luciferase mutant protein expressed in the nuclei of transfected mammalian cells. The assay involves the treatment of cells expressing the fluorescent mutant protein with a translation inhibitor and a proteasome inhibitor to assess the proteasome-mediated degradation of the misfolded protein.

Fluorescence Microplate-Based Cycloheximide Chase Assay: A Technique to Monitor the Degradation Kinetics of Fluorescent Nuclear Misfolded Proteins

Cellular viability depends on timely and efficient management of protein misfolding.  Here we describe a method for visualizing the different potential fates of a misfolded protein:  refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

4D Imaging of Protein Aggregation in Live Cells

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae.  Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

Assays for the Degradation of Misfolded Proteins in Cells

This protocol describes a method to sensitively measure the nucleocytoplasmic transport rate within motor neuron-like NSC-34 cells by quantifying the real-time change in the nuclear import of a NLS-NES-GFP protein.

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

This protocol describes a method to determine protein half-lives in single living adherent cells, using pulse labeling and fluorescence time-lapse imaging of SNAP-tag fusion proteins.

Single-Cell Quantification of Protein Degradation Rates by Time-Lapse Fluorescence Microscopy in Adherent Cell Culture

To overcome the limitations of classical site-directed mutagenesis, proline analogs with specific modifications were incorporated into several fluorescent proteins. We show how the replacement of hydrogen by fluorine or of the single by double bonds in proline residues ("molecular surgery") affects fundamental protein properties, including their folding and interaction with light.

Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability

Learn radioactive pulse-chase methods using 35S-labeled methionine and cysteine to analyze protein folding, transport, and degradation in live cells. This sensitive assay tracks protein biosynthesis kinetics over time.

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

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