Name: analysis of protein folding, transport, and degradation in living cells by radioactive pulse chase
Uploaded: 2019-02-12T13:30:00
Description: Watch this Scientific Journal Video about Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase at JoVE.com

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 p...

<ol>
	<li>Ellgaard, L., McCaul, N., Chatsisvili, A., Braakman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-+and+Post-Translational+Protein+Folding+in+the+ER.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Five+Questions+(with+their+Answers)+on+ER-Associated+Degradation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-Glycan-based+ER+Molecular+Chaperone+and+Protein+Quality+Control+System:+The+Calnexin+Binding+Cycle.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+topology+around+the+cleavage+site+regulate+post-translational+cleavage+of+the+HIV-1+gp160+signal+peptide.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+of+influenza+hemagglutinin+in+the+endoplasmic+reticulum.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinated+nonvectorial+folding+in+a+newly+synthesized+multidomain+protein.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+of+the+human+immunodeficiency+virus+type+1+envelope+glycoprotein+in+the+endoplasmic+reticulum.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+Transduction+of+Helper+T+Cells+as+a+Genetic+Approach+to+Study+Mechanisms+Controlling+their+Differentiation+and+Function.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+evolves+into+a+nonsyncytium-inducing+virus+upon+prolonged+culture+in+vitro.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cotranslational+folding+and+calnexin+binding+during+glycoprotein+synthesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-linked+glycans+direct+the+cotranslational+folding+pathway+of+influenza+hemagglutinin.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+Hsp47,+Grp78,+and+Grp94+with+procollagen+supports+the+successive+or+coupled+action+of+molecular+chaperones.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+primary+folding+defect+and+rescue+of+DeltaF508+CFTR+emerge+during+translation+of+the+mutant+domain.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+of+CFTR+is+predominantly+cotranslational.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posttranscriptional+Regulation+of+Glycoprotein+Quality+Control+in+the+Endoplasmic+Reticulum+Is+Controlled+by+the+E2+Ub-Conjugating+Enzyme+UBC6e.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+influenza+hemagglutinin+trimers+and+its+role+in+intracellular+transport.">Assembly of influenza hemagglutinin trimers and its role in intracellular transport.</a> Journal of Cell Biology. 103 (4), 1179-1191 (1986).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" style="width:707px;" width="707">
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			<td height="42" style="height:42px;width:216px;">1.5 mL safeseal microcentrifuge tubes</td>
			<td style="width:71px;">Sarstedt</td>
			<td style="width:119px;">72.706.400</td>
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			<td height="21" style="height:21px;width:216px;">Acetic Acid</td>
			<td style="width:71px;">Sigma</td>
			<td>A6283</td>
			<td style="width:301px;">glacial acetic acid</td>
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			<td>B8026</td>
			<td style="width:301px;">Molecular biology grade</td>
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			<td height="42" style="height:42px;width:216px;">Cell-culture media</td>
			<td style="width:71px;">Various</td>
			<td>N/A</td>
			<td style="width:301px;">Normal cell culture media for specific cell-lines used</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Cell-culture media, no methionine/cysteine</td>
			<td style="width:71px;">Various</td>
			<td style="width:119px;">N/A</td>
			<td style="width:301px;">Same media formulation as normal culture media e.g DMEM/MEM/RPMI, lacking methionine and cysteine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Charcoal filter paper</td>
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			<td style="width:71px;">Sigma</td>
			<td>112,553</td>
			<td style="width:301px;">for electrophoresis</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cysteine</td>
			<td style="width:71px;">Sigma</td>
			<td>C7352</td>
			<td style="width:301px;">Molecular biology grade, Make 500 mM stock, store at -20&#160;</td>
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			<td height="21" style="height:21px;width:216px;">Dithiothreitol (DTT)</td>
			<td style="width:71px;">Sigma</td>
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			<td style="width:301px;">Molecular biology grade</td>
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			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:71px;">Sigma</td>
			<td>E1644</td>
			<td style="width:301px;">Molecular biology grade</td>
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			<td height="21" style="height:21px;width:216px;">Gel-drying equipment</td>
			<td style="width:71px;">Various</td>
			<td style="width:119px;">N/A</td>
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			<td height="21" style="height:21px;width:216px;">Glycerol</td>
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			<td height="42" style="height:42px;width:216px;">Methionine</td>
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			<td style="width:301px;">Molecular biology grade, Make 1M stock in 100% ethanol, store at -20</td>
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			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
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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...

Watch this Scientific Journal Video about Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase at JoVE.com

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

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 ...

The radioactive pulse chase using 35 S-labeled methionine and cysteine is still the only biochemical methods to investigate protein biosynthesis with time in live cells. Protein biosynthesis covers both translation, folding and assembly, and trafficking and degradation. Combining the radioactive pulse chase with analysis of co-and post-translation protein modifications, such as disulfide bond formation and acetylation provides a sensitive and quantitative assay to investigate the protein's faith with time.Good focus and experimental handling is required for this procedure. Good preparation is crucial, like buffers, your radioactive workspace, and a clear schedule. This video provides a clear over the shoulder view of the radioactive pulse chase protocol.Demonstrating the procedure will be Hui Ying Yeoh, a pHd candidate from our laboratory. To begin this procedure, seed transfect and culture cells as outlined in the text protocol. Before the pulse chase, inspect your cells through the microscope.Next, wash the dishes with two milliliters of wash buffer. And two milliliters of starvation medium to the cells and place the dishes in a humidified incubator at 37 degrees Celsius with 5%carbon dioxide for 15 minutes. Make sure that your pulse chase set up is in order.Transfer the dishes to the racks in the water bath, pre-warmed to 37 degrees Celsius. Make sure the dishes are in contact with water, but do not float. Then, start a timer.At 40 seconds, aspirate the starvation medium. Using a micro pipet, draw up 600 microliters of pulse solution. At exactly one minute, gently add the pulse solution to the center of the dish.Repeat this process of removing the starvation medium and adding the pulse solution for the remaining dishes at one minute intervals. At exactly 11 minutes and for all following dishes, according to the pulse chase scheme, except for the zero minute chase sample, add two milliliters of chase medium directly to the dish. Aspirate the chase medium and replace it with two milliliters of fresh chase medium.Repeat this process for all remaining dishes at one minute intervals. When the timer reads exactly 16 minutes, add two militers of chase medium directly to the zero minute chase dish, on top of the pulse medium, to stop labeling. Immediately aspirate the medium.Transfer the dish to a cooled aluminum plate and add two militers of ice cold stop buffer. Transfer all other dishes to an incubator at 37 degrees Celsius. Transfer each chase dish back from the incubator to the water bath two minutes before the end of each chase time.At the exact chase time for each dish, aspirate the chase medium and transfer the dish to a cooled aluminum plate. Add two milliliters of ice cold stop buffer. Leave all dishes on ice and stop buffer until there is time to lyse the cells.Then aspirate the stop solution and wash all dishes. Three at the same time with two milliliters of ice cold stop solution. Aspirate the stop solution completely and add 600 microliters of lysis buffer to two dishes at a time.Using a cell scraper, scrape the surface of the dish thoroughly, but gently to mix the lysate. Transfer lysate from each dish to a fresh 1.5 milliliter micro centrifuge tube. Centrifuge at 15, 000 to 20, 000 G at four degree Celsius for 10 minutes to pellet the nuclei.For the pulse chase on suspension cells collection five million cells per time point in one 50 milliliter tube. Perform the starvation procedure as described in the text. After the starvation procedure in the water bath for 15 minutes, start the timer.At exactly one minute, add 275 microcuries of undiluted label directly to the tube containing cells and swirl to mix. At exactly 11 minutes, add four milliliters of chase media to stop the labeling. And immediately transfer one milliliter to a 15 milliliter tube on ice that contains nine milliliters of ice cold stop solution to stop labeling.Repeat this process of transferring one milliliter of chase to a tube on ice containing nine milliliters of stop solution for each successive chase time point. After the immunoprecipitation, aspirate the last wash completely. Re-suspend the beads in 20 microliters of TE buffer and vortex.Add 20 microliters of two X sample buffer without the reducing agent. Vortex the samples and then heat them at 95 degrees Celsius for five minutes. Vortex the samples again.Centrifuge at 12, 000 times G at room temperature for one minute. Transfer 19 microliters of the non-reduced supernatant to a fresh microcentrifuge tube that contains one microliter of 500 millimolar DDT. If needed, centrifuge the sample quickly so that all liquids are at the bottom.And heat at 95 degrees Celsius for five minutes. Let the reduced sample cool down and then vortex. Centrifuge both the reduced and non-reduced samples at 12, 000 times G at room temperature for one minute, and then add 1.1 microliters of one molar NEM to all the samples.In this study, a radioactive pulse chase approach is used to analyze protein folding and transport in intact cells. The folding and secretion of HIV one GP120 from an adherent pulse chase is shown here. The non-reducing gel shows the oxidative folding of GP120.Immediately after the pulse labeling, it appears as a diffuse band higher in the gel. As the chase progresses, the band migrates down the gel through even more diffuse folding intermediates until it accumulates in the tight band that represents natively folded at GP120. This occurs as the formation of disulfide bonds increases the compactness of the protein, causing it to migrate faster than the fully reduced protein.On the reducing gel, the disulfide bonds and all molecules have been reduced and do not affect mobiilty. As such, differences in mobility are only the result of changes in molecular mass. The shift over time from the reduced signal peptide uncleaved form to the reduced signal peptide cleaved form represents the post-translational signal peptide cleavage of GP120.The mobility increases due to the loss of the signal peptide which increases during the chase as more proteins attain the native fold and lose their signal peptide. On both the non-reducing and reducing gel, the signal begins to decrease from approximately one hour onward due to the secretion of GP120. This can be monitored by analyzing the media.This method is applicable to any cell line, including organoid cell models. But the success greatly depends on the expression level of the protein of interest. Before beginning this procedure, mark your dishes and keep this order throughout the experiment.Make sure everything is prepared before adding starvation medium. This is the start of the experiment. And your lab time is your friend, but can be your biggest foe when you're not prepared.Bill's chase can be combined with limited proteolysis to investigate the full estate of proteins with time. Also different elected freezers methods will allow analysis of rinse or rigormorization or charge differences. To protect the researcher and apparatus, all local radiation safety rules must be obeyed.And protective measures should be taken according to protocol. Note that your acrylamide, used for your STH page is neurotoxic.

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Cell Biology

Biochemistry

Unit 1

Transmembrane Transport in Endoplasmic Reticulum and Peroxisomes

SCIENTISTS IN ACTION

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins.
Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.

A streamlined protocol for performing an extensive biochemical and structural characterization of a carbohydrate substrate binding protein from Streptococcus pneumoniae is presented.

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in understanding how such pathways are organized and how they function. There are many methods to detect protein-protein interactions in cells, but relatively few can be used to detect interactions between endogenous proteins. One such method, the proximity ligation assay (PLA), has several advantages to recommend its use. Compared to other common methods of protein-protein interaction analysis, PLA has relatively high sensitivity and specificity, can be performed with minimal cell manipulation, and, in the protocol described herein, requires only two target-specific antibodies derived from different species (e.g., from mouse and rabbit) and one specialized reagent: a set of secondary antibodies that are covalently linked to specific oligonucleotides that, when brought in close proximity of one another, create an amplifiable platform for in situ PCR or rolling circle amplification. In this presentation, we show how to apply the PLA technique to visualize changes in MST1 and MST2 proximity in fixed cells. The technique described in this manuscript is particularly applicable for the analysis of cell signaling studies.

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

A protocol for the elucidation of plant thylakoid protein complex organization and composition with blue native polyacrylamide gel electrophoresis (BN-PAGE) and 2D-SDS-PAGE is described. The protocol is optimized for Arabidopsis thaliana, but can be used for other plant species with minor modifications.

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

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

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

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

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

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells

Transport of proteins and membranes from the cell surface to the Golgi and beyond is essential for homeostasis, organelle identity and physiology. To study retrograde protein traffic, we have recently developed a versatile nanobody-based toolkit to analyze transport from the cell surface to the Golgi complex, either by fixed and live cell imaging, by electron microscopy, or biochemically. We engineered functionalized anti-green fluorescent protein (GFP) nanobodies &#8212; small, monomeric, high-affinity protein binders &#8212; that can be applied to cell lines expressing membrane proteins of interest with an extracellular GFP moiety. Derivatized nanobodies bound to the GFP reporters are specifically internalized and transported piggyback along the reporters&#39; sorting routes. Nanobodies were functionalized with fluorophores to follow retrograde transport by fluorescence microscopy and live imaging, with ascorbate peroxidase 2 (APEX2) to investigate the ultrastructural localization of reporter-nanobody complexes by electron microscopy, and with tyrosine sulfation (TS) motifs to assess kinetics of trans-Golgi network (TGN) arrival. In this methodological article, we outline the general procedure to bacterially express and purify functionalized nanobodies. We illustrate the powerful use of our tool using the mCherry- and TS-modified nanobodies to analyze endocytic uptake and TGN arrival of cargo proteins.

Retrograde transport of proteins from the cell surface to the Golgi is essential to maintain membrane homeostasis. Here, we describe a method to biochemically analyze cell surface-to-Golgi transport of recombinant proteins using functionalized nanobodies in HeLa cells.

Transport of proteins and membranes from the cell surface to the Golgi and beyond is essential for homeostasis, organelle identity and physiology. To ...

Using the Open-Source MALDI TOF-MS IDBac Pipeline for Analysis of Microbial Protein and Specialized Metabolite Data

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, we developed IDBac&#8212;a low-cost and high-throughput matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) bioinformatics pipeline. IDBac software is designed for non-experts, is freely available, and capable of analyzing a few to thousands of bacterial colonies. Here, we present procedures for the preparation of bacterial colonies for MALDI-TOF MS analysis, MS instrument operation, and data processing and visualization in IDBac. In particular, we instruct users how to cluster bacteria into dendrograms based on protein MS fingerprints and interactively create Metabolite Association Networks (MANs) from specialized metabolite data.

IDBac is an open-source mass spectrometry-based bioinformatics pipeline that integrates data from both intact protein and specialized metabolite spectra, collected on cell material scraped from bacterial colonies. The pipeline allows researchers to rapidly organize hundreds to thousands of bacterial colonies into putative taxonomic groups, and further differentiate them based on specialized metabolite production.

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, ...