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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		<tr height="42">
			<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>
			<td style="width:301px;"></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
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			<td height="42" style="height:42px;width:216px;">BAS Storage phosphor screen 20x25 cm</td>
			<td style="width:71px;">GE Life Sciences</td>
			<td align="right" style="width:119px;">28956475</td>
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			<td height="21" style="height:21px;width:216px;">Bromophenol Blue</td>
			<td style="width:71px;">Sigma</td>
			<td>B8026</td>
			<td style="width:301px;">Molecular biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Carestream Biomax MR films</td>
			<td style="width:71px;">Kodak</td>
			<td>Z350370-50EA</td>
			<td></td>
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		<tr height="42">
			<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>
			<td style="width:71px;">Whatman</td>
			<td align="right" style="width:119px;">1872047</td>
			<td></td>
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			<td height="84" style="height:84px;width:216px;">Charcoal filtered pipette tips</td>
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			<td>5069B</td>
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			<td align="right" style="width:119px;">67221001</td>
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			<td height="21" style="height:21px;width:216px;">Coomassie Brilliant Blue R250</td>
			<td style="width:71px;">Sigma</td>
			<td>112,553</td>
			<td style="width:301px;">for electrophoresis</td>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dithiothreitol (DTT)</td>
			<td style="width:71px;">Sigma</td>
			<td>10197777001</td>
			<td style="width:301px;">Molecular biology grade</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">EasyTag Express35S Protein Labeling Mix</td>
			<td style="width:71px;">Perkin Elmer</td>
			<td>NEG772014MC</td>
			<td style="width:301px;">Other size batches of label are available depending on useage</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>
		</tr>
		<tr height="21">
			<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>
			<td style="width:301px;"></td>
		</tr>
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			<td height="21" style="height:21px;width:216px;">Glycerol</td>
			<td style="width:71px;">Sigma</td>
			<td>G5516</td>
			<td style="width:301px;">Molecular biology grade</td>
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			<td height="42" style="height:42px;width:216px;">Grade 3 chromatography paper</td>
			<td style="width:71px;">GE Life Sciences</td>
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			<td height="42" style="height:42px;width:216px;">HEPES</td>
			<td style="width:71px;">Sigma</td>
			<td>H4034</td>
			<td style="width:301px;">Molecular biology grade, Make 1M stock pH 7.4, store at 4&#730;C</td>
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			<td style="width:119px;">M3671</td>
			<td style="width:301px;">Molecular biology grade</td>
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			<td height="21" style="height:21px;width:216px;">Methanol</td>
			<td style="width:71px;">Sigma</td>
			<td>MX0490&#160;</td>
			<td style="width:301px;"></td>
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			<td height="42" style="height:42px;width:216px;">Methionine</td>
			<td style="width:71px;">Sigma</td>
			<td>M5308</td>
			<td style="width:301px;">Molecular biology grade, Make 250 mM stock, store at -20</td>
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			<td height="42" style="height:42px;width:216px;">Minigel casting/running 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;">NaCl</td>
			<td style="width:71px;">Sigma</td>
			<td>S7653</td>
			<td style="width:301px;">Molecular biology grade</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">N-ethylmaleimide</td>
			<td style="width:71px;">Sigma</td>
			<td>E3876</td>
			<td style="width:301px;">Molecular biology grade, Make 1M stock in 100% ethanol, store at -20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P5368</td>
			<td style="width:301px;">Molecular biology grade</td>
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			<td height="21" style="height:21px;width:216px;">Sodium Dodecyl Sulfate (SDS)</td>
			<td style="width:71px;">Sigma</td>
			<td>L3771</td>
			<td style="width:301px;">Molecular biology grade</td>
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			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:71px;">Sigma</td>
			<td>T8787</td>
			<td style="width:301px;">Molecular biology grade</td>
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			<td height="21" style="height:21px;width:216px;">Trizma base (Tris)</td>
			<td style="width:71px;">Sigma</td>
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</table>

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 ...

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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 ...

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 ...

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 ...

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 ...

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 ...

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, ...