This work was supported by funds from the Research Grants Council Hong Kong, CUHK direct grant scheme, United College endowment fund, and the TUYF Charitable Trust. The figures in this work were adapted from our previous publication, &#34;ARF6-Rac1 signaling-mediated neurite outgrowth is potentiated by the neuronal adaptor FE65 through orchestrating ARF6 and ELMO1&#34; published in the FASEB Journal in October 2020.

1. Mammalian cell culture and transfection
<ol>
	<li>Plate 2 x 106 cells in a 100 mm culture dish. Use four dishes for each transfection. 
	NOTE: The number of cells required may vary for different cell lines. Optimization may be necessary before proceeding to the isolation step.</li>
	<li>The next day, transfect the cells with Lipofectamine according to the manufacturer&#39;s instructions.</li>
</ol>
2. Cell harvest
<ol>
	<li>Discard the culture medium 48 h post-transfection.</li>
	<li>Wash the cells with ice-cold PBS (10 mM sodium phosphates, 2.68 mM potassium chloride, 140 mM sodium chloride) twice.</li>
	<li>Add 1 mL of ice-cold PBS+ (PBS supplemented with 0.5x protease inhibitor cocktail and 0.5x phosphatase inhibitor cocktail) to each dish.</li>
	<li>Collect the cells with a cell scraper and transfer the cell suspension to a 15 mL centrifuge tube.</li>
	<li>Pellet the cells by centrifugation using a swing bucket rotor at 400 x g for 5 min.</li>
	<li>Discard the supernatant and resuspend the cell pellet gently in 5 mL of homogenization buffer (HB; 250 mM sucrose, 3 mM imidazole at pH 7.4, 1 mM EDTA supplemented with 0.03 mM cycloheximide, 1x protease inhibitor cocktail, and 1x phosphatase inhibitor cocktail).</li>
	<li>Collect the cells by centrifugation at 1,300 x g for 10 min.</li>
	<li>Resuspend the cell pellet in 1 mL of HB.</li>
	<li>Homogenize the cells with a Dounce homogenizer for 15-20 strokes. 
	NOTE: Other homogenization methods, for example, passing the sample through a syringe, could be used. The homogenization efficiency could be revealed by observing the homogenate under a phase-contrast microscope.</li>
	<li>Transfer the homogenate to a 2 mL centrifugation tube. 
	NOTE: Harvest 50 &#181;L of homogenate with 12.5 &#181;L of 5x sample buffer and label it as total lysate.</li>
	<li>Add 0.7 mL of HB to the homogenate.</li>
	<li>Spin the diluted homogenate at 2,000 x g for 10 min at 4 &#176;C. 
	NOTE: The pellet contains nuclei and unbroken cells.</li>
	<li>Collect 1.5 mL of the supernatant and repeat step 2.12 once.</li>
	<li>Collect 1.4 mL of the supernatant and label it as post-nuclear supernatant (PNS).</li>
</ol>
3. Density gradient column preparation
<ol>
	<li>Transfer 1.2 mL of PNS to an ultracentrifuge tube.</li>
	<li>Add 1 mL of 62% sucrose solution (2.351 M sucrose, 3 mM imidazole at pH 7.4) to the sample and mix well by gentle pipetting. 
	NOTE: The resultant solution is a 40.6% sucrose solution.</li>
	<li>Add 3.3 mL of 35% sucrose solution (1.177 M sucrose, 3 mM imidazole at pH 7.4) carefully on top of the sample.</li>
	<li>Add 2.2 mL of 25% sucrose solution (0.806 M sucrose, 3 mM imidazole at pH 7.4) carefully on top of the 35% sucrose solution. 
	NOTE: The refractive index of the 62%, 40.6%, 35%, and 25% sucrose solutions at room temperature are 1.44, 1.40, 1.39, and 1.37, respectively. The refractive indexes of the sucrose solutions&#160;could be checked with a refractometer to ensure the precision and consistency of the experiment.</li>
	<li>Fill up the ultracentrifugation tube with HB. 
	&#8203;NOTE: Temporarily store the prepared density gradient column at 4 &#176;C.</li>
</ol>
4. Fractionation and recovery of recycling endosome-containing fraction
<ol>
	<li>Centrifuge the column at 210,000 x g for 3 h at 4 &#176;C.</li>
	<li>Collect 12 fractions (1 mL each) carefully, starting from the top of the gradient. 
	NOTE: The recycling endosomes should be found at the interface between 35% and 25% sucrose solutions. The collected fractions can be snap-frozen in liquid nitrogen and stored at -80 &#176;C.</li>
	<li>Dilute all the fractions with 1 mL of dilution buffer (3 mM imidazole at pH 7.4, 1 mM EDTA).</li>
	<li>Centrifuge the diluted sample at 100,000 x g for 1 h at 4 &#176;C.</li>
	<li>Aspirate the supernatant and add 50 &#181;L of 1x sample buffer to harvest the fractions.</li>
	<li>Analyze the protein contents in the fractions by western blotting.</li>
</ol>

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytic+pathways+and+endosomal+trafficking:+a+primer.">Endocytic pathways and endosomal trafficking: a primer.</a> Wiener Medizinische Wochenschrift. 166 (7-8), 196-204 (2016).</li><li>Kumari, S., Mg, S., Mayor, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytosis+unplugged:+multiple+ways+to+enter+the+cell.">Endocytosis unplugged: multiple ways to enter the cell.</a> Cell Research. 20 (3), 256-275 (2010).</li><li>Naslavsky, N., Caplan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enigmatic+endosome+-+sorting+the+ins+and+outs+of+endocytic+trafficking.">The enigmatic endosome - sorting the ins and outs of endocytic trafficking.</a> Journal of Cell Science. 131 (13), (2018).</li><li>Cullen, P. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Explosion+in+the+complexity+of+membrane+protein+recycling.">Explosion in the complexity of membrane protein recycling.</a> American Journal of Physiology. Cell Physiology. 320 (4), 483-494 (2021).</li><li>O'Sullivan, M. J., Lindsay, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Endosomal+Recycling+pathway-at+the+crossroads+of+the+cell.">The Endosomal Recycling pathway-at the crossroads of the cell.</a> International Journal of Molecular Sciences. 21 (17), 6074 (2020).</li><li>D'Souza-Schorey, C., Li, G., Colombo, M. I., Stahl, P. 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T., 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=Sphingolipids+inhibit+endosomal+recycling+of+nutrient+transporters+by+inactivating+ARF6.">Sphingolipids inhibit endosomal recycling of nutrient transporters by inactivating ARF6.</a> Journal of Cell Science. 131 (12), (2018).</li><li>Lu, 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=APE1+upregulates+MMP-14+via+redox-sensitive+ARF6-mediated+recycling+to+promote+cell+invasion+of+esophageal+adenocarcinoma.">APE1 upregulates MMP-14 via redox-sensitive ARF6-mediated recycling to promote cell invasion of esophageal adenocarcinoma.</a> Cancer Research. 79 (17), 4426-4438 (2019).</li><li>Qi, S., 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=Arf6-driven+endocytic+recycling+of+CD147+determines+HCC+malignant+phenotypes.">Arf6-driven endocytic recycling of CD147 determines HCC malignant phenotypes.</a> Journal of Experimental and Clinical Cancer Research. 38 (1), 471 (2019).</li><li>Crupi, M. 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The authors declare that they have no conflicts of interest with the contents of this article.

The above protocol outlines the procedures for isolating recycling endosomes from cultured cells by ultracentrifugation. The reliability of this method has been demonstrated by the latest publication22, proving that recycling endosomes are successfully isolated from other organelles (Figure 1), such as the Golgi apparatus and mitochondria. Some critical steps need to be paid attention to for obtaining a good separation result. While preparing the sucrose solutions, it...

Endosomal trafficking is an essential physiological process that implicates various biological events1, for example, the transportation of signaling receptors, ion channels, and adhesion molecules. Proteins localized at the plasma membrane are internalized by endocytosis2. The internalized proteins are then sorted by the early endosome3. Some of the proteins are targeted to lysosomes for degradation4. However, a significant amount of proteins are recycled back to the cell surface by fast recycling and slow recycling processes. In fast recycling, proteins leave the early endosomes and directly return to the plasma membrane. Conversely, in slow recycling, proteins are first sorted to the endocytic recycling compartment and then transported back to the plasma membrane. Various cargo proteins, for example, clathrin, retromer complex, retriever complex and Wiskott-Aldrich syndrome protein, and SCAR Homologue (WASH) complex, participate in such membrane recycling processes4,5,6,7,8,9. The balance of the endocytosis and recycling event is crucial for cell survival and contributes to various cellular events10, for instance, cell adhesion, cell migration, cell polarity, and signal transduction.
ARF6, a small GTPase, is a reported regulator of endocytic trafficking7,11,12. Of interest, various research groups have illustrated the importance of ARF6 in endocytic recycling13,14,15,16,17. The study aims to investigate the relationship between ARF6-mediated neurite outgrowth and endocytic recycling. The previous report suggests that the activation of ARF6 is upstream to Rac1 activity through acting on ELMO1-dedicator of cytokinesis 1 (DOCK180) complex18. However, how ARF6 triggers ELMO1-DOCK180 mediated Rac1 signaling remains unclear. Density gradient ultracentrifugation was employed to investigate the role of ARF6-mediated endocytic recycling in such process. By using that, the recycling endosome-containing fraction was obtained from cell lysates19. The fraction was subjected to Western blotting for protein content analysis. The immunoblot results revealed that under the presence of FE65, a brain-enriched adaptor protein, active ARF6 substantially increased the level of ELMO1 in the recycling endosome-containing fraction. The following protocol includes the procedures for (1) transfecting mammalian cells; (2) preparing the samples and density gradient columns; and (3) obtaining the recycling endosome-containing fraction.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL, Open-Top Thickwall Polypropylene Tube, 11 x 34 mm</td>
			<td>Beckman Coulter</td>
			<td>347287</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm tissue culture dish</td>
			<td>SPL</td>
			<td>20100</td>
			<td></td>
		</tr>
		<tr>
			<td>13.2 mL, Certified Free Open-Top Thinwall Ultra-Clear Tube, 14 x 89 mm</td>
			<td>Beckman Coulter</td>
			<td>C14277</td>
			<td></td>
		</tr>
		<tr>
			<td>5x Sample Buffer</td>
			<td>GenScript</td>
			<td>MB01015</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete, EDTA-free Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>COX IV (3E11) Rabbit mAb</td>
			<td>Cell Signaling Technology</td>
			<td>4850S</td>
			<td>Rabbit monoclonal antibody for detecting COX IV.</td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma-Aldrich</td>
			<td>C1988</td>
			<td></td>
		</tr>
		<tr>
			<td>Dounce Tissue Grinder, 7 mL</td>
			<td>DWK Life Sciences</td>
			<td>357542</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM) with low glucose</td>
			<td>HyClone</td>
			<td>SH30021.01</td>
			<td></td>
		</tr>
		<tr>
			<td>ELMO1 antibody (B-7)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-271519</td>
			<td>Mouse monoclonal antibody for detecting ELMO1.</td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit</td>
			<td>QIAGEN</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr>
			<td>FE65 antibody (E-20)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-19751</td>
			<td>Goat polyclonal antibody for detecting FE65.</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, Research Grade</td>
			<td>HyClone</td>
			<td>SV30160.03</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH Monoclonal Antibody (6C5)</td>
			<td>Ambion</td>
			<td>AM4300</td>
			<td>Mouse monoclonal antibody for detecting GAPDH.</td>
		</tr>
		<tr>
			<td>ImageLab Software</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>Measurement of band intensity</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>I2399</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 Transfection Reagent</td>
			<td>Invitrogen</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal Anti-&#946;-COP antibody</td>
			<td>Sigma</td>
			<td>G6160</td>
			<td>Mouse monoclonal antibody for detecting &#946;-COP.</td>
		</tr>
		<tr>
			<td>Myc-tag (9B11) mouse mAb</td>
			<td>Cell Signaling Technology</td>
			<td>2276S</td>
			<td>Mouse monoclonal antibody for detecting myc tagged proteins.</td>
		</tr>
		<tr>
			<td>OmniPur EDTA, Disodium Salt, Dihydrate</td>
			<td>Calbiochem</td>
			<td>4010-OP</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima L-100 XP</td>
			<td>Beckman Coulter</td>
			<td>392050</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima MAX-TL</td>
			<td>Beckman Coulter</td>
			<td>A95761</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Media</td>
			<td>Gibco</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS Tablets</td>
			<td>Gibco</td>
			<td>18912014</td>
			<td></td>
		</tr>
		<tr>
			<td>PhosSTOP</td>
			<td>Roche</td>
			<td>4906845001</td>
			<td></td>
		</tr>
		<tr>
			<td>RAB11A-Specific Polyclonal antibody</td>
			<td>Proteintech</td>
			<td>20229-1-AP</td>
			<td>Rabbit polyclonal antibody for detecting Rab11.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Affymetrix</td>
			<td>AAJ21931A4</td>
			<td></td>
		</tr>
		<tr>
			<td>SW 41 Ti Swinging-Bucket Rotor</td>
			<td>Beckman Coulter</td>
			<td>331362</td>
			<td></td>
		</tr>
		<tr>
			<td>TLA-120.2 Fixed-Angle Rotor</td>
			<td>Beckman Coulter</td>
			<td>362046</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>Gibco</td>
			<td>25300062</td>
			<td></td>
		</tr>
	</tbody>
</table>

density gradient ultracentrifugation for investigating endocytic recycling in mammalian cells

Endosomal trafficking is an essential cellular process that regulates a broad range of biological events. Proteins are internalized from the plasma membrane and then transported to the early endosomes. The internalized proteins could be transited to the lysosome for degradation or recycled back to the plasma membrane. A robust endocytic recycling pathway is required to balance the removal of membrane materials from endocytosis. Various proteins are reported to regulate the pathway, including ADP-ribosylation factor 6 (ARF6). Density gradient ultracentrifugation is a classical method for cell fractionation. After the centrifugation, organelles are sedimented at their isopycnic surface. The fractions are collected and used for other downstream applications. Described here is a protocol to obtain a recycling endosome-containing fraction from transfected mammalian cells using density gradient ultracentrifugation. The isolated fractions were subjected to standard Western blotting for analyzing their protein contents. By employing this method, we identified that the plasma membrane targeting of engulfment and cell motility 1 (ELMO1), a Ras-related C3 botulinum toxin substrate 1 (Rac1) guanine nucleotide exchange factor, is through ARF6-mediated endocytic recycling.

After fractionating the untransfected HEK293 cells by density gradient ultracentrifugation, 12 fractions were collected starting from the top of the gradient. The harvested fractions were diluted with the dilution buffer in a 1:1 ratio and subjected to a second round of centrifugation. The samples were then subjected to western blotting for analyzing their protein contents. As shown in Figure 1, the recycling endosome marker Rab11 is detected in fraction 720. Other su...

Watch this Scientific Journal Video about Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells at JoVE.com

Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells

This paper aims to present a protocol for preparing recycling endosomes from mammalian cells using sucrose density gradient ultracentrifugation.

Endosomal trafficking is an essential cellular process that regulates a broad range of biological events. Proteins are internalized from the plasma ...

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Biochemistry

3.9K Views.  The Chinese University of Hong Kong. This paper aims to present a protocol for preparing recycling endosomes from mammalian cells using sucrose density gradient ultracentrifugation.

Video: Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most interestingly, sRNAs are also found outside of cells and are stably present in all biological fluids. As such, extracellular sRNAs represent a novel class of disease biomarkers and are likely involved in cell signaling and intercellular communication networks. To assess their potential as biomarkers, sRNAs can be quantified in plasma, urine, and other fluids. Nevertheless, to fully understand the impact of extracellular sRNAs as endocrine signals, it is important to determine which carriers are transporting and protecting them in biological fluids (e.g., plasma), which cells and tissues contribute to extracellular sRNA pools, and cells and tissues capable of accepting and utilizing extracellular sRNA. To accomplish these goals, it is critical to isolate highly pure populations of extracellular carriers for sRNA profiling and quantification. We have previously demonstrated that lipoproteins, particularly high-density lipoproteins (HDL), transport functional microRNAs (miRNA) between cells and HDL-miRNAs are significantly altered in disease. Here, we detail a new protocol that utilizes tandem HDL isolation with density-gradient ultracentrifugation (DGUC) and fast-protein-liquid chromatography (FPLC) to obtain highly pure HDL for downstream profiling and quantification of all sRNAs, including miRNAs, using both high-throughput sequencing and real-time PCR approaches. This protocol will be a valuable resource for the investigation of sRNAs on HDL.

This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most ...

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

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 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 ...

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in proteins and generating redox stress. Pathological conditions that lead to metal depletion or accumulation are causal agents of different human diseases. Some examples include anemia, acrodermatitis enteropathica, and Wilson&#8217;s and Menkes&#8217; diseases. It is therefore important to be able to measure the levels and transport of transition metals in biological samples with high sensitivity and accuracy in order to facilitate research exploring how these elements contribute to normal physiological functions and toxicity. Zinc (Zn), for example, is a cofactor in many mammalian proteins, participates in signaling events, and is a secondary messenger in cells. In excess, Zn is toxic and can inhibit absorption of other metals, while in deficit, it can lead to a variety of potentially lethal conditions.

Graphite furnace atomic absorption spectroscopy (GF-AAS) provides a highly sensitive and effective method for determining Zn and other transition metal concentrations in diverse biological samples. Electrothermal atomization via GF-AAS quantifies metals by atomizing small volumes of samples for subsequent selective absorption analysis using wavelength of excitation of the element of interest. Within the limits of linearity of the Beer-Lambert Law, the absorbance of light by the metal is directly proportional to concentration of the analyte. Compared to other methods of determining Zn content, GF-AAS detects both free and complexed Zn in proteins and possibly in small intracellular molecules with high sensitivity in small sample volumes. Moreover, GF-AAS is also more readily accessible than inductively coupled plasma mass spectrometry (ICP-MS) or synchrotron-based X-ray fluorescence. In this method, the systematic sample preparation of different cultured cell lines for analyses in a GF-AAS is described. Variations in this trace element were compared in both whole cell lysates and subcellular fractions of proliferating and differentiated cells as proof of principle.

Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance spectroscopy.

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in ...

Isolation and Analysis of Plasma Lipoproteins by Ultracentrifugation

Analysis of plasma lipoproteins and apolipoproteins is an essential part for the diagnosis of dyslipidemia and studies of lipid metabolism and atherosclerosis. Although there are several methods for analyzing plasma lipoproteins, ultracentrifugation is still one of the most popular and reliable methods. Because of its intact separation procedure, the lipoprotein fractions isolated by this method can be used for analysis of lipoproteins, apolipoproteins, proteomes, and functional study of lipoproteins with cultured cells in vitro. Here, we provide a detailed protocol to isolate seven lipoprotein fractions including VLDL (d&#60;1.006 g/mL), IDL (d=1.02 g/mL), LDLs (d=1.04 and 1.06 g/mL), HDLs (d=1.08, 1.10, and 1.21 g/mL) from rabbit plasma using sequential floating ultracentrifugation. In addition, we introduce the readers how to analyze apolipoproteins such as apoA-I, apoB, and apoE by SDS-PAGE and Western blotting and show representative results of lipoprotein and apolipoprotein profiles using hyperlipidemic rabbit models. This method can become a standard protocol for both clinicians and basic scientists to analyze lipoprotein functions.

Several methods have been used for analyzing plasma lipoproteins; however, ultracentrifugation is still one of the most popular and reliable methods. Here, we describe a method regarding how to isolate lipoproteins from plasma using sequential density ultracentrifugation and how to analyze the apolipoproteins for both diagnostic and research purposes.

Analysis of plasma lipoproteins and apolipoproteins is an essential part for the diagnosis of dyslipidemia and studies of lipid metabolism and ...

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and isopycnic density gradient centrifugation. The major advantage of this procedure is that it does not rely on the sole use of solubilizing detergents to obtain subcellular fractions. This makes it possible to separate the plasma membrane from other membrane-bound organelles of the cell. This procedure will facilitate the determination of protein localization in cells with a reproducible, scalable, and selective method. This method has been successfully used to isolate cytosolic, nuclear, mitochondrial, and plasma membrane proteins from the human monocyte cell line, U937. Although optimized for this cell line, this procedure may serve as a suitable starting point for the subcellular fractionation of other cell lines. Potential pitfalls of the procedure and how to avoid them are discussed as are alterations that may need to be considered for other cell lines.

This fractionation protocol will allow researchers to isolate cytoplasmic, nuclear, mitochondrial, and membrane proteins from mammalian cells. The latter two subcellular fractions are further purified via isopycnic density gradient.

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and ...

Polysome Profiling without Gradient Makers or Fractionation Systems

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs being translated by ribosomes, and analyze polysome associated factors. While automated gradient makers and gradient fractionation systems are commonly used with this technique, these systems are generally expensive and can be cost-prohibitive for laboratories that have limited resources or cannot justify the expense due to their infrequent or occasional need to perform this method for their research. Here, a protocol is presented to reproducibly generate polysome profiles using standard equipment available in most molecular biology laboratories without specialized fractionation instruments. Moreover, a comparison of polysome profiles generated with and without a gradient fractionation system is provided. Strategies to optimize and produce reproducible polysome profiles are discussed. Saccharomyces cerevisiae is utilized as a model organism in this protocol. However, this protocol can be easily modified and adapted to generate ribosome profiles for many different organisms and cell types.

This protocol describes how to generate a polysome profile without using automated gradient makers or gradient fractionation systems.

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs ...

Efficient and Scalable Production of Full-length Human Huntingtin Variants in Mammalian Cells using a Transient Expression System

Full-length huntingtin (FL HTT) is a large (aa 1-3,144), ubiquitously expressed, polyglutamine (polyQ)-containing protein with a mass of approximately 350 kDa. While the cellular function of FL HTT is not entirely understood, a mutant expansion of the polyQ tract above ~36 repeats is associated with Huntington's disease (HD), with the polyQ length correlating roughly with the age of onset. To better understand the effect of structure on the function of mutant HTT (mHTT), large quantities of the protein are required. Submilligram production of FL HTT in mammalian cells was achieved using doxycycline-inducible stable cell line expression. However, protein production from stable cell lines has limitations that can be overcome with transient transfection methods. 
This paper presents a robust method for low-milligram quantity production of FL HTT and its variants from codon-optimized plasmids by transient transfection using polyethylenimine (PEI). The method is scalable (&gt;10 mg) and consistently yields 1-2 mg/L of cell culture of highly purified FL HTT. Consistent with previous reports, the purified solution state of FL HTT was found to be highly dynamic; the protein has a propensity to form dimers and high-order oligomers. A key to slowing oligomer formation is working quickly to isolate the monomeric fractions from the dimeric and high-order oligomeric fractions during size exclusion chromatography. 
Size exclusion chromatography with multiangle light scattering (SEC-MALS) was used to analyze the dimer and higher-order oligomeric content of purified HTT. No correlation was observed between FL HTT polyQ length (Q23, Q48, and Q73) and oligomer content. The exon1-deleted construct (aa 91-3,144) showed comparable oligomerization propensity to FL HTT (aa 1-3,144). Production, purification, and characterization methods by SEC/MALS-refractive index (RI), sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot, Native PAGE, and Blue Native PAGE are described herein.

We provide scalable protocols covering construct design, transient transfection, and expression and purification of full-length human huntingtin protein variants in HEK293 cells.

Full-length huntingtin (FL HTT) is a large (aa 1-3,144), ubiquitously expressed, polyglutamine (polyQ)-containing protein with a mass of approximately 350 ...

Quantifying Bacterial Surface Swarming Motility on Inducer Gradient Plates

Bacterial swarming motility is a common microbiological phenotype that bacterial communities use to migrate over semisolid surfaces. In investigations of induced swarming motility, specific concentration&#160;of an inducer may not be able to report events occurring within the optimal concentration range to elicit the desired responses from a species. Semisolid plates containing multiple concentrations are commonly used to investigate the response within an inducer concentration range. However, separate semisolid plates increase variations in medium viscosity and moisture content within each plate due to nonuniform solidification time.
This paper describes a one-step method to simultaneously test surface swarming motility on a single gradient plate, where the isometrically arranged test wells allow the simultaneous acquisition of multiconcentration responses. In the present work, the surface swarming of Escherichia coli K12 and Pseudomonas aeruginosa PAO1 were evaluated in response to a concentration gradient of inducers such as resveratrol and arabinose. Periodically, the swarm morphologies were imaged using an imaging system to capture the entire surface swarming process.
The quantitative measurement of the swarm morphologies was acquired using ImageJ software, providing analyzable information of the swarm area. This paper presents a simple gradient swarm plate method that provides qualitative and quantitative information about the inducers&#39; effects on surface swarming, which can be extended to study the effects of other inducers on a broader range of motile bacterial species.

Here, we describe the use of inducer gradient plates to evaluate bacterial swarming motility while simultaneously obtaining multiple concentration responses.

Bacterial swarming motility is a common microbiological phenotype that bacterial communities use to migrate over semisolid surfaces. In investigations of ...